CN116606251A - Fluorescent probe and preparation method and application thereof - Google Patents
Fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN116606251A CN116606251A CN202310354400.1A CN202310354400A CN116606251A CN 116606251 A CN116606251 A CN 116606251A CN 202310354400 A CN202310354400 A CN 202310354400A CN 116606251 A CN116606251 A CN 116606251A
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- fluorescent probe
- compound
- golgi
- chloride ions
- fluorescent
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 109
- 238000002360 preparation method Methods 0.000 title abstract description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims abstract description 73
- 150000001875 compounds Chemical class 0.000 claims abstract description 41
- 238000003756 stirring Methods 0.000 claims abstract description 18
- HFDLDPJYCIEXJP-UHFFFAOYSA-N 6-methoxyquinoline Chemical compound N1=CC=CC2=CC(OC)=CC=C21 HFDLDPJYCIEXJP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 23
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 claims description 11
- LSTRKXWIZZZYAS-UHFFFAOYSA-N 2-bromoacetyl bromide Chemical compound BrCC(Br)=O LSTRKXWIZZZYAS-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
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- 238000012360 testing method Methods 0.000 description 12
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 239000007995 HEPES buffer Substances 0.000 description 9
- 239000000975 dye Substances 0.000 description 9
- 238000002189 fluorescence spectrum Methods 0.000 description 9
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- 230000004044 response Effects 0.000 description 8
- 150000001450 anions Chemical class 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000012981 Hank's balanced salt solution Substances 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 6
- 150000001768 cations Chemical class 0.000 description 6
- 230000008045 co-localization Effects 0.000 description 6
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- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
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- JZNKNTMOKKSWRT-UHFFFAOYSA-N 2-bromo-n-(4-sulfamoylphenyl)acetamide Chemical compound NS(=O)(=O)C1=CC=C(NC(=O)CBr)C=C1 JZNKNTMOKKSWRT-UHFFFAOYSA-N 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 2
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- 210000003712 lysosome Anatomy 0.000 description 2
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- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 2
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- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 2
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
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- 238000006206 glycosylation reaction Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
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- 230000003834 intracellular effect Effects 0.000 description 1
- -1 iodide ions Chemical class 0.000 description 1
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- 229940055695 pancreatin Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D215/20—Oxygen atoms
-
- 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
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a fluorescent probe and a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The preparation method of the fluorescent probe provided by the invention comprises the following steps: the preparation method comprises the steps of stirring, reacting, separating and purifying the compound I shown in the formula I and 6-methoxyquinoline in DMF to obtain the fluorescent probe compound. The fluorescence of the fluorescent probe synthesized by the invention can be quenched selectively by chloride ions, and the Stern-Volmer constant of the chloride ions is not influenced by pH in the vicinity of physiological conditions. The compound has good golgi targeting ability, and the fluorescence intensity changes along with the change of the chloride ion concentration in the golgi, which proves that the probe can be used for detecting the change of the chloride ion concentration in the golgi.
Description
Technical Field
The invention relates to the technical field of novel small molecular fluorescent probes, in particular to a fluorescent probe and a preparation method and application thereof.
Background
The chloride ion is the anion with the highest content in the human body, is distributed in all cell types, and plays an important role in regulating the physiological processes of cell volume, membrane potential, maintaining the stability of the pH value of the cells and the like. Typically, the extracellular chloride concentration (120 mM) is much higher than the chloride concentration in the cytoplasm (5-40 mM).
The golgi apparatus, also known as golgi apparatus or golgi complex, is a weakly acidic subcellular apparatus that is ubiquitous in eukaryotic cells and has a pH between 6.0 and 6.5 under physiological conditions. The main function of the golgi apparatus is to process, classify and package various proteins synthesized by the endoplasmic reticulum, and then transport them to specific sites of cells or secrete them outside the cells, respectively. As a final processing and packaging site for cell secretions (e.g. proteins), golgi apparatus is involved in a variety of biological processes such as cell polarization, stress response, directional migration, mitosis, metabolism, autophagy, apoptosis, DNA repair, and the like. When the cell is subjected to external stimulus or damage to cause the content of substances in the organelle to change, the main function of the organelle is affected, thereby affecting the physiological function of the whole cell. Imbalance in chloride ion concentration in the golgi apparatus may impair its function of precisely processing and sorting corresponding proteins into specific regions of neurons to induce apoptosis. Meanwhile, the imbalance of chloride ion concentration causes the pH change in the golgi apparatus, which can directly reduce glycosylation and change the structure and function of the golgi apparatus so as to cause a series of diseases related to the golgi apparatus, such as Alzheimer's disease, cystic fibrosis, parkinson's disease, liver diseases and the like.
Therefore, the design and synthesis of the fluorescent probe capable of detecting the change of the chloride ion concentration in the high-definition matrix in real time have very important significance.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a fluorescent probe which can detect chloride ions through fluorescence quenching caused by collision with the chloride ions, can specifically target a golgi body, and can detect the change of the concentration of the chloride ions in the golgi body.
The invention also provides a preparation method of the fluorescent material.
The invention also provides application of the fluorescent material.
The first aspect of the invention provides a fluorescent probe, and the structural formula of the fluorescent probe is shown as II:
the fluorescent probe according to the embodiment of the first aspect of the invention has at least the following beneficial effects:
the fluorescent probe provided by the invention is synthesized by taking 6-methoxyquinoline as a chloride ion recognition site and p-aminobenzene sulfonamide as a Golgi body targeting group. Specific: interaction of quinoline groups with chloride ions causes fluorescence quenching of quinoline structures, i.e., fluorescence of the fluorescent probe of the invention can be selectively quenched by chloride ions, the fluorescent probe has moderate-intensity affinity to chloride ions, the Stern-Volmer constant of chloride ions is not affected by pH, and the Stern-Volmer constant is about 60M under physiological pH conditions -1 。
The fluorescent probe provided by the invention has the advantages of very rapid response to chloride ions, good specificity and anti-interference performance, good biocompatibility, low toxicity and small damage to cells.
The fluorescent probe provided by the invention has good stability and good golgi targeting capability, and the fluorescence intensity of the fluorescent probe changes along with the change of the concentration of chloride ions in the golgi.
Therefore, the probe provided by the invention can detect chloride ions through fluorescence quenching caused by collision with the chloride ions, can specifically target the Golgi apparatus, and can detect the change of the chloride ion concentration in the Golgi apparatus.
The second aspect of the present invention provides a method for producing a fluorescent material, the method comprising the steps of:
the preparation method comprises the steps of stirring, reacting, separating and purifying a compound I shown as the I and 6-methoxyquinoline in DMF to obtain the fluorescent probe compound;
the preparation method of the fluorescent material according to the embodiment of the second aspect of the present invention has at least the following advantages:
the preparation method of the fluorescent probe has simple and mild conditions, can be completed in two steps, and has simpler subsequent treatment.
According to some embodiments of the invention, the method of synthesizing compound I as shown in I comprises reacting p-aminobenzenesulfonamide and bromoacetyl bromide in acetone with stirring.
According to some embodiments of the invention, the compound I is 2-bromo-N- (4-sulfamoylphenyl) acetamide, CAS number 5332-70-7.
According to some embodiments of the invention, the yield of compound i is about 30-36%.
Preferably, the yield of compound i is about 35%.
According to some embodiments of the invention, the molar ratio of p-aminobenzenesulfonamide to bromoacetyl bromide is 1: (0.9-1).
Preferably, the molar ratio of p-aminobenzenesulfonamide to bromoacetyl bromide is about 1:0.95.
according to some embodiments of the invention, the mass to volume ratio of the para-aminobenzenesulfonamide to the acetone is (0.08-0.2) g:1ml.
Preferably, the mass to volume ratio of p-aminobenzenesulfonamide to acetone is about 0.17g:1mL.
According to some embodiments of the invention, the temperature of the stirring reaction in the synthesis of compound I is 50-55 ℃.
Preferably, the temperature of the stirring reaction in the synthesis method of the compound I is 52 ℃.
According to some embodiments of the invention, the stirring reaction time in the synthesis method of the compound I is 10-30 min.
Preferably, the stirring reaction time in the synthesis method of the compound I is 20min.
According to some embodiments of the invention, the method for synthesizing the compound I further comprises the step of adding 10-20 mL of water after stirring and reacting for 8-12 h.
Preferably, the method for synthesizing the compound I further comprises the step of adding 15mL of water after stirring and reacting for 10 hours.
According to some embodiments of the invention, the method for synthesizing compound I further comprises a purification and separation operation after the stirring reaction.
According to some embodiments of the invention, the purification and separation method comprises: the solid in the system is recrystallized by ethanol after being washed by water with the temperature of 2-10 ℃.
According to some embodiments of the invention, the solid after recrystallization from ethanol further comprises the steps of filtration and vacuum drying.
According to some embodiments of the invention, the water comprises at least one of distilled water and deionized water.
According to some embodiments of the invention, the temperature of the water is 2-10 ℃ at standard atmospheric pressure.
Preferably, the temperature of the water is 4 ℃ at standard atmospheric pressure.
According to some embodiments of the invention, the molar ratio of compound i to 6-methoxyquinoline is 1: (2-4).
Preferably, the molar ratio of compound i to 6-methoxyquinoline is about 1:2.
according to some embodiments of the invention, the mass to volume ratio of the 6-methoxyquinoline to the DMF solution is (0.02-0.09) g:1ml.
Preferably, the mass to volume ratio of the 6-methoxyquinoline to DMF solution is about 0.04g:1ml.
According to some embodiments of the invention, the temperature of the stirring reaction in the preparation method of the fluorescent probe compound is 90-110 ℃.
Preferably, the temperature of the stirring reaction in the preparation method of the fluorescent probe compound is 100 ℃.
According to some embodiments of the invention, the stirring reaction time in the preparation method of the fluorescent probe compound is 8-12 hours.
Preferably, the stirring reaction time in the preparation method of the fluorescent probe compound is 10 hours.
According to some embodiments of the invention, the method for separating and purifying the fluorescent probe compound comprises: and extracting with dichloromethane and water, collecting a water layer, and freeze-drying to obtain the fluorescent probe compound.
According to some embodiments of the invention, the yield of the fluorescent probe compound is about 80-85%.
Preferably, the yield of the fluorescent probe compound is about 83%.
In a third aspect, the invention provides the use of a fluorescent probe for detecting chloride ions in an organelle.
According to some embodiments of the invention, the use comprises detecting a change in the concentration of chloride ions within the cell.
According to some embodiments of the invention, the organelle comprises at least one of golgi, mitochondrial, lysosomal, and endoplasmic reticulum.
Preferably, the organelle is the golgi apparatus.
The application of the fluorescent probe according to the embodiment of the third aspect of the invention has at least the following beneficial effects:
the fluorescent probe can detect the concentration change of chloride ions in the golgi in real time, and has great significance for elucidating the physiological functions of the golgi and researching the occurrence and development mechanism of diseases related to the golgi.
The term "about" as used herein, unless otherwise specified, means that the tolerance is within + -2%, for example, about 100 is actually 100 + -2%. Times.100.
Additional features and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a synthetic route for the preparation method of example 1 of the present invention;
FIG. 2 is a schematic diagram of a fluorescent probe prepared by the method of example 1 of the present invention 1 H NMR spectrum;
FIG. 3 shows a fluorescent probe prepared by the method of example 1 of the present invention 13 C NMR spectrum;
FIG. 4 is a HR-ESI-MS spectrum of a fluorescent probe prepared by the method for preparing example 1 of the present invention;
FIG. 5 shows a compound I prepared by the method of example 1 of the present invention 1 H NMR spectrum;
FIG. 6 shows a compound I prepared by the method of example 1 of the present invention 13 C NMR spectrum;
FIG. 7 is a spectrum of HR-ESI-MS of compound I prepared by the procedure in example 1 of the present invention;
FIG. 8 is an ultraviolet visible absorption spectrum and a fluorescence emission spectrum of a fluorescent probe according to the present invention in the presence or absence of chloride ions;
(a) An ultraviolet visible absorption spectrum, (b) a fluorescence emission spectrum; lambda (lambda) ex ＝350nm；
FIG. 9 is a graph showing the results of the test of the photostability and response to chloride ions of the fluorescent probe of the present invention, lambda ex /λ em ＝350/453nm；
FIG. 10 is a graph showing the results of fluorescence spectra and fluorescence intensity changes of the fluorescent probe according to the present invention in the absence of chloride ions at different pH;
(a) A fluorescence spectrum, (b) a fluorescence intensity change; lambda (lambda) ex ＝453nm；
FIG. 11 shows the fluorescence probe of the present invention at different pH and different Cl - Fluorescence spectrum in 100mM phosphate buffer at concentration;
FIG. 12 is a fluorescent quilt Cl of the fluorescent probe of the present invention - A quenched Stern-Volmer diagram;
FIG. 13 is a graph showing the results of the selectivity and specificity of the fluorescent probe according to the present invention for common anions and cations in an organism;
FIG. 14 shows Cl concentrations of the fluorescent probe according to the present invention - The fluorescence spectrum below;
FIG. 15 shows the fluorescence intensity at 453nm of the fluorescent probe according to the present invention with Cl - A concentration profile;
FIG. 16 shows the fluorescence intensity at 453nm with Cl of the fluorescent probe of the present invention - A linear plot between concentrations;
FIG. 17 is a graph showing the results of cell viability after 12h of the fluorescent probe of the present invention was applied to HeLa cells;
FIG. 18 is a graph showing the results of a co-localization imaging experiment performed with fluorescent probes of the present invention and HeLa cells;
FIG. 19 is a photograph showing the staining of HeLa cells with different concentrations of chloride ions using the fluorescent probe of the present invention;
(a) Chloride ion concentration was 0mM; (b) chloride ion concentration 9mM; (c) chloride ion concentration is 18mM; (d) chloride ion concentration is 36mM; (e) chloride ion concentration is 72mM; (f) chloride ion concentration was 144mM.
FIG. 20 is a bar graph of relative fluorescence intensity after staining HeLa cells with fluorescent probes of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The preparation raw materials and instruments used in the examples of the present invention:
instrument model and company:
fluorescence spectrometer: perkin Elmer FL8500 fluorescence spectrophotometer, perkin Elmer company, usa;
ultraviolet visible photometer: u-3900, HITACHI;
nuclear magnetic resonance spectrometer: bruker Avance AV 500MHz NMR spectrometer, bruker Inc. of Switzerland;
mass spectrometer: thermo Scientific TM Orbitrap Fusion TM Mass spectrometer, company Thermo Fisher Scientific usa;
laser confocal microscope: LEICA/TCS SP8, leica, germany;
reagent source and manufacturer:
Golgi-Tracker Red, mitochondrial dye Mito-Tracker Red, lysosomal dye Lyso-Tracker Red, endoplasmic reticulum dye ER-Tracker Red: beyotime, shanghai Biyundian biotechnology limited;
para-aminobenzenesulfonamide: CAS:63-74-1; guangzhou Master Spectrum Biotechnology Co., ltd;
bromoacetyl bromide: CAS:598-21-0; beijing carboline technologies Co., ltd;
acetone: CAS:67-64-1; guangdong test agent technology Co., ltd;
ethanol: CAS:64-17-5; anhui Zernike technologies Co., ltd;
6-methoxyquinoline: CAS:5263-87-6; beijing carboline technologies Co., ltd;
DMF: CAS:68-12-2; taiwanese technologies (Guangzhou) limited;
dichloromethane: CAS:75-09-2; taiwanese technologies (Guangzhou) Inc.
Example 1
The invention prepares a fluorescent probe, which comprises the following steps:
paraminobenzenesulfonamide (1.72 g,10 mmol) was weighed and suspended in acetone (10 mL), and bromoacetyl bromide (0.82 mL,9.5 mmol) was added dropwise at 0deg.C. The reaction mixture was stirred at 55℃for 10min, 20ml of ice water was added thereto, and the reaction was stirred for 8h. The collected solid was washed with ice water and recrystallized from ethanol. The solid was collected by filtration and dried in vacuo to give compound i in a yield of 980mg in 35%.
The test results of the specific obtained compounds are: 1 H NMR(500MHz,DMSO-d 6 )δ10.72(s,1H),7.79(d,J＝8.9Hz,2H),7.74(d,J＝8.9Hz,2H),7.29(s,2H),4.07(s,2H)； 13 C NMR(125MHz,DMSO-d 6 )δ172.0,141.9,138.9,127.0,119.6,62.3；HR-ESI-MS for C 8 H 9 N 2 O 3 S([M+H] + ) Calcd 292.9592, found 292.9590. Specific results of the compound I thus obtained are shown in FIGS. 2 to 4.
Compound I (235 mg,0.8 mmol) was weighed into a solution of 6-methoxyquinoline (255 mg,1.6 mmol) in DMF (6 mL) and reacted at 100℃for 8h with stirring. The aqueous layer was collected by extraction with dichloromethane and water and finally weighed after freeze-drying to give 300mg of solid product in 83% yield. The synthetic route of the fluorescent probe of this example is shown in FIG. 1.
The test results of the product obtained in this example are: 1 H NMR(500MHz,DMSO-d 6 )δ11.20(s,1H),9.39(d,J＝4.3Hz),9.23(d,J＝8.1Hz,1H),8.41(d,J＝9.6Hz,1H),8.22(dd,J＝8.1,4.3Hz,1H),7.95(d,J＝2.9Hz,1H),7.90(dd,J＝9.6,2.9Hz,1H),7.79(d,J＝8.9Hz,2H),7.75(d,J＝8.9Hz,2H),7.31(s,2H),6.08(s,2H),4.01(s,3H)； 13 C NMR(500MHz,DMSO-d 6 )δ164.0,159.7,148.8,147.0,141.4,139.7,134.8,131.9,128.6,127.4,122.8,121.0,119.5,108.6,59.9,56.9；HR-ESI-MS calcd for C 18 H 18 N 3 O 4 S + ([M-Br] + ) 372.1012,found 372.1010. As is clear from this, the fluorescent probe compounds of formula II were obtained in the present example, and the specific results are shown in FIGS. 5 to 7.
Test case
Ultraviolet absorption spectrum and fluorescence emission spectrum test of fluorescent probe
Preparing a solution:
(1) HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer solution: weighing a certain amount of HEPES, dissolving with ultrapure water to make the final concentration of the HEPES be 50mM, and adjusting the pH to 7.4 with sodium hydroxide;
(2) fluorescent probe stock solution: dissolving the fluorescent probe compound with HEPES buffer solution (50 mM, pH 7.4) to give a final concentration of the fluorescent probe mother liquor of 10mM;
(3) sodium chloride solution: an amount of sodium chloride was weighed and dissolved in HEPES buffer (50 mM, pH 7.4) to give a final concentration of 5M sodium chloride solution.
And adding a proper amount of fluorescent probe mother solution into HEPES buffer solution (50 mM, pH 7.4) with or without chloride ions to ensure that the concentration of the fluorescent probe in a test system is 100 mu M, the concentration of the chloride ions is 0mM or 50mM respectively, and testing the ultraviolet absorption spectrum and the fluorescence emission spectrum of the fluorescent probe. The fluorescence probe has stronger absorption at 320nm and 350nm, and the absorption spectrum is not changed obviously after the fluorescence probe reacts with chloride ions, as shown in fig. 8 (a). The fluorescence intensity of the fluorescent probe at 453nm was significantly reduced after chloride ion addition under excitation at 350nm, as shown in FIG. 8 (b), relative to the ultraviolet absorption spectrum. This result demonstrates that chloride ions can quench the fluorescence of the fluorescent probe.
Response time and light stability test
A suitable amount of fluorescent probe stock solution was prepared as a test solution containing a fluorescent probe (100. Mu.M) with HEPES buffer solution (50 mM, pH 7.4), and the fluorescence intensity of the HEPES buffer solution (50 mM, pH 7.4) of the fluorescent probe (100. Mu.M) was time-tracked with a fluorescence spectrophotometer. At 30min, a sodium chloride solution was added to bring the chloride ion concentration to 50mM, and the change in fluorescence intensity was continuously observed. After adding chloride ions under excitation of 350nm, the fluorescence intensity of the fluorescent probe is rapidly reduced and instantly stabilized, which indicates that the response of the fluorescent probe to the chloride ions is very rapid, and can be used for monitoring the instant change of the chloride ions, as shown in fig. 9. On the other hand, the fluorescence intensity at 453nm did not significantly change during the monitoring time of 30min in the presence or absence of chloride ion, indicating that the fluorescence probe has good light stability.
Influence of pH
Preparing a solution:
phosphate buffer:
and (3) solution A: 23.996g NaH 2 PO 4 Dissolving in 1L of ultrapure water to obtain a final concentration of 0.2M;
and (2) liquid B: 28.392g Na 2 HPO 4 Dissolving in 1L of ultrapure water to obtain a final concentration of 0.2M;
mixing a certain volume of solution A and solution B, diluting with ultrapure water to make the concentration of phosphate in the test system be 0.1M, and respectively using 1. 1M H 3 PO 4 And 2M NaOH to adjust pH to 3, 4, 5, 6, 7, 8.
To confirm that the fluorescent probe was able to operate at pH conditions within the golgi, the fluorescent probe was tested for fluorescence spectra in phosphate buffer solutions (100 μm) at different pH, as shown in fig. 10 (a). The fluorescence intensity of the fluorescent probe decreases with an increase in pH, as shown in FIG. 10 (b).
To quantitatively characterize the response of the fluorescent probe to chloride ions at different pH's, by gradually varying the concentration of chloride ions in the range of 0 to 250mM with a concentration of the anchoring compound of 100. Mu.M, a regular decrease in the fluorescent intensity of the fluorescent probe was observed, as shown in FIG. 11, and the Stern-Volmer constant was obtained from the Stern-Volmer equation.
F 0 /F＝1+K sv [Cl - ]；
Wherein F is 0 The fluorescence intensity of the fluorescent probe without adding chloride ions is shown, and F is the fluorescence intensity after adding chloride ions. By F 0 F vs [ Cl - ]A straight line is plotted and fitted linearly, as shown in fig. 12, and the slope of the straight line equation is the stem-Volmer constant under the test conditions, as shown in table 1. In summary, the response capacity of fluorescent probes to chloride ions at each pH is substantially equivalent.
TABLE 1 fluorescent probe pairs Cl - Stern-Volmer constant of (F)
Selectivity and interference resistance experiments
And selecting common anions and cations in organisms to perform specificity test of the fluorescent probe. The anions and cations comprise Cl - 、Br - 、I - 、F - 、SO 4 2- 、NO 3 - 、Na + 、Ca 2+ 、Mg 2+ NH (NH) 4 + . 4mL (50 mM HEPES, pH 7.4) of the solution to be tested was prepared with the fluorescent probe, the final concentration of the fluorescent probe was 100. Mu.M, and the final concentration of each of the anions and cations was 100mM. Each set of experiments was repeated three times in parallel and the assay was performed.
Orange-yellow bars represent F of fluorescent probe in the presence of various ions 0 the/F ratio (mean±s.d., n=3). The green bar indicates F of the fluorescent probe in the presence of chloride and competitor ions 0 The ratio of/F (mean±s.d., n=3), as shown in fig. 13, shows that the fluorescent probe can be quenched by chloride, iodide and bromide, while other anions and cations have little quenching effect on the fluorescence of the fluorescent probe. When chloride ions and other anions and cations coexist, the response of the fluorescent probe to the chloride ions is not interfered. In general, the concentration of chloride ions in cytoplasm can reach 5-40 mM, and the concentration of iodide ions and bromide ions in cells is in the order of mu M, which is far lower than the concentration of chloride ions, so the probe is expected to be applied to detecting chloride ions in cells. In conclusion, the fluorescent probe has good specificity and anti-interference performance on chloride ions.
Detection limit
To test the response of the fluorescent probe to chloride ions, the fluorescent probe was mixed with chloride ions of different concentrations and subjected to fluorescence spectrometry. As can be seen from fig. 14, the fluorescence intensity of the fluorescent probe gradually decreases as the chloride ion concentration increases; fluorescence intensity with Cl - The concentration increases and decreases when Cl - As can be seen from FIG. 15, fluorescence is almost completely quenched at a concentration exceeding 150 mM.
The Cl added was subjected to a fluorescence intensity at 453nm - Concentration was plotted and a linear fit was performed, fluorescence intensity versus Cl at pH7.4 - The concentration of 0.1-10 mM has good linear relation, and the linear equation is y= -1307[ Cl ] - ]+51201, the detection limit was calculated using the formula lod=3σ/k, where σ represents the standard deviation of the blank probe tested 15 times in parallel and k represents the slope of the fitted curve, as shown in fig. 16. The detection limit of fluorescent probe for chloride ions lod=34 μm.
Cytotoxicity test
After HeLa cells were cultured to the logarithmic growth phase, they were digested with pancreatin to prepare cell suspensions. 96-well plates (8000 cells/well) were plated. After 24 hours, the medium was aspirated and replaced with 100. Mu.L of medium containing fluorescent probes of different concentrations (1% DMSO content per well, 4 multiplex wells were set for fluorescent probes), and after a further incubation for 12 hours 10. Mu.L of MTT solution (5 mg/mL) was added to each well, after 4 hours the medium in the well was aspirated, and 100. Mu.L of DMSO was added to each well, and after shaking, absorbance at 570nm was measured using an enzyme-labeled instrument.
Even if the concentration of the fluorescent probe is higher than 3mM, the cell viability is as high as 90%, and as shown in FIG. 17, the cytotoxicity of the fluorescent probe is small.
Cell co-localization experiments
In order to verify whether the fluorescent probe can specifically target the Golgi area, four subcellular organelle specific dyes, including Golgi-Tracker Red, mitochondrial dye Mito-Tracker Red, lysosome dye Lyso-Tracker Red and endoplasmic reticulum dye ER-Tracker Red, are co-transfected with HeLa cells with the fluorescent probe, and a co-localization imaging experiment is performed to explore the targeting localization capability of the fluorescent probe for each subcellular organelle. The concentration of the fluorescent probe was 2mM, the concentration of Golgi-Tracker Red was 150. Mu.g/mL, and the concentrations of the remaining three dyes were 1. Mu.M.
The fluorescent probe and Golgi-Tracker Red exhibit good co-localization coefficients, which are 0.814. The co-localization effect was relatively poor with all three other dyes, with co-localization coefficients of 0.401 for mitochondria, 0.627 for lysosomes, and 0.314 for endoplasmic reticulum, as shown in fig. 18. These results demonstrate that the fluorescent probes have good golgi targeting ability.
Detection of chloride ion concentration change in golgi
HeLa cells were grown at 5X 10 4 The density of the spots/wells was inoculated into a confocal dedicated petri dish and incubated for 24h. After the incubation, the supernatant was removed and washed with a chloride ion-free HBSS solution (1 mL. Times.3). Then, 1mL of a chloride ion-free HBSS solution containing 10% FBS was added and incubated in an incubator at 37℃for 6 hours. After 6h, the supernatant was removed and washed with a chloride ion-free HBSS solution (1 mL. Times.3). HBSS solutions containing fluorescent probes (2 mM) at different chloride ion concentrations were then added to the dishes, respectively, and incubated for 2.5h at 37 ℃. After 2.5 hours, the dishes were removed, the supernatants were washed with HBSS solutions (1 mL. Times.3) corresponding to the concentrations of chloride ions, and 1mL of HBSS solution corresponding to the concentrations of chloride ions was added, respectively. Finally observe under the laser confocal microscopeStaining of cells and selection of appropriate areas for photographing, as shown in fig. 19.
The blue fluorescence intensity was quantitatively calculated on the photographed cell picture using ImageJ software, and the higher the chloride ion concentration, the lower the intracellular fluorescence intensity, and the concentration-dependent decrease was seen from fig. 20. This result demonstrates that the fluorescent probe can be applied to detect changes in chloride ion concentration in the golgi apparatus.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The fluorescent probe is characterized by having a structural formula shown as II:
2. the method for preparing the fluorescent probe according to claim 1, wherein the method comprises the steps of stirring, reacting, separating and purifying a compound I shown in the formula I and 6-methoxyquinoline in DMF to obtain the fluorescent probe compound;
3. the method for preparing a fluorescent probe according to claim 2, wherein the method for synthesizing the compound I shown in the formula I comprises the step of stirring and reacting p-aminobenzenesulfonamide and bromoacetyl bromide in acetone.
4. The method for preparing a fluorescent probe according to claim 3, wherein the molar ratio of the p-aminobenzenesulfonamide to bromoacetyl bromide is 1: (0.9-1); preferably, the mass volume ratio of the p-aminobenzene sulfonamide to the acetone is (0.08-0.2) g:1ml.
5. The method for preparing a fluorescent probe according to claim 2, wherein the molar ratio of the compound i to 6-methoxyquinoline is 1: (2-4).
6. The method for preparing a fluorescent probe according to claim 2, wherein the mass volume ratio of the 6-methoxyquinoline to the DMF solution is (0.02-0.04) g:1ml.
7. The method for preparing a fluorescent probe according to claim 2, wherein the stirring reaction conditions are: the reaction temperature is 90-110 ℃; the reaction time is 8-12 h.
8. The method for preparing a fluorescent probe according to claim 2, wherein the method for separation and purification comprises the following steps: and extracting with dichloromethane and water, collecting a water layer, and freeze-drying to obtain the fluorescent probe compound.
9. Use of the fluorescent probe of claim 1 for detecting chloride ions in a cell.
10. The use of claim 10, wherein the organelle comprises at least one of golgi, mitochondrial, lysosomal, and endoplasmic reticulum.
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