CN112898344A - Preparation method and application of targeted mitochondrial fluorescein derivative fluorescent probe - Google Patents
Preparation method and application of targeted mitochondrial fluorescein derivative fluorescent probe Download PDFInfo
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- CN112898344A CN112898344A CN202110087700.9A CN202110087700A CN112898344A CN 112898344 A CN112898344 A CN 112898344A CN 202110087700 A CN202110087700 A CN 202110087700A CN 112898344 A CN112898344 A CN 112898344A
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- fluorescein
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- hydrazine
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 title claims abstract description 178
- 230000002438 mitochondrial effect Effects 0.000 title claims abstract description 82
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 84
- -1 fluorescein hydrazine compound Chemical class 0.000 claims abstract description 58
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Substances NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims abstract description 49
- 230000008685 targeting Effects 0.000 claims abstract description 49
- 239000000126 substance Substances 0.000 claims abstract description 46
- 150000001413 amino acids Chemical class 0.000 claims abstract description 44
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims abstract description 37
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims abstract description 37
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 239000003446 ligand Substances 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 18
- MLOSJPZSZWUDSK-UHFFFAOYSA-N 4-carboxybutyl(triphenyl)phosphanium;bromide Chemical compound [Br-].C=1C=CC=CC=1[P+](C=1C=CC=CC=1)(CCCCC(=O)O)C1=CC=CC=C1 MLOSJPZSZWUDSK-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 11
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 10
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 10
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical group [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims description 9
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 abstract description 92
- 238000001514 detection method Methods 0.000 abstract description 21
- 210000001700 mitochondrial membrane Anatomy 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 156
- 239000000243 solution Substances 0.000 description 108
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 102
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- 235000001014 amino acid Nutrition 0.000 description 39
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 30
- 238000005406 washing Methods 0.000 description 21
- LHIJANUOQQMGNT-UHFFFAOYSA-N aminoethylethanolamine Chemical compound NCCNCCO LHIJANUOQQMGNT-UHFFFAOYSA-N 0.000 description 18
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- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 5
- VRVRGVPWCUEOGV-UHFFFAOYSA-N 2-aminothiophenol Chemical compound NC1=CC=CC=C1S VRVRGVPWCUEOGV-UHFFFAOYSA-N 0.000 description 5
- JKIFPWHZEZQCQA-UHFFFAOYSA-N 2-nitrobenzenethiol Chemical compound [O-][N+](=O)C1=CC=CC=C1S JKIFPWHZEZQCQA-UHFFFAOYSA-N 0.000 description 5
- 239000012300 argon atmosphere Substances 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- DFUXHKDPVJEPOX-UHFFFAOYSA-N hydroxycarbamoyl acetate Chemical compound CC(=O)OC(=O)NO DFUXHKDPVJEPOX-UHFFFAOYSA-N 0.000 description 5
- 239000012299 nitrogen atmosphere Substances 0.000 description 5
- 238000002390 rotary evaporation Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 4
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- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
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- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 2
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
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- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 1
- WDCYWAQPCXBPJA-UHFFFAOYSA-N 1,3-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC([N+]([O-])=O)=C1 WDCYWAQPCXBPJA-UHFFFAOYSA-N 0.000 description 1
- DHEJDHPRYHYLKS-UHFFFAOYSA-N 2-(3,6-dihydroxy-9h-xanthen-9-yl)benzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 DHEJDHPRYHYLKS-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 1
- 231100000111 LD50 Toxicity 0.000 description 1
- 208000029549 Muscle injury Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
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- 125000002587 enol group Chemical group 0.000 description 1
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- 239000003344 environmental pollutant Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- ADAUKUOAOMLVSN-UHFFFAOYSA-N gallocyanin Chemical compound [Cl-].OC(=O)C1=CC(O)=C(O)C2=[O+]C3=CC(N(C)C)=CC=C3N=C21 ADAUKUOAOMLVSN-UHFFFAOYSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-O hydrazinium(1+) Chemical compound [NH3+]N OAKJQQAXSVQMHS-UHFFFAOYSA-O 0.000 description 1
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
- C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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Abstract
The invention discloses a preparation method of a targeted mitochondrial fluorescein derivative fluorescent probe, belonging to the technical field of biology, wherein fluorescein hydrazine hydrate is obtained by reacting fluorescein with hydrazine hydrate; adding the fluorescein hydrazine compound into an amino acid absolute ethyl alcohol solution for reaction to obtain a fluorescein hydrazine derivative; reacting the fluorescein hydrazine derivative with a mitochondrial targeting ligand to obtain a fluorescein hydrazine derivative-mitochondrial targeting substance; fluorescein hydrazinization derivatization-mitochondrial targeting substances react with recognition molecules to obtain the targeted mitochondrial fluorescein derivative fluorescent probe. The targeted mitochondrial fluorescein derivative fluorescent probe obtained by the invention has high identification on thiophenol and strong anti-interference detection capability; the positioning effect on mitochondria is good, and the Pearson coefficient obtained by detecting the mitochondria is 0.96; mitochondrial membrane potential drops significantly.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a targeted mitochondrial fluorescein derivative fluorescent probe.
Background
Mitochondria are the "energy mill" of most cells, which produce Reactive Oxygen Species (ROS) by respiration. Normally, mitochondria produce normal amounts of oxide through aerobic metabolism, but when they are attacked by trace amounts of toxic substances, they quickly produce large amounts of oxide, breaking the intracellular oxidative-antioxidant balance, a phenomenon known as poison-induced oxidative stress. It is known that thiophenol is the most toxic of all environmental pollutants, has a median lethal dose of only 0.01-0.4mM for fish and 46.2mg/kg for rats, and causes serious diseases in humans after prolonged exposure to thiophenol atmosphere, such as: muscle damage, asthma, nervous system damage and even death. Although oxidative stress damage caused by trace amounts of thiophenol residues is very extensive, it is often overlooked by humans due to lack of obvious symptoms. Because thiophenol preferentially attacks mitochondria in cells and mitochondria are associated with the production of many pathogenic reactive oxygen species, it is desirable to detect thiophenol in mitochondria.
The small molecule fluorescent probe method has become the most effective method for detecting thiophenol in organisms due to the advantages of simple operation, high sensitivity, low cost and the like. The fluorescent probe mainly comprises three parts, namely a fluorophore, a recognition group and a connecting arm. The fluorophore may act as a signal converter to convert environmental changes into a fluorescent signal representing a change in the properties of the fluorophore. The type of fluorophore determines a number of properties of the probe, for example: fluorescence intensity, excitation and emission wavelength. Commonly used fluorophores are: rhodamine (Rhodamine), Coumarin (Coumarin), Fluorescein (Fluorescein), cyanine (Gallocyanine), BODIPY (BODIPY), and the like.
Some small molecule fluorescent probes of thiophenol have been developed, but since some biological thiols include cysteine, glutathione, and homocysteine, which have similar chemical properties to thiophenol, the probes are easily interfered by these biological thiols when detecting thiophenol. The method realizes the specific detection of the thiophenol in the mitochondria, and has important significance for monitoring the cell damage caused by the thiophenol in real time and deeply knowing the oxidative stress process induced by toxic substances.
Disclosure of Invention
The invention aims to provide a fluorescein derivative which can be used for a fluorescent probe, can detect thiophenol in mitochondria of cells and has good specificity.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a fluorescein derivative having the formula:
Preferably, R1Comprises the following steps:
preferably, R2Comprises the following steps:
preferably, R3Comprises the following steps:
the invention also aims to provide a preparation method of the targeted mitochondrial fluorescein derivative fluorescent probe, which has good selectivity on the thiophenol, good mitochondrial positioning, capability of reducing the mitochondrial membrane potential and high yield.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a preparation method of a targeted mitochondrial fluorescein derivative fluorescent probe comprises the following steps: reacting fluorescein with hydrazine hydrate to obtain fluorescein hydrazine compound; adding the fluorescein hydrazine compound into an amino acid absolute ethyl alcohol solution for reaction to obtain a fluorescein hydrazine derivative; reacting the fluorescein hydrazine derivative with a mitochondrial targeting ligand to obtain a fluorescein hydrazine derivative-mitochondrial targeting substance; fluorescein hydrazinization derivatization-mitochondrial targeting substances react with recognition molecules to obtain targeted mitochondrial fluorescein derivative fluorescent probes; the mitochondrion targeting ligand is 4-carboxybutyl triphenyl phosphonium bromide; the recognition molecule is 2, 4-dinitrofluorobenzene; the amino acid is tyrosine. 4-carboxybutyl triphenyl phosphonium bromide is introduced into fluorescein molecules, so that the fluorescent probe has stronger recognition on mitochondria and better selectivity; by introducing recognition molecules, the specific recognition of the fluorescent probe is improved; the amino acid is introduced into fluorescein molecules, the fluorescence intensity is improved through intermolecular conjugation effect and enol structure transformation, and the introduction of the 4-carboxybutyltriphenylphosphonium bromide and the amino acid stimulates the activity of cells, improves the activity degree of mitochondria, reduces the mitochondrial membrane potential and improves the detection efficiency.
Preferably, fluorescein hydrazide preparation: dissolving fluorescein in absolute ethyl alcohol, adding an absolute ethyl alcohol solution containing hydrazine hydrate, stirring and refluxing for 12-36h, removing the solvent by rotary evaporation, adding a hydrochloric acid solution, adding sodium hydroxide to adjust the pH value to 8-9, precipitating, filtering, washing with deionized water, and drying in vacuum to obtain fluorescein hydrazine hydrate; the adding amount of the fluorescein is 1-5 wt% of the anhydrous ethanol, the mass fraction of the hydrazine hydrate is 60-90 wt%, the proportion of the hydrazine hydrate in the anhydrous ethanol solution of the hydrazine hydrate is 5-20 wt%, the adding amount of the anhydrous ethanol solution containing the hydrazine hydrate is 30-80 wt% of the anhydrous ethanol solution containing the fluorescein, the concentration of the hydrochloric acid solution is 0.01-1M, and the adding amount of the hydrochloric acid solution is 50-100 wt% of the anhydrous ethanol solution containing the fluorescein.
More preferably, the dropping rate of the anhydrous ethanol solution containing hydrazine hydrate is 0.5-3 mL/min.
Preferably, the fluorescein hydrazinized derivative is prepared by: dissolving amino acid and sodium hydroxide in absolute ethyl alcohol at the temperature of 30-60 ℃ to obtain an amino acid absolute ethyl alcohol solution, adding fluorescein hydrazine compound, carrying out reflux reaction at the temperature of 60-75 ℃ for 3-12h, cooling to 20-30 ℃, carrying out suction filtration, washing with absolute ethyl alcohol for 2-3 times, and carrying out vacuum drying to obtain a fluorescein hydrazine derivative; the amino acid is tyrosine, the mass fraction of the amino acid is 1-10 wt% of the amino acid absolute ethyl alcohol solution, the mass fraction of the sodium hydroxide is 0.5-3 wt% of the amino acid absolute ethyl alcohol solution, and the addition amount of the fluorescein hydrazine compound is 2-30 wt% of the amino acid absolute ethyl alcohol solution; the preparation of the fluorescein hydrazine derivative is carried out under the protection of nitrogen atmosphere.
Preferably, fluorescein hydrazinization derivative-mitochondrial targeting preparation: dissolving the fluorescein hydrazine derivative in DMSO, adding a mitochondrial target, reacting for 6-24h, adding absolute ethanol to precipitate, filtering, washing with absolute ethanol for 2-3 times, and vacuum drying to obtain the fluorescein hydrazine derivative; the addition amount of the fluorescein hydrazine compound is 0.5-4 wt% of DMSO, the mitochondrial target is 4-carboxybutyltriphenylphosphonium bromide, and the addition amount of the 4-carboxybutyltriphenylphosphonium bromide is 80-130 wt% of the fluorescein hydrazine compound.
Preferably, the preparation of the fluorescence probe of the targeted mitochondrial fluorescein derivative comprises the following steps: dissolving fluorescein hydrazinization derivative-mitochondrial target in dichloromethane, adding dichloromethane solution containing triethylamine, stirring for 10-60min, adding recognition molecules, reacting at the temperature of 20-50 ℃ for 6-24h, cooling to minus 10-5 ℃ to separate out precipitate, performing suction filtration, washing with ethyl dichloride, and drying to obtain a targeted mitochondrial fluorescein derivative fluorescent probe; the mass fraction of the fluorescein hydrazinization-mitochondrial targeting substance in the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance is 1-6 wt%, the mass fraction of the triethylamine in the dichloromethane solution of the triethylamine is 1-4 wt%, the addition amount of the dichloromethane solution of the triethylamine is 60-120 wt% of the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance, the addition amount of the recognition molecule is 2, 4-dinitrofluorobenzene, and the addition amount of the recognition molecule is 0.5-2 wt%.
More preferably, the fluorescein derivative fluorescent probe is prepared under an argon atmosphere.
More preferably, the addition of methylene chloride containing triethylamine is in ice bath conditions.
More preferably, the dropwise addition rate of the methylene chloride solution containing triethylamine is 0.5 to 3 mL/min.
Preferably, the preparation of the fluorescence probe of the targeted mitochondrial fluorescein derivative comprises the following steps: dissolving fluorescein hydrazinization derivative-mitochondrial target in dichloromethane, adding hydroxyethyl ethylenediamine and acetoxyhydroxamic acid, adding dichloromethane solution containing triethylamine, stirring for 10-60min, adding recognition molecules, reacting at 20-50 ℃ for 6-24h, cooling to-10 to 5 ℃ to separate out precipitate, performing suction filtration, washing with glacial dichloroethane, and drying to obtain the targeted mitochondrial fluorescein derivative fluorescent probe; the mass fraction of the fluorescein hydrazinization-mitochondrial targeting substance in the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance is 1-6 wt%, the addition amount of hydroxyethyl ethylenediamine is 0.2-1.2 wt% of the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance, the addition amount of acetohydroxamic acid is 0.1-0.8 wt% of the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance, the mass fraction of triethylamine in the dichloromethane solution of triethylamine is 1-4 wt%, the addition amount of the dichloromethane solution of triethylamine is 60-120 wt% of the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance, the identification molecule is 2, 4-dinitrofluorobenzene, and the addition amount of the identification molecule is 0.5-2 wt%. Hydroxyethyl ethylenediamine and fluorescein hydrazinization derivative-mitochondrial targeting substance molecules form mutual attraction force under the action of polar groups, so that the binding force of active groups is weakened, and the yield of the targeted mitochondrial fluorescein derivative fluorescent probe is improved. The molecule of the acetoxyhydroxamic acid is small and cannot form stable acting force with fluorescein hydrazinization derivative-mitochondrial targeting substance molecules, when hydroxyethyl ethylenediamine exists, the polarity of the hydroxyethyl ethylenediamine is enhanced through electrostatic action, the polarity acting force of the hydroxyethyl ethylenediamine and the fluorescein hydrazinization derivative-mitochondrial targeting substance molecules is improved, and therefore the yield of the targeted mitochondrial fluorescein derivative fluorescent probe is further improved.
The invention adopts hydrazine hydrate, tyrosine, 4-carboxybutyltriphenyl phosphonium bromide and 2, 4-dinitrofluorobenzene to modify and derive fluorescein to obtain the fluorescent probe of the targeted mitochondrial fluorescein derivative, thereby having the following beneficial effects: the recognition of the p-thiophenol is high, and the anti-interference detection capability is strong; the positioning effect on mitochondria is good, and the Pearson coefficient obtained by detecting the mitochondria is 0.96; mitochondrial membrane potential drops significantly. By adopting hydroxyethyl ethylenediamine and acetohydroxamic acid in the preparation process of the fluorescent probe of the targeted mitochondrial fluorescein derivative, the yield of the obtained fluorescent probe at least reaches 30 percent. Therefore, the preparation method of the fluorescence probe of the targeted mitochondrial fluorescein derivative has good selectivity to the thiophenol, good mitochondrial positioning, capability of reducing the mitochondrial membrane potential and high yield.
Drawings
FIG. 1 is a graph of the selectivity of a fluorescent probe for targeting mitochondrial fluorescein derivatives;
FIG. 2 is a graph of interference selectivity for a fluorescent probe targeting mitochondrial fluorescein derivatives;
FIG. 3 is a map of the localization of a fluorescent probe targeting mitochondrial fluorescein derivatives;
FIG. 4 is a graph of the effect of a fluorescent probe targeting mitochondrial fluorescein derivatives on mitochondrial potential;
FIG. 5 is a graph showing the yield of fluorescent probes targeting mitochondrial fluorescein derivatives.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
a preparation method of a fluorescence probe of a targeted mitochondrion fluorescein derivative,
preparation of fluorescein hydrazine: dissolving fluorescein in absolute ethyl alcohol, adding an absolute ethyl alcohol solution containing hydrazine hydrate, stirring and refluxing for 24 hours, removing the solvent by rotary evaporation, adding a hydrochloric acid solution, adding sodium hydroxide to adjust the pH value of 8 for precipitation, filtering, washing with deionized water, and drying in vacuum to obtain a fluorescein hydrazine compound; the adding amount of the fluorescein is 4 wt% of the absolute ethyl alcohol, the mass fraction of the hydrazine hydrate is 90 wt%, the proportion of the hydrazine hydrate in the absolute ethyl alcohol solution of the hydrazine hydrate is 6 wt%, the adding amount of the absolute ethyl alcohol solution containing the hydrazine hydrate is 50 wt% of the absolute ethyl alcohol solution containing the fluorescein, the dropping speed of the absolute ethyl alcohol solution containing the hydrazine hydrate is 1mL/min, the concentration of the hydrochloric acid solution is 0.3M, and the adding amount of the hydrochloric acid solution is 80 wt% of the absolute ethyl alcohol solution containing the fluorescein.
Preparation of fluorescein hydrazinized derivative: dissolving amino acid and sodium hydroxide in absolute ethyl alcohol at the temperature of 50 ℃ to obtain an amino acid absolute ethyl alcohol solution, adding fluorescein hydrazine compound, carrying out reflux reaction at the temperature of 70 ℃ for 6 hours, cooling to 20 ℃, carrying out suction filtration, washing with absolute ethyl alcohol for 2 times, and carrying out vacuum drying to obtain a fluorescein hydrazine derivative; the amino acid is tyrosine, the mass fraction of the amino acid is 4 wt% of the amino acid absolute ethyl alcohol solution, the mass fraction of the sodium hydroxide is 1.2 wt% of the amino acid absolute ethyl alcohol solution, and the addition amount of the fluorescein hydrazine compound is 15 wt% of the amino acid absolute ethyl alcohol solution; the preparation of the fluorescein hydrazine derivative is carried out under the protection of nitrogen atmosphere.
Preparing fluorescein hydrazinization derivative-mitochondrion target: dissolving the fluorescein hydrazine derivative in DMSO, adding a mitochondrial target, reacting for 12h, adding absolute ethanol to precipitate, filtering, washing for 2 times with the absolute ethanol, and vacuum drying to obtain the fluorescein hydrazine derivative; the addition amount of the fluorescein hydrazine compound is 2 wt% of DMSO, the mitochondrial target is 4-carboxybutyltriphenylphosphonium bromide, and the addition amount of the 4-carboxybutyltriphenylphosphonium bromide is 100 wt% of the fluorescein hydrazine compound.
Preparing a targeted mitochondrial fluorescein derivative fluorescent probe: dissolving fluorescein hydrazinization derivative-mitochondrial target in dichloromethane, adding dichloromethane containing triethylamine, stirring for 30min, adding recognition molecules, reacting at 40 ℃ for 12h, cooling to 0 ℃ to precipitate, performing suction filtration, washing with ice dichloroethane, and drying to obtain a targeted mitochondrial fluorescein derivative fluorescent probe; the mass fraction of the fluorescein hydrazinization-mitochondrial targeting substance in the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance is 3 wt%, the mass fraction of triethylamine in the dichloromethane solution of the triethylamine is 2 wt%, the addition amount of the dichloromethane solution of the triethylamine is 80 wt% of the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance, the dripping speed of the dichloromethane containing triethylamine is 1mL/min, the solution is in an ice bath condition when being added with the dichloromethane containing triethylamine, the identification molecule is 2, 4-dinitrofluorobenzene, and the addition amount of the identification molecule is 2 wt%; the preparation of the fluorescein derivative fluorescent probe is carried out in an argon atmosphere.
Example 2:
a preparation method of a fluorescence probe of a targeted mitochondrion fluorescein derivative,
preparation of fluorescein hydrazine: dissolving fluorescein in absolute ethyl alcohol, adding an absolute ethyl alcohol solution containing hydrazine hydrate, stirring and refluxing for 24 hours, removing the solvent by rotary evaporation, adding a hydrochloric acid solution, adding sodium hydroxide to adjust the pH value of 8 for precipitation, filtering, washing with deionized water, and drying in vacuum to obtain a fluorescein hydrazine compound; the adding amount of the fluorescein is 4 wt% of the absolute ethyl alcohol, the mass fraction of the hydrazine hydrate is 90 wt%, the proportion of the hydrazine hydrate in the absolute ethyl alcohol solution of the hydrazine hydrate is 6 wt%, the adding amount of the absolute ethyl alcohol solution containing the hydrazine hydrate is 50 wt% of the absolute ethyl alcohol solution containing the fluorescein, the dropping speed of the absolute ethyl alcohol solution containing the hydrazine hydrate is 1mL/min, the concentration of the hydrochloric acid solution is 0.3M, and the adding amount of the hydrochloric acid solution is 80 wt% of the absolute ethyl alcohol solution containing the fluorescein.
Preparation of fluorescein hydrazinized derivative: dissolving amino acid and sodium hydroxide in absolute ethyl alcohol at the temperature of 50 ℃ to obtain an amino acid absolute ethyl alcohol solution, adding fluorescein hydrazine compound, carrying out reflux reaction at the temperature of 70 ℃ for 6 hours, cooling to 20 ℃, carrying out suction filtration, washing with absolute ethyl alcohol for 2 times, and carrying out vacuum drying to obtain a fluorescein hydrazine derivative; the amino acid is tyrosine, the mass fraction of the amino acid is 8 wt% of the amino acid absolute ethyl alcohol solution, the mass fraction of the sodium hydroxide is 1.2 wt% of the amino acid absolute ethyl alcohol solution, and the addition amount of the fluorescein hydrazine compound is 15 wt% of the amino acid absolute ethyl alcohol solution; the preparation of the fluorescein hydrazine derivative is carried out under the protection of nitrogen atmosphere.
Preparing fluorescein hydrazinization derivative-mitochondrion target: dissolving the fluorescein hydrazine derivative in DMSO, adding a mitochondrial target, reacting for 12h, adding absolute ethanol to precipitate, filtering, washing for 2 times with the absolute ethanol, and vacuum drying to obtain the fluorescein hydrazine derivative; the addition amount of the fluorescein hydrazine compound is 2 wt% of DMSO, the mitochondrial target is 4-carboxybutyltriphenylphosphonium bromide, and the addition amount of the 4-carboxybutyltriphenylphosphonium bromide is 100 wt% of the fluorescein hydrazine compound.
Preparing a targeted mitochondrial fluorescein derivative fluorescent probe: dissolving fluorescein hydrazinization derivative-mitochondrial target in dichloromethane, adding dichloromethane containing triethylamine, stirring for 30min, adding recognition molecules, reacting at 40 ℃ for 12h, cooling to 0 ℃ to precipitate, performing suction filtration, washing with ice dichloroethane, and drying to obtain a targeted mitochondrial fluorescein derivative fluorescent probe; the mass fraction of the fluorescein hydrazinization-mitochondrial targeting substance in the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance is 3 wt%, the mass fraction of triethylamine in the dichloromethane solution of the triethylamine is 2 wt%, the addition amount of the dichloromethane solution of the triethylamine is 80 wt% of the dichloromethane solution of the fluorescein hydrazinization-mitochondrial targeting substance, the dripping speed of the dichloromethane containing triethylamine is 1mL/min, the solution is in an ice bath condition when being added with the dichloromethane containing triethylamine, the identification molecule is 2, 4-dinitrofluorobenzene, and the addition amount of the identification molecule is 2 wt%; the preparation of the fluorescein derivative fluorescent probe is carried out in an argon atmosphere.
Example 3:
a preparation method of a fluorescence probe of a targeted mitochondrion fluorescein derivative,
preparation of fluorescein hydrazine: dissolving fluorescein in absolute ethyl alcohol, adding an absolute ethyl alcohol solution containing hydrazine hydrate, stirring and refluxing for 24 hours, removing the solvent by rotary evaporation, adding a hydrochloric acid solution, adding sodium hydroxide to adjust the pH value of 8 for precipitation, filtering, washing with deionized water, and drying in vacuum to obtain a fluorescein hydrazine compound; the adding amount of the fluorescein is 4 wt% of the absolute ethyl alcohol, the mass fraction of the hydrazine hydrate is 90 wt%, the proportion of the hydrazine hydrate in the absolute ethyl alcohol solution of the hydrazine hydrate is 6 wt%, the adding amount of the absolute ethyl alcohol solution containing the hydrazine hydrate is 50 wt% of the absolute ethyl alcohol solution containing the fluorescein, the dropping speed of the absolute ethyl alcohol solution containing the hydrazine hydrate is 1mL/min, the concentration of the hydrochloric acid solution is 0.3M, and the adding amount of the hydrochloric acid solution is 80 wt% of the absolute ethyl alcohol solution containing the fluorescein.
Preparation of fluorescein hydrazinized derivative: dissolving amino acid and sodium hydroxide in absolute ethyl alcohol at the temperature of 50 ℃ to obtain an amino acid absolute ethyl alcohol solution, adding fluorescein hydrazine compound, carrying out reflux reaction at the temperature of 70 ℃ for 6 hours, cooling to 20 ℃, carrying out suction filtration, washing with absolute ethyl alcohol for 2 times, and carrying out vacuum drying to obtain a fluorescein hydrazine derivative; the amino acid is tyrosine, the mass fraction of the amino acid is 8 wt% of the amino acid absolute ethyl alcohol solution, the mass fraction of the sodium hydroxide is 1.2 wt% of the amino acid absolute ethyl alcohol solution, and the addition amount of the fluorescein hydrazine compound is 15 wt% of the amino acid absolute ethyl alcohol solution; the preparation of the fluorescein hydrazine derivative is carried out under the protection of nitrogen atmosphere.
Preparing fluorescein hydrazinization derivative-mitochondrion target: dissolving the fluorescein hydrazine derivative in DMSO, adding a mitochondrial target, reacting for 12h, adding absolute ethanol to precipitate, filtering, washing for 2 times with the absolute ethanol, and vacuum drying to obtain the fluorescein hydrazine derivative; the addition amount of the fluorescein hydrazine compound is 2 wt% of DMSO, the mitochondrial target is 4-carboxybutyltriphenylphosphonium bromide, and the addition amount of the 4-carboxybutyltriphenylphosphonium bromide is 100 wt% of the fluorescein hydrazine compound.
Preparing a targeted mitochondrial fluorescein derivative fluorescent probe: dissolving fluorescein hydrazinization derivative-mitochondrial target in dichloromethane, adding hydroxyethyl ethylenediamine and acetoxyhydroxamic acid, adding dichloromethane containing triethylamine, stirring for 30min, adding recognition molecules, reacting at 40 ℃ for 12h, cooling to 0 ℃ to separate out precipitate, performing suction filtration, washing with glacial dichloroethane, and drying to obtain a targeted mitochondrial fluorescein derivative fluorescent probe; the mass fraction of fluorescein hydrazinization-mitochondrial targeting substance in dichloromethane solution of fluorescein hydrazinization-mitochondrial targeting substance is 3 wt%, the addition amount of hydroxyethyl ethylenediamine is 0.4 wt% of the fluorescein hydrazinization-mitochondrial targeting substance dichloromethane solution, the addition amount of acetohydroxamic acid is 0.2 wt% of the fluorescein hydrazinization-mitochondrial targeting substance dichloromethane solution, the mass fraction of triethylamine in dichloromethane solution of triethylamine is 2 wt%, the addition amount of dichloromethane solution of triethylamine is 80 wt% of the fluorescein hydrazinization-mitochondrial targeting substance dichloromethane solution, the dripping speed of dichloromethane containing triethylamine is 1mL/min, and the solution is in ice bath condition when dichloromethane containing triethylamine is added, the identification molecule is 2, 4-dinitrofluorobenzene, and the addition amount of the identification molecule is 2 wt%; the preparation of the fluorescein derivative fluorescent probe is carried out in an argon atmosphere.
Example 4:
a preparation method of a fluorescence probe of a targeted mitochondrion fluorescein derivative,
preparation of fluorescein hydrazine: dissolving fluorescein in absolute ethyl alcohol, adding an absolute ethyl alcohol solution containing hydrazine hydrate, stirring and refluxing for 24 hours, removing the solvent by rotary evaporation, adding a hydrochloric acid solution, adding sodium hydroxide to adjust the pH value of 8 for precipitation, filtering, washing with deionized water, and drying in vacuum to obtain a fluorescein hydrazine compound; the adding amount of the fluorescein is 4 wt% of the absolute ethyl alcohol, the mass fraction of the hydrazine hydrate is 90 wt%, the proportion of the hydrazine hydrate in the absolute ethyl alcohol solution of the hydrazine hydrate is 6 wt%, the adding amount of the absolute ethyl alcohol solution containing the hydrazine hydrate is 50 wt% of the absolute ethyl alcohol solution containing the fluorescein, the dropping speed of the absolute ethyl alcohol solution containing the hydrazine hydrate is 1mL/min, the concentration of the hydrochloric acid solution is 0.3M, and the adding amount of the hydrochloric acid solution is 80 wt% of the absolute ethyl alcohol solution containing the fluorescein.
Preparation of fluorescein hydrazinized derivative: dissolving amino acid and sodium hydroxide in absolute ethyl alcohol at the temperature of 50 ℃ to obtain an amino acid absolute ethyl alcohol solution, adding fluorescein hydrazine compound, carrying out reflux reaction at the temperature of 70 ℃ for 6 hours, cooling to 20 ℃, carrying out suction filtration, washing with absolute ethyl alcohol for 2 times, and carrying out vacuum drying to obtain a fluorescein hydrazine derivative; the amino acid is tyrosine, the mass fraction of the amino acid is 8 wt% of the amino acid absolute ethyl alcohol solution, the mass fraction of the sodium hydroxide is 1.2 wt% of the amino acid absolute ethyl alcohol solution, and the addition amount of the fluorescein hydrazine compound is 15 wt% of the amino acid absolute ethyl alcohol solution; the preparation of the fluorescein hydrazine derivative is carried out under the protection of nitrogen atmosphere.
Preparing fluorescein hydrazinization derivative-mitochondrion target: dissolving the fluorescein hydrazine derivative in DMSO, adding a mitochondrial target, reacting for 12h, adding absolute ethanol to precipitate, filtering, washing for 2 times with the absolute ethanol, and vacuum drying to obtain the fluorescein hydrazine derivative; the addition amount of the fluorescein hydrazine compound is 2 wt% of DMSO, the mitochondrial target is 4-carboxybutyltriphenylphosphonium bromide, and the addition amount of the 4-carboxybutyltriphenylphosphonium bromide is 100 wt% of the fluorescein hydrazine compound.
Preparing a targeted mitochondrial fluorescein derivative fluorescent probe: dissolving fluorescein hydrazinization derivative-mitochondrial target in dichloromethane, adding hydroxyethyl ethylenediamine and acetoxyhydroxamic acid, adding dichloromethane containing triethylamine, stirring for 30min, adding recognition molecules, reacting at 40 ℃ for 12h, cooling to 0 ℃ to separate out precipitate, performing suction filtration, washing with glacial dichloroethane, and drying to obtain a targeted mitochondrial fluorescein derivative fluorescent probe; the mass fraction of fluorescein hydrazinization-mitochondrial targeting substance in dichloromethane solution of fluorescein hydrazinization-mitochondrial targeting substance is 3 wt%, the addition amount of hydroxyethyl ethylenediamine is 1 wt% of the dichloromethane solution of fluorescein hydrazinization-mitochondrial targeting substance, the addition amount of acetohydroxamic acid is 0.6 wt% of the dichloromethane solution of fluorescein hydrazinization-mitochondrial targeting substance, the mass fraction of triethylamine in dichloromethane solution of triethylamine is 2 wt%, the addition amount of dichloromethane solution of triethylamine is 80 wt% of the dichloromethane solution of fluorescein hydrazinization-mitochondrial targeting substance, the dripping speed of dichloromethane containing triethylamine is 1mL/min, and the solution is in ice bath condition when dichloromethane containing triethylamine is added, the identification molecule is 2, 4-dinitrofluorobenzene, and the addition amount of the identification molecule is 2 wt%; the preparation of the fluorescein derivative fluorescent probe is carried out in an argon atmosphere.
Comparative example 1:
this comparative example is different from example 2 only in that the fluorescein hydrazinized derivative preparation step was not performed.
Comparative example 2:
the comparative example is different from example 4 only in that acetohydroxamic acid is not added in the preparation step of the fluorescent probe for the targeted mitochondrial fluorescein derivative.
Comparative example 3:
this comparative example is different from example 4 only in that hydroxyethylethylenediamine was not added in the procedure for preparing the fluorescent probe for a mitochondrial fluorescein derivative.
Test example 1:
1. mass spectrometric detection
The fluorescein, fluorescein hydrazine derivative-mitochondrial targeting substance and the fluorescent probe of the targeted mitochondrial fluorescein derivative in example 2 were dissolved in DMSO-d 6, and the product was characterized by a nuclear magnetic resonance spectrometer1H NMR, TMS internal standard.
Of fluorescein1In H NMR, chemical shifts of carboxyl hydrogens appeared at 11.24ppm and those of hydroxyl hydrogens appeared at 5.16ppmThe shift is the chemical shift of hydrogen on the conjugated ring between 6.21 ppm and 7.87 ppm;
method for preparing fluorescein hydrazine1Compared with fluorescein, chemical shifts of carboxyl hydrogen at 11.24ppm disappear, chemical shifts of amide hydrogen at 8.05ppm appear, and chemical shifts of amine hydrogen at the upper end of hydrazine appear at 2.12ppm by H NMR, which indicates that fluorescein hydrazine hydrate is obtained.
Process for preparing hydrazinized derivatives of fluorescein1H NMR showed 11.28ppm chemical shift of carboxyl hydrogen compared to fluorescein hydrazinium, indicating successful acquisition of fluorescein hydrazinium derivative.
Method for preparing fluorescein hydrazinization derivative-mitochondrion target1Compared with fluorescein hydrazinization derivatives, chemical shifts of hydrogen on the No. 2 and No. 3 carbons of butyl in 4-carboxybutyltriphenylphosphonium bromide appear at 1.26-1.62ppm, and chemical shifts of hydrogen on the No. 1 and No. 4 carbons connected with P and carbonyl respectively in butyl in 4-carboxybutyltriphenylphosphonium bromide appear at 2.34-2.58ppm, which indicates that fluorescein hydrazinization derivative-mitochondrial targeting substances are successfully obtained.
Targeting mitochondrial fluorescein derivative fluorescent probes1Compared with fluorescein hydrazinization derivative-mitochondrial targeting substances, chemical shift of hydrogen on benzene rings adjacent to nitro on 2, 4-dinitrobenzene appears between 8.15 ppm and 8.96ppm in H NMR, and the targeted mitochondrial fluorescein derivative fluorescent probe is successfully obtained.
Test example 2:
1. thiophenol selectivity detection
Preparing standard solutions of thiophenol, o-aminothiophenol and o-nitrothiophenol:
accurately measuring 5 mu L of thiophenol, 5 mu L of o-aminothiophenol and 6 mu L of o-nitrothiophenol, respectively dissolving the thiophenol, the o-aminothiophenol and the o-nitrothiophenol in DMSO, and then fixing the volume in a 50mL volumetric flask.
Preparing standard solutions of phenol, aniline and o-nitrophenol:
accurately measuring 9 mu L of phenol, 9 mu L of aniline and 8 mu L of o-nitrophenol, dissolving the mixture by DMSO, and fixing the volume in a 10mL volumetric flask.
The fluorescence detection was performed using the fluorescence probe for the targeted mitochondrial fluorescein derivative obtained in example 2.
The selective detection result of the fluorescent probe of the targeted mitochondrial fluorescein derivative on the thiophenol is shown in figure 1, wherein the blank is DMSO solution; a is phenol; b is aniline; c is o-nitrophenol; d is o-nitrothiophenol; e is o-aminothiophenol; f is thiophenol; as can be seen from the figure, the fluorescence intensity of the fluorescent probe of the targeted mitochondrial fluorescein derivative obtained in the embodiment 2 is the maximum for detecting the thiophenol, and the detection intensity of other components is equivalent to the blank intensity and is far lower than the detection fluorescence intensity of the thiophenol, which shows that the fluorescent probe of the targeted mitochondrial fluorescein derivative obtained in the embodiment 2 has good specificity and high recognition on the thiophenol.
The anti-interference detection result of the fluorescent probe of the targeted mitochondrial fluorescein derivative on the thiophenol is shown in figure 2, wherein A is phenol; b is aniline; c is o-nitrophenol; d is o-nitrothiophenol; e is o-aminothiophenol; f is thiophenol; g is A + B + C + D + E; as shown in FIG. 2, the increase of fluorescence intensity is not caused by the detection of A, B, C, D, E, and after thiophenol is added, the fluorescence intensity is equivalent to that of F (thiophenol), which shows that A, B, C, D, E does not influence the detection of the inventive fluorescent probe on thiophenol basically, and A, B, C, D, E is mixed together, which does not influence the detection of thiophenol, and shows that the anti-interference capability of the fluorescent probe of the targeted mitochondrial fluorescein derivative is strong.
2. Localization detection
The co-localization ability of the targeted mitochondrial fluorescein derivative fluorescent probe obtained in example 2 and MitoTracker Green was studied, cultured HepG2 cells were taken out, washed 3 times with PBS, 30. mu.M of the probe targeted mitochondrial fluorescein derivative fluorescent probe solution was added to a 1mL cell culture dish, incubated at 37 ℃ for 10min, then 200nM MitoTracker Green FM was added and incubation continued for 20min, washed 2 times with PBS, 60. mu.M thiophenol solution was added, and then a cell fluorescence imaging experiment was performed. Green channel: lambda [ alpha ]ex＝488nm，λem490 and 530 nm; and (3) red channel: lambda [ alpha ]ex＝561nm，λem＝570-650nm。
The result of the localization detection of the targeted mitochondrial fluorescein derivative fluorescent probe to the organelles is shown in fig. 3, wherein the pearson coefficient obtained by the detection of mitochondria is 0.96, the pearson coefficient obtained by the detection of lysosomes is 0.51, the pearson coefficient obtained by the detection of endoplasmic reticulum is 0.54, and the pearson coefficient obtained by the detection of golgi is 0.31; the fluorescent probe of the targeted mitochondrial fluorescein derivative obtained by the invention has a good positioning effect on mitochondria.
3. Detection of Effect on mitochondrial Membrane potential
JC-1 is a fluorescent probe for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, the mitochondrial membrane assumes an aggregation state in a matrix of mitochondria, thereby generating red fluorescence; when the mitochondrial membrane potential is low, the membrane is in a monomer state, and green fluorescence is generated. The red-green fluorescence ratio can detect the level of the membrane potential.
(1) L6 cells were seeded in blackboard clear bottom 96-well plates at a density of 6W/mL (DMEM, 10% FBS), and when the density reached 70% (next day), they were fluid-changed and differentiated (DMEM, 2% FBS), and used for the experiment five days after differentiation.
(2) Diluting the targeted mitochondrial fluorescein derivative fluorescent probe to 10 mu M, adding the targeted mitochondrial fluorescein derivative fluorescent probe into 1ml DMEM-2% FBS according to 1000X, discarding the old culture medium in a 96-well plate, adding the targeted mitochondrial fluorescein derivative fluorescent probe, and then culturing for 1h at 37 ℃.
(3) 100 mu L of DMEM-2% FBS containing JC-1 (mother solution concentration of 1mg/mL and working concentration of 1 mu g/mL) was added 20min before the compound treatment was finished, and the 96-well plate was incubated for 20min in the dark.
(4) The culture medium in the 96-well plate was spun off, placed on absorbent paper, and then washed three times with KRPH buffer, 200. mu.L per well. Then 100. mu.L of KRPH was added for detection.
(5) The detection values of examples and comparative examples were compared with a blank group of DMSO, i.e., the amount of decrease in membrane potential.
The result of detecting the effect of the fluorescent probe of the targeted mitochondrial fluorescein derivative on the mitochondrial membrane potential is shown in fig. 4, and when the mitochondrial membrane potential of the blank group is 100, the membrane potential of example 1 is reduced, the membrane potential of example 2 is reduced more, and the membrane potential of comparative example 1 is not reduced, which indicates that the fluorescent probe of the targeted mitochondrial fluorescein derivative obtained by tyrosine modification reduces the mitochondrial membrane potential; example 2 compared to example 1, shows that the higher the amount of tyrosine modification, the more pronounced the mitochondrial membrane potential drop.
4. Yield of fluorescent probes targeting mitochondrial fluorescein derivatives
Theoretical yields of all the produced mitochondrial fluorescein derivative-targeted fluorescent probes in each example and comparative example were calculated, yields of the mitochondrial fluorescein derivative-targeted fluorescent probes actually obtained in each example and comparative example were weighed, and yields were calculated.
Yield ═ actual yield/theoretical yield × 100%
The yield results of the fluorescent probe for the targeted mitochondrial fluorescein derivative are shown in fig. 5, with the lowest yield obtained in example 2 and the highest yield obtained in example 4; example 4 compared with example 2, the yield was increased by 4.9%, indicating that the addition of hydroxyethylethylenediamine and acetohydroxamic acid increased the yield of the targeted mitochondrial fluorescein derivative fluorescent probe; example 4 compared with example 3, the yield was increased by 2.2%, indicating that the yield of the fluorescent probe of the targeted mitochondrial fluorescein derivative was further increased when the addition amounts of hydroxyethylethylenediamine and acetoxyhydroxamic acid were higher; example 4 compared with comparative example 2, the yield was improved by 2.8%, indicating that the effect of the co-use of hydroxyethyl ethylenediamine and acetohydroxamic acid is better than that of hydroxyethyl ethylenediamine; example 4 compared with comparative example 3, the yield was improved by 4.6%, indicating that the effect of the co-use of hydroxyethyl ethylenediamine and acetohydroxamic acid is better than that of acetohydroxamic acid; the yield of comparative example 2 was increased by 2.1% compared to example 2, indicating that the use of hydroxyethylethylenediamine alone could increase the yield of the mitochondrial fluorescein derivative-targeted fluorescent probe, and the use of acetohydroxamic acid alone did not increase the yield of the mitochondrial fluorescein derivative-targeted fluorescent probe compared to example 2 in comparative example 3. The yield of the fluorescent probe of the targeted mitochondrial fluorescein derivative at least reaches 30 percent.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (10)
1. A fluorescein derivative having the formula:
2. A fluorescein derivative as defined in claim 1 wherein: the R is1Comprises the following steps:
3. a fluorescein derivative as defined in claim 1 wherein: the R is2Comprises the following steps:
4. a fluorescein derivative as defined in claim 1 wherein: the R is3Comprises the following steps:
5. use of a fluorescein derivative as claimed in any one of claims 1 to 4 in a fluorescent probe.
6. A preparation method of a targeted mitochondrial fluorescein derivative fluorescent probe comprises the following steps: reacting fluorescein with hydrazine hydrate to obtain fluorescein hydrazine compound; adding the fluorescein hydrazine compound into an amino acid absolute ethyl alcohol solution for reaction to obtain a fluorescein hydrazine derivative; reacting the fluorescein hydrazine derivative with a mitochondrial targeting ligand to obtain a fluorescein hydrazine derivative-mitochondrial targeting substance; fluorescein hydrazinization derivatization-mitochondrial targeting substances react with recognition molecules to obtain targeted mitochondrial fluorescein derivative fluorescent probes; the mitochondrion targeting ligand is 4-carboxybutyl triphenyl phosphonium bromide.
7. The method for preparing a fluorescence probe of targeted mitochondrial fluorescein derivative as claimed in claim 6, wherein the fluorescence probe comprises: the recognition molecule is dinitrofluorobenzene.
8. The method for preparing a fluorescence probe of targeted mitochondrial fluorescein derivative as claimed in claim 6, wherein the fluorescence probe comprises: the amino acid is tyrosine.
9. The method for preparing a fluorescence probe of targeted mitochondrial fluorescein derivative as claimed in claim 6, wherein the fluorescence probe comprises: the mass fraction of the amino acid is 1-10 wt% of the amino acid absolute ethyl alcohol solution.
10. The fluorescence probe of the targeted mitochondrial fluorescein derivative prepared by the method of any one of claims 6-9.
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