CN106432312B - A kind of Mitochondrially targeted fluorescence probe and its preparation method and application - Google Patents
A kind of Mitochondrially targeted fluorescence probe and its preparation method and application Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 229940125782 compound 2 Drugs 0.000 claims description 11
- HZNVUJQVZSTENZ-UHFFFAOYSA-N 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(C#N)=C(C#N)C1=O HZNVUJQVZSTENZ-UHFFFAOYSA-N 0.000 claims description 10
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 229940125904 compound 1 Drugs 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- 238000004440 column chromatography Methods 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- MFFMQGGZCLEMCI-UHFFFAOYSA-N 2,4-dimethyl-1h-pyrrole Chemical compound CC1=CNC(C)=C1 MFFMQGGZCLEMCI-UHFFFAOYSA-N 0.000 claims description 5
- PAPNRQCYSFBWDI-UHFFFAOYSA-N DMP Natural products CC1=CC=C(C)N1 PAPNRQCYSFBWDI-UHFFFAOYSA-N 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 229910015900 BF3 Inorganic materials 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 3
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 3
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical compound OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 claims description 3
- KDCIHNCMPUBDKT-UHFFFAOYSA-N hexane;propan-2-one Chemical compound CC(C)=O.CCCCCC KDCIHNCMPUBDKT-UHFFFAOYSA-N 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000003068 molecular probe Substances 0.000 claims description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- BXXWFOGWXLJPPA-UHFFFAOYSA-N 2,3-dibromobutane Chemical compound CC(Br)C(C)Br BXXWFOGWXLJPPA-UHFFFAOYSA-N 0.000 claims 1
- AQZMINLSVARCSL-UHFFFAOYSA-N 4-chloro-3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile Chemical class ClC1=CC(=O)C(C#N)=C(C#N)C1=O AQZMINLSVARCSL-UHFFFAOYSA-N 0.000 claims 1
- 210000003470 mitochondria Anatomy 0.000 abstract description 17
- 238000003384 imaging method Methods 0.000 abstract description 13
- 238000004061 bleaching Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000090 biomarker Substances 0.000 abstract description 2
- 230000007812 deficiency Effects 0.000 abstract description 2
- 238000006862 quantum yield reaction Methods 0.000 abstract description 2
- 210000001082 somatic cell Anatomy 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 27
- 239000000243 solution Substances 0.000 description 17
- 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 13
- 238000002474 experimental method Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 239000012930 cell culture fluid Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 230000002438 mitochondrial effect Effects 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000000295 emission spectrum Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- ULTHEAFYOOPTTB-UHFFFAOYSA-N 1,4-dibromobutane Chemical compound BrCCCCBr ULTHEAFYOOPTTB-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- TUFFYSFVSYUHPA-UHFFFAOYSA-M rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C(C=CC(N)=C2)C2=[O+]C2=C1C=CC(N)=C2 TUFFYSFVSYUHPA-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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Abstract
The invention discloses a kind of Mitochondrially targeted fluorescence probes and its preparation method and application, the probe has structure shown in formula (I), overcome many deficiencies of traditional commodities probe, it is small with fluorescence quantum yield height, the influence of good water solubility, environment polarity and acid-base property to probe, the advantages such as that in addition there are mitochondria imaging effects is good, toxicity is low, anti-light bleaching power is strong, to have a good application prospect in the imaging of long-time active somatic cell mitochondria and biomarker field.
Description
Technical field
The invention belongs to light technical field of analysis and detection, and in particular to a kind of Mitochondrially targeted fluorescence probe and its preparation side
Method and application.
Background technique
Mitochondria is organelle important in cell, is the energy plants of cell, and in addition to this mitochondria is gone back in cell
There are many metabolism, calcium ion regulatory and the Apoptosis accesses of important function, such as control cell to control, mitochondria
Dysfunction will lead to a variety of diseases, such as Parkinson's disease, Alzheimer disease, therefore mitochondria imaging is to subcellsular level
Diagnosis and treatment have great importance.
Existing frequently-used mitochondrial probe has Rhodamine 123, JC-1, Mitotracker series etc., although they have made
It is that commodity probe is imaged for mitochondria, but still remains that cytotoxicity is big, anti-light bleaching power is weak, is not suitable for long-time line
Many disadvantages such as plastochondria imaging, it is therefore necessary to develop a kind of new mitochondria fluorescence probe for active somatic cell mitochondria at
Picture.
Summary of the invention
In order to overcome many deficiencies of traditional commodities probe, good, toxicity that the present invention provides a kind of mitochondria imaging effects
The strong Mitochondrially targeted fluorescence probe and its preparation method and application of low, anti-light bleaching power.
Technical solution provided by the invention is specific as follows:
A kind of fluorescent molecular probe (hereinafter referred to as TEA-BODIPY) with Mitochondrially targeted function has formula (I) institute
The structure shown:
A method of preparing the above-mentioned fluorescence probe with Mitochondrially targeted function, comprising the following steps:
(1) parahydroxyben-zaldehyde is dissolved in anhydrous DMF, Anhydrous potassium carbonate and Isosorbide-5-Nitrae-dibromobutane is added, reacted at room temperature,
Crude product is obtained, crude by column chromatography purifying obtains compound 1；The compound 1 is
(2) by compound 1 and 2,4- dimethyl pyrrole is dissolved in anhydrous methylene chloride together, under nitrogen protection, is added
Trifluoroacetic acid is stirred at room temperature 12 hours as catalyst, and 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone, room temperature item is then added
Continue stirring 2 hours under part, triethylamine and boron trifluoride ether are added after stirring, is stirred overnight, after reaction, reaction
Liquid successively uses saturated sodium bicarbonate solution and water washing, is then extracted with dichloromethane, then dry with anhydrous sodium sulfate, will slightly produce
Column chromatographic purifying is used after object concentration, obtains compound 2；The compound 2 is
(3) compound 2 is flowed back 48 hours in dry toluene together with triethylamine, is chromatographed after reaction solution is concentrated through column
Purifying obtains the compound of structure shown in formula (I).
In step (1), parahydroxyben-zaldehyde, potassium carbonate and Isosorbide-5-Nitrae-dibromobutane molar ratio are 1:1.5:2.
In step (2), mole of compound 1,2,4- dimethyl pyrrole and 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
Than for 1:2:1.
In step (3), the molar ratio of compound 2 and triethylamine is 1:10.
In step (1), the leacheate of column chromatography is n-hexane-acetone mixture that volume ratio is 8:1；In step (2), column
The leacheate of chromatography is the petroleum ether-ethyl acetate mixed liquor that volume ratio is 8:1；In step (3), the leacheate of column chromatography is body
Product is than the methanol-ethyl acetate mixed liquor for 4:1.
Application of the above-mentioned fluorescence probe with Mitochondrially targeted function in biomarker field.
The above-mentioned fluorescence probe with Mitochondrially targeted function is marked and is imaged for living cells mitochondria.
A method of changed using the above-mentioned fluorescence probe detection line particle shape looks with Mitochondrially targeted function, by altogether
The pattern of focal imaging technology detection line grain changes.
The present invention has the following advantages and beneficial effects:
(1) Mitochondrially targeted fluorescence probe prepared by the present invention, have fluorescence quantum yield high (0.80), good water solubility,
Environment polarity and acid-base property influence the advantages such as small, anti-light bleaching power is strong, cytotoxicity is low to probe.
(2) synthetic method of the present invention is simple, can be mass produced.
(3) Mitochondrially targeted fluorescence probe prepared by the present invention can be used for bio-imaging, mitochondrial markers, be good life
Substance markers probe.
Detailed description of the invention:
Fig. 1 is the synthetic route chart of 1 middle probe TEA-BODIPY of embodiment.
Fig. 2 is Absorption and emission spectra figure of the 2 middle probe TEA-BODIPY of embodiment in acetonitrile.
Fig. 3 is the Absorption and emission spectra figure of 2 middle probe TEA-BODIPY of embodiment in aqueous solution.
Fig. 4 is the test chart of 3 probe TEA-BODIPY fluorescence intensity under condition of different pH of embodiment.
The MTT cytotoxicity in SGC7901 cell and HeLa cell that Fig. 5 is 4 middle probe TEA-BODIPY of embodiment is real
Test result figure；Wherein, Fig. 5 (a) is SGC7901 cell, and Fig. 5 (b) is HeLa cell.
Fig. 6 is 5 middle probe TEA-BODIPY of embodiment and commodity mitochondrial dye Mito-Tracker red to living cells
The common location imaging experiment result figure of mitochondria；Wherein, Fig. 6 (a) is image of the TEA-BODIPY to living cells mitochondria, figure
6 (b) be Mito-Tracker red to the image of living cells mitochondria, and Fig. 6 (c) is TEA-BODIPY and Mito-Tracker
Red is overlapped the image of living cells mitochondria, and Fig. 6 (d) is the emitting area and TEA-BODIPY of Mito-Tracker red
Emitting area superposition.
Fig. 7 is the anti-light bleaching experiment result figure of 6 middle probe TEA-BODIPY of embodiment, wherein 1~16 indicates scanning sequence
Number.
Fig. 8 is the anti-light bleaching experiment result figure of commodity probe Mito-Tracker red in embodiment 6, wherein 1~16
Indicate scanning serial number.
Specific embodiment
Below in conjunction with specific embodiment, the technical scheme of the present invention will be explained in further detail.Implement of the invention
Process, condition, reagent, experimental method etc. are in addition to what is specifically mentioned below the universal knowledege of this field and known
Common sense, there are no special restrictions to content by the present invention.
Embodiment 1: the preparation of the fluorescent organic molecule (TEA-BODIPY) of Mitochondrially targeted function
It is anhydrous that parahydroxyben-zaldehyde (1.22g, 10mmol) and 1,4- dibromobutane (4.30g, 20mmol) are dissolved in 20mL
In n,N-Dimethylformamide (DMF), Anhydrous potassium carbonate (K is added2CO3) (2.07g, 15mmol) as alkali, room temperature reaction is obtained
To crude product, chromatographic column is crossed, obtains compound 1, leacheate is the n-hexane-acetone mixing of n-hexane/acetone (v/v)=8/1
The structural formula of liquid, compound 1 is
It is dry that compound 1 (0.512g, 2mmol) and 2,4- dimethyl pyrrole (0.380g, 4mmol) is dissolved in 200mL together
In dry methylene chloride, under nitrogen protection, use a small amount of trifluoroacetic acid (TFA) as catalyst, after being stirred at room temperature 12 hours
It is added 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (2.27g, 20mmol), then continues stirring 2 at room temperature
Hour, 5mL triethylamine (Et is added after stirring3) and 8mL boron trifluoride ether (BF N3-OEt2), it is stirred overnight, reaction knot
Shu Hou, reaction solution are successively used saturated sodium bicarbonate solution and water washing, are then extracted with dichloromethane, and anhydrous sodium sulfate is dry,
Rear pillar chromatographic purifying is concentrated in crude product, and leacheate is that the petroleum ether-ethyl acetate of petrol ether/ethyl acetate (v/v)=8/1 mixes
Liquid, obtains compound 2, and the structural formula of compound 2 is
By compound 2 (0.237g, 0.5mmol) and triethylamine (0.505g, 5mmol), reflux 48 is small in dry toluene
When, reaction solution evaporating column chromatographic purifying obtains target product TEA-BODIPY, and leacheate is methanol/ethyl acetate (v/v)=4/1
Methanol-ethyl acetate mixed liquor；1H NMR (300MHz, DMSO) δ=7.23-7.26 (d, J=8.6Hz, 2H), 7.08-
7.10 (d, J=8.5Hz, 2H), 6.14 (s, 2H), 4.06 (s, 2H), 3.20-3.22 (s, 2H), 3.20 (s, 6H), 2.40 (s,
6H),1.76(s,4H),1.36(s,6H),1.13(s,15H)；ESI: the theoretical value of elemental analysis: C41H41BBrF2N2OP+(M+)
657.30；The numerical value that actual measurement arrives: 657.40.
Embodiment 2: Absorption and emission spectra of the Mitochondrially targeted fluorescence probe TEA-BODIPY in opposed polarity solution
Test
For the spectrum test concentration that the present invention uses for 10 μM, the solvent of test is aqueous solution and acetonitrile solution, TEA-
Absorption and emission spectra of the BODIPY in different solvents is as shown in Figures 2 and 3.Absorption and transmitting of the polarity of solvent to probe
The change very little of spectrum, Stokes shift is almost without changing, it is seen that solvent polarity is to probe TEA-BODIPY's
Influence very little.
Embodiment 3: the test of Mitochondrially targeted fluorescence probe TEA-BODIPY fluorescence intensity under condition of different pH
The spectrum test concentration that the present invention uses is 10 μM, and the test of TEA-BODIPY fluorescence intensity under the conditions of pH is as schemed
Shown in 4.PH influences very little to the fluorescence intensity of probe, illustrates that the probe can be used normally under the conditions of cell physiological pH.
Embodiment 4: the MTT cytotoxicity experiment of Mitochondrially targeted fluorescence probe TEA-BODIPY
By postdigestive cell inoculation on 96 orifice plates, cell-seeding-density 104A/hole, in 37 DEG C, 5%CO2Condition
Lower culture 24 hours, is sucked out culture solution, is then continued to cultivate cell 24 with the culture solution of the various concentration containing TEA-BODIPY
Hour, then 10 μ L MTT (5mg/mL) are added in every hole, terminate culture after continuing culture 4 hours, and culture solution is sucked out, and every hole is added
150 μ L dimethyl sulfoxides, shaking table shake after ten minutes, tested using multi-function microplate reader, MTT cytotoxicity result as shown in figure 5,
Two kinds of cell HeLa and the SGC-7901 cells tested increase to 200 μm of ol/L in TEA-BODIPY concentration, cultivate 24 hours
After, cell survival rate is all larger than 90%, it was demonstrated that the probe has lower cytotoxicity, can be used for cell imaging.
Embodiment 5: Mitochondrially targeted fluorescence probe TEA-BODIPY and commodity mitochondrial dye Mito-Tracker red
Common location experiment to living cells mitochondria
The cell that the present invention uses is HeLa cell, by postdigestive cell inoculation in culture dish, in 37 DEG C, 5%CO2
Under the conditions of, culture is allowed to adherent in 24 hours, uses TEA-BODIPY (5 μM) after washing away stale cell culture fluid with PBS solution
Cell culture fluid incubated cell 12 hours, then continue to cultivate with containing Mito-Tracker red (0.5 μM) cell culture fluid
It 30 minutes, is washed with PBS solution and is imaged afterwards three times, common location imaging results are as shown in fig. 6, TEA-BODIPY is swashed by 488nm laser
Hair, Mito-Tracker red is by 561nm laser excitation, by the emitting area of commodity mitochondrial dye Mito-Tracker red
It is superimposed with the emitting area of TEA-BODIPY, it is found that the registration of the two is very high and imaging effect is good, it was demonstrated that line grain of the invention
Body fluorescence probe TEA-BODIPY can be used for living body mitochondrial markers.
Embodiment 6: Mitochondrially targeted fluorescence probe TEA-BODIPY comparison commodity probe Mito-Tracker red's is anti-
Photobleaching experiment
The anti-light bleaching experiment of TEA-BODIPY is specific as follows: test the cell used as HeLa cell, it will be postdigestive
Cell inoculation is in culture dish, in 37 DEG C, 5%CO2Under the conditions of, culture is allowed to adherent in 24 hours, is washed away with PBS solution stale
Cell culture fluid after, with cell culture fluid incubated cell 12 hours of TEA-BODIPY (5 μM), after being washed three times with PBS solution
Then imaging carries out photobleaching experiment with laser confocal microscope, laser intensity is (65 μ W), and excitation wavelength is (488nm),
Scanning 13.6 seconds every time, experimental result is as shown in Figure 7.
The anti-light bleaching experiment of Mito-Tracker red is specific as follows: testing the cell used as HeLa cell, will disappear
Cell inoculation after change is in culture dish, in 37 DEG C, 5%CO2Under the conditions of, culture is allowed to adherent in 24 hours, is washed away with PBS solution
After stale cell culture fluid, continues culture 30 minutes with containing Mito-Tracker red (0.5 μM) cell culture fluid, use
PBS solution is washed to be imaged afterwards three times, then carries out photobleaching experiment with laser confocal microscope, and laser intensity is (65 μ W), is swashed
Sending out wavelength is (561nm), every time scanning 13.6 seconds, and experimental results are shown in figure 8.
The experimental results showed that fluorescence probe TEA-BODIPY shown in the present invention compares commodity probe Mito-Tracker
Red has very excellent anti-light bleaching characteristic, can be used for the imaging of long-time mitochondria.
Protection content of the invention is not limited to above embodiments, without departing from the spirit and scope of the invention, this
Field technical staff it is conceivable that variation and advantage be all included in the present invention, and with appended claims be protect
Protect range.
Claims (6)
1. a kind of fluorescent molecular probe with Mitochondrially targeted function, which is characterized in that have structure shown in formula (I):
2. a kind of prepare the method described in claim 1 with Mitochondrially targeted function fluorescence probe, which is characterized in that including
Following steps:
(1) parahydroxyben-zaldehyde is dissolved in anhydrous DMF, Anhydrous potassium carbonate and Isosorbide-5-Nitrae-dibromobutane is added, room temperature reaction obtains
Crude product, crude by column chromatography purifying, obtains compound 1；The compound 1 is
(2) by compound 1 and 2,4- dimethyl pyrrole is dissolved in anhydrous methylene chloride together, under nitrogen protection, trifluoro is added
Acetic acid is stirred at room temperature 12 hours as catalyst, 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone is then added, under room temperature
Continue stirring 2 hours, triethylamine and boron trifluoride ether be added after stirring, is stirred overnight, after reaction, reaction solution according to
It is secondary to use saturated sodium bicarbonate solution and water washing, it is then extracted with dichloromethane, then dry with anhydrous sodium sulfate, crude product is dense
Column chromatographic purifying is used after contracting, obtains compound 2；The compound 2 is
(3) compound 2 is flowed back 48 hours in dry toluene together with triethylamine, through column chromatographic purifying after reaction solution is concentrated
Obtain the compound of structure shown in formula (I).
3. according to the method described in claim 2, it is characterized by: in step (1), parahydroxyben-zaldehyde, potassium carbonate and Isosorbide-5-Nitrae-
The molar ratio of dibromobutane is 1:1.5:2.
4. according to the method described in claim 2, it is characterized by: in step (2), compound 1,2,4- dimethyl pyrrole and 2,
The molar ratio of the chloro- 5,6- dicyano -1,4- benzoquinones of 3- bis- is 1:2:1.
5. according to the method described in claim 2, it is characterized by: in step (3), compound 2 and the molar ratio of triethylamine are
1:10.
6. according to the method described in claim 2, it is characterized by: it is 8 that the leacheate of column chromatography, which is volume ratio, in step (1):
1 n-hexane-acetone mixture；In step (2), the leacheate of column chromatography is that the petroleum ether-ethyl acetate that volume ratio is 8:1 is mixed
Close liquid；In step (3), the leacheate of column chromatography is the methanol-ethyl acetate mixed liquor that volume ratio is 4:1.
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| CN107722055B (en) * | 2017-10-09 | 2020-04-03 | 天津理工大学 | Low-power white-light-driven mitochondrion-targeted fluorescent probe photosensitizer and synthesis method and application thereof |
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| CN111995619B (en) * | 2020-08-06 | 2022-02-25 | 广州医科大学附属第二医院 | Mitochondrial-targeted thioredoxin reductase fluorescent probe and preparation method and application thereof |
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