CN106279062B - A kind of benzothiazole 2- acetonitrile derivatives and its application - Google Patents
A kind of benzothiazole 2- acetonitrile derivatives and its application Download PDFInfo
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- CN106279062B CN106279062B CN201610575769.5A CN201610575769A CN106279062B CN 106279062 B CN106279062 B CN 106279062B CN 201610575769 A CN201610575769 A CN 201610575769A CN 106279062 B CN106279062 B CN 106279062B
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- KICQMCOLHFQZFC-RVPDKONISA-N CN(C=CC1/C=C/C=C/c2[n+](C)c3ccccc3[s]2)c2c1cccc2 Chemical compound CN(C=CC1/C=C/C=C/c2[n+](C)c3ccccc3[s]2)c2c1cccc2 KICQMCOLHFQZFC-RVPDKONISA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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Abstract
The present invention relates to a kind of 2 acetonitrile derivative of benzothiazole, chemical constitution such as following formula (I) is shown.2 acetonitrile derivative of benzothiazole of the present invention is to obtain 4 (diethylamide) benzaldehydes and 2 acetonitrile reaction of benzothiazole.2 acetonitrile derivative of benzothiazole of the present invention can carry out fluorescent marker to intracellular mitochondrial.
Description
Technical field
The invention belongs to organic chemistry fileds, are related to benzothiazole 2- cyanide compounds, further relate to benzothiazole 2- acetonitriles
Purposes.
Background technology
Mitochondria is the position that eucaryote carries out oxidative metabolism, is that carbohydrate, fat and amino acid finally aoxidize release energy
The place of amount.Mitochondria can provide place for the vital movement of cell, be the main field of Cellular Oxidation phosphorylation and formation ATP
Institute.In addition, mitochondria has the DNA and genetic system of itself, but the gene dosage of mitochondrial genomes is limited, therefore, mitochondria
A kind of simply semi-autonomous organelle.In addition to being energized for cell, mitochondria also participates in such as cell differentiation, cellular informatics biography
Pass with the processes such as Apoptosis, and possess regulating cell growth and the ability of cell cycle.Therefore, it is intracellular to design synthesizing new
Mitochondria fluorescence probe has important scientific meaning.
Benzothiazole compound has good fluorescent characteristic, can apply as fluorescence probe material, detection hydrogen sulfide etc..
Such as Cui Jing south etc. discloses a kind of hydrogen sulfide fluorescent detection probe (Publication No. based on benzothiazoles
CN103923640A).Chen Xiuying designs have synthesized benzothiazoles asymmetry trimethine cyanine, are applied as nucleic acid fluorescent
Detection probe (Chen Xiuying, Guo Lin, Zheng Changge, high petrel, An Wen, the synthesis of benzothiazoles nucleic acid molecules fluorescence probe and light
Spectral property,《Applied chemistry》, 2012,29 (8):892-897).But benzothiazole fluorochrome is easily quenched in the solution,
Do not possess with aggregation-induced emission effect.
The content of the invention
The object of the present invention is to provide a kind of benzothiazole 2- acetonitrile derivatives, benzothiazole 2- acetonitrile derivatives have
Aggregation-induced emission effect can carry out fluorescence imaging to intracellular mitochondria.
Technical proposal that the invention solves the above-mentioned problems is：
A kind of benzothiazole 2- acetonitrile derivatives, structural formula is shown in lower formula (I)：
Benzothiazole 2- acetonitrile derivatives of the present invention are synthesized using method commonly used in the art, e.g., by 4- (diethyl
Amine) benzaldehyde obtains with benzothiazole -2- acetonitrile reactions, and the reaction equation such as following formula (II) of this method represents：
Benzothiazole 2- acetonitrile derivatives of the present invention have aggregation-induced emission effect (Aggregation-
Induced emission, AIE), i.e., fluorescence is not shown under solvent state, but is had strongly under coherent condition or solid state
Fluorescence.Therefore, can be entirely applied in water solution system, fluorescence spectrum has a hyperfluorescence peak at 550nm, in 365nm light
The lower display yellow-green fluorescence of excitation.
Benzothiazole 2- acetonitrile derivatives of the present invention can be used for marking intracellular mitochondrial.
In above application, the specific method of the mark intracellular mitochondrial comprises the following steps：
Take the logarithm growth period cell inoculation in being equipped in culture plate, add in and contain 5~10 μM of benzos of the present invention
The 1640 culture medium of thiazole 2- acetonitrile derivatives continues after cultivating 30min, cleans 6 orifice plate 3 times with phosphate buffer, removed
The benzothiazole 2- acetonitrile derivatives of amount, cell is placed under laser confocal microscope and is observed, shows green fluorescence position
As mitochondria.
Experiment results proved benzothiazole 2- acetonitrile derivatives of the present invention can mark into the cell with high selectivity
Mitochondria.Benzothiazole 2- acetonitrile derivatives of the present invention have the advantages that good biocompatibility.
Description of the drawings
Fig. 1 is fluorescence of the benzothiazole 2- acetonitrile derivatives of the present invention in water/acetonitrile mixed solution of different ratios,
Wherein benzothiazole 2- acetonitrile derivatives concentration is 5 μM.
Fig. 2 be benzothiazole 2- acetonitrile derivatives of the present invention different ratios water/DMSO mixed solutions in 550nm
The fluorescence intensity at place, wherein 2- hydroxy phenyl-benzothiazole derivant concentration are 5 μM.
Fig. 3 be 2- hydroxy phenyls-benzothiazole derivant different ratios water/alcohol mixed solution at 550nm
Fluorescence intensity, wherein 2- hydroxy phenyl-benzothiazole derivant concentration are 5 μM.
The cytotoxicity of Fig. 4 benzothiazole 2- acetonitrile derivatives.
Specific embodiment
Embodiment 1
1. the preparation of benzothiazole 2- acetonitrile derivatives
1.77g (10mmol) 4- (diphenylamines) benzaldehyde, 1.74g (10mmol) benzothiazoles 2- are added in 50mL flasks
Then acetonitrile -2- acetonitriles and 0.77g (10mmol) ammonium acetate add in 20mL absolute ethyl alcohols.At room temperature after reaction overnight, precipitated
Filter, is recrystallized to give orange/yellow solid 2.36g in ethanol.Yield 71%.
2. the characterization of compound
1H NMR (400MHz, CDCl3)δ(ppm):7.98 (s, 1H), 7.92 (d, 1H), 7.87 (d, 2H), 7.77 (d,
1H), 7.39 (t, 1H), 7.27 (t, 1H), 6.61 (d, 2H), 3.36 (dd, 4H), 1.64 (s, IH), 1.14 (t, 6H)
13C NMR (100MHz, CDCl3)δ(ppm):163.88,152.83,149.82,145.86,132.42,125.49,
123.96,121.73,120.42,118.58,110.28,95.88,43.68,28.68,11.57.
IR (v-1, KBr):3396,2973,2357,2208,1590,1569,1515,1468,1405,1352,1268,
1179,1146,1069,986,819,762,724,670,584,519.
HR-MS(ESI):C20H19N3S m/z, 333.1300for [M+Na]+:356.1203
Mp:191.5-193.℃
Above-mentioned testing result confirms that the compound prepared is the benzothiazole 2- acetonitrile derivatives shown in chemical formula (I).
Embodiment 2 (fluorescent characteristic of benzothiazole 2- acetonitrile derivatives)
Compound concentration is the benzothiazole 2- acetonitrile derivative acetonitrile solutions of 5mM, and 10 μ L benzothiazole 2- acetonitriles is taken to derive
Object acetonitrile solution adds in the volumetric flask of 10mL, is separately added into 1,2,3,4,5,6,7,8,9mL distilled water, then adds in acetonitrile tune
Whole liquor capacity is 10mL, respectively obtains benzothiazole 2- acetonitrile derivatives water/acetonitrile solution (9/1, v/v) that concentration is 5 μM,
Concentration is 5 μM of benzothiazole 2- acetonitrile derivatives water/acetonitrile solution (8/2, v/v), and concentration is 5 μM of benzothiazole 2- acetonitriles
Derivative water/acetonitrile solution (7/3, v/v), concentration are 1 μM of benzothiazole 2- acetonitrile derivatives water/acetonitrile solution (6/4, v/
V), concentration is 5 μM of benzothiazole 2- acetonitrile derivatives water/acetonitrile solution (5/5, v/v), and concentration is 5 μM of benzothiazole 2-
Acetonitrile derivative water/acetonitrile solution (4/6, v/v), concentration are 5 μM of benzothiazole 2- acetonitrile derivatives water/acetonitrile solution (3/
7, v/v), concentration is 5 μM of benzothiazole 2- acetonitrile derivatives water/acetonitrile solution (2/8, v/v), and concentration is 5 μM of benzo thiophene
Azoles 2- acetonitrile derivatives water/acetonitrile solution (1/9, v/v),.
Compound concentration is the benzothiazole 2- acetonitrile derivative acetonitrile solutions of 5mM, and 10 μ L benzothiazole 2- acetonitriles is taken to derive
Object acetonitrile solution, adds in the volumetric flask of 10mL, and 10mL is diluted to distilled water, and it is water-soluble to obtain benzothiazole 2- acetonitrile derivatives
Liquid.
Compound concentration is the benzothiazole 2- acetonitrile derivative acetonitrile solutions of 5mM, and 10 μ L benzothiazole 2- acetonitriles is taken to derive
Object acetonitrile solution adds in the volumetric flask of 10mL, and with dilution in acetonitrile to 10mL, it is molten to obtain benzothiazole 2- acetonitrile derivative acetonitriles
Liquid.
The fluorescence of above-mentioned benzothiazole 2- acetonitrile derivative solution is detected, as a result as depicted in figs. 1 and 2.Benzothiazole 2-
The acetonitrile solution of acetonitrile derivative does not have fluorescence, and when the volume ratio of water in mixed solution is more than 70%, one is shown in 550nm
Fluorescence peak.The fluorescence intensity of benzothiazole 2- acetonitrile derivative aqueous solutions is the acetonitrile solution of benzothiazole 2- acetonitrile derivatives
43 times of fluorescence intensity.Therefore, benzothiazole 2- acetonitrile derivatives have AIE characteristics.
Foundation document (Chen Xiuying, Guo Lin, Zheng Changge, high petrel, An Wen, benzothiazoles nucleic acid molecules fluorescence probe
Synthesis and spectral quality,《Applied chemistry》, 2012,29 (8):892-897) benzothiazoles asymmetry is prepared in the method
Trimethine cyanine, structural formula are lower shown.
By the benzothiazoles asymmetry trimethine cyanine dissolving shown in chemical formula (III) in ethanol, a system is prepared
Row concentration is 5 μM of ethyl alcohol/H2O mixed solutions, detection benzothiazoles asymmetry cyanine dye ethyl alcohol/H2O mixed solutions it is glimmering
Light spectrum.The results are shown in Figure 3 for it.The results show is with water content increase, benzothiazoles asymmetry front three in mixed solution
The fluorescence of river cyanines declines.
Detectable concentration is that the benzothiazoles asymmetry trimethine cyanine shown in 5 μM of chemical formula (III) is water-soluble respectively
The fluorescence light of benzothiazoles asymmetry trimethine cyanine ethanol solution shown in liquid and the chemical formula (III) that concentration is 5 μM
Spectrum.It turns out that benzothiazoles asymmetry trimethine cyanine aqueous solution fluorescence shown in chemical formula (III) almost without.Change
The fluorescence intensity of benzothiazoles asymmetry trimethine cyanine aqueous solution shown in formula (III) is shown in chemical formula (III)
0.028 times of fluorescence intensity of ethanol solution of benzothiazoles asymmetry trimethine cyanine.
Therefore, document (Chen Xiuying, Guo Lin, Zheng Changge, high petrel, An Wen, benzothiazoles nucleic acid molecules fluorescence probe
Synthesis and spectral quality,《Applied chemistry》, 2012,29 (8):Benzothiazoles asymmetry cyanine dye dye described in 892-897)
Material does not have AIE performances.
Embodiment 3 (cell culture and fluorescence imaging)
Take the logarithm growth period HeLa cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 10 μM of embodiments 1
The 1640 culture medium of obtained benzothiazole 2- acetonitrile derivatives continues after cultivating 30min, and 6 orifice plates are cleaned with phosphate buffer
3 times, remove excessive benzothiazole 2- acetonitrile derivatives.The 1640 culture medium that cell continuously adds DAPI (10g/ml) continues to train
After supporting 30min, clean 6 orifice plate 3 times with phosphate buffer, remove excessive DAPI.It is micro- that cell is placed in laser co-focusing
Microscopic observation.Yellow-green fluorescence is shown in cytoplasm.
Embodiment 4 (mtt assay detection cells growth activity)
By HeLa cells with every hole 6x103A cell inoculation uses the benzene containing embodiment 1 instead in 96 orifice plates, overnight incubation
And thiazole 2- acetonitrile derivative concentration is respectively 5,10,15,20 μM of culture solution, after continuing culture for 24 hours, supernatant is abandoned in suction, often
Hole adds in 200 μ l MTT reagents (5mg/ml is prepared with PBS), continues to cultivate 4h, discards culture solution, 150 μ are added in per hole
LDMSO is placed in 10min in cell shaking table, until blue particle is completely dissolved.With microplate reader (the full-automatic microplate reader of ELX800, the U.S.
Bao Te Instrument Ltd.) under the conditions of excitation wavelength is 490nm, each hole absorbance is measured, with culture solution containing cell
With MTT be control group, using only plus equivalent culture solution and MTT as blank well.Cell survival rate is calculated according to the following equation:Cell
Survival rate (%)=(experimental port absorbance-blank well absorbance)/(control wells absorbance-blank well absorbance) ×
100%.Each concentration sets 5 parallel holes, and experiment is repeated 3 times.The results are shown in Figure 2.Fig. 2 shows that benzothiazole 2- acetonitriles spread out
The cytotoxicity of biology is low.
Embodiment 5
Take the logarithm growth period HeLa cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 5 μM of embodiments 1
To benzothiazole 2- acetonitrile derivatives 1640 culture medium continue cultivate 30min after, clean 6 orifice plates 3 with phosphate buffer
It is secondary, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
It is observed under light Laser Scanning Confocal Microscope.The red distributed areas with fluorescence probe of the present invention in the cell of mitochondria are similar.Altogether
Orientation factor is high, and Pearson ' s coefficients (Rr) are that 0.8, overlap coefficient are 0.94.Illustrate of the present invention
Fluorescence probe main accumulation is in living cells mitochondria.
Embodiment 6
Take the logarithm growth period HeLa cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 10 μM of embodiments 1
The 1640 culture medium of obtained benzothiazole 2- acetonitrile derivatives continues after cultivating 30min, and 6 orifice plates are cleaned with phosphate buffer
3 times, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
Continuous exposure is observed under light Laser Scanning Confocal Microscope.After continuous scanning 360s, under the fluorescence intensity of fluorescence probe of the present invention
Drop~23% and MTR has dropped~32%.Therefore, fluorescence probe of the present invention has good photostability.
Embodiment 7
Take the logarithm growth period LoVo cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 8 μM of embodiments 1
To benzothiazole 2- acetonitrile derivatives 1640 culture medium continue cultivate 30min after, clean 6 orifice plates 3 with phosphate buffer
It is secondary, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
It is observed under light Laser Scanning Confocal Microscope.The red distributed areas with fluorescence probe of the present invention in the cell of mitochondria are similar.
Pearson ' s coefficients (Rr) are that 0.84, overlap coefficient are 0.96.Illustrate fluorescence probe master of the present invention
It accumulates in living cells mitochondria.
Embodiment 8
Take the logarithm growth period A549 cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 8 μM of embodiments 1
To benzothiazole 2- acetonitrile derivatives 1640 culture medium continue cultivate 30min after, clean 6 orifice plates 3 with phosphate buffer
It is secondary, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
It is observed under light Laser Scanning Confocal Microscope.The red distributed areas with fluorescence probe of the present invention in the cell of mitochondria are similar.
Pearson ' s coefficients (Rr) are that 0.83, overlap coefficient are 0.92.Illustrate fluorescence probe master of the present invention
It accumulates in living cells mitochondria.
Embodiment 9
Take the logarithm growth period PC3 cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 10 μM of embodiments 1
To benzothiazole 2- acetonitrile derivatives 1640 culture medium continue cultivate 30min after, clean 6 orifice plates 3 with phosphate buffer
It is secondary, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
It is observed under light Laser Scanning Confocal Microscope.The red distributed areas with fluorescence probe of the present invention in the cell of mitochondria are similar.
Pearson ' s coefficients (Rr) are that 0.86, overlap coefficient are 0.94.Illustrate fluorescence probe master of the present invention
It accumulates in living cells mitochondria.
Embodiment 10
Take the logarithm growth period LNCaP cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead containing 10 μM of embodiments 1
The 1640 culture medium of obtained benzothiazole 2- acetonitrile derivatives continues after cultivating 30min, and 6 orifice plates are cleaned with phosphate buffer
3 times, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
It is observed under light Laser Scanning Confocal Microscope.The red distributed areas with fluorescence probe of the present invention in the cell of mitochondria are similar.
Pearson ' s coefficients (Rr) are that 0.84, overlap coefficient are 0.95.Illustrate fluorescence probe master of the present invention
It accumulates in living cells mitochondria.
Embodiment 11
Take the logarithm growth period 145 cell inoculations of D μ in being equipped in 6 orifice plates, overnight incubation is used instead containing 10 μM of embodiments 1
The 1640 culture medium of obtained benzothiazole 2- acetonitrile derivatives continues after cultivating 30min, and 6 orifice plates are cleaned with phosphate buffer
3 times, remove excessive benzothiazole 2- acetonitrile derivatives, then with red (MTR) solution of mitochondria of the diluted 250nM of PBS in CO2
30min is cultivated in incubator.6 orifice plate is cleaned with phosphate buffer 3 times, and it is red to remove excessive mitochondria.Cell is placed in sharp
It is observed under light Laser Scanning Confocal Microscope.The red distributed areas with fluorescence probe of the present invention in the cell of mitochondria are similar.
Pearson ' s coefficients (Rr) are that 0.82, overlap coefficient are 0.93.Illustrate fluorescence probe master of the present invention
It accumulates in living cells mitochondria.
Embodiment 12 (comparative example)
Take the logarithm growth period HeLa cell inoculations in being equipped in 6 orifice plates, overnight incubation is used instead (old containing 10 μM of documents
Elegant English, Guo Lin, Zheng Changge, high petrel, An Wen, the synthesis of benzothiazoles nucleic acid molecules fluorescence probe and spectral quality,《Using
Chemistry》, 2012,29 (8):892-897) 1640 of the benzothiazoles asymmetry trimethine cyanine shown in chemical formula (III)
Culture solution continues after cultivating 30min, cleans 6 orifice plate 3 times with phosphate buffer, removes excessive benzothiazoles asymmetry three
Methine cyanine dyes, then with the Mito-Tracker Green solution of the diluted 250nM of PBS 30min is cultivated in CO2 incubators.
6 orifice plate is cleaned with phosphate buffer 3 times, removes excessive Mito-Tracker Green.Cell is placed in laser co-focusing
Micro- Microscopic observation.Distributed areas of the Mito-Tracker Green with fluorescence probe of the present invention in the cell are similar.
Pearson ' s coefficients (Rr) are that 0.54, overlap coefficient are 0.63.Illustrate that the benzothiazoles are asymmetric
The dyeing site of trimethine cyanine and Mito-Tracker Green are different, do not have to the fluorescent staining of mitochondria special
Property probe.
Claims (2)
1. application of the benzothiazole 2- acetonitrile derivatives in intracellular mitochondrial is marked, wherein the benzothiazole 2- acetonitriles
The structural formula of derivative for shown in lower formula (I),
2. application according to claim 1, which is characterized in that the method for the mark intracellular mitochondrial includes following step
Suddenly：
Take the logarithm growth period cell inoculation in 6 orifice plates, add in containing 5~10 μM of benzothiazole 2- described in claim 1
The 1640 culture medium of acetonitrile derivative continues after cultivating 30min, cleans 6 orifice plate 3 times with phosphate buffer, removes excessive benzene
And thiazole 2- acetonitrile derivatives, cell is placed under laser confocal microscope and is observed, it is line to show green fluorescence position
Plastochondria.
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| CN103788940A (en) * | 2012-11-02 | 2014-05-14 | 国家纳米科学中心 | Aggregation-induced emission fluorescent molecule as well as preparation method and fluorescent dye composition, and application of aggregation-induced emission fluorescent molecule and fluorescent dye composition in mitochondria dyeing |
| CN103893172A (en) * | 2014-03-28 | 2014-07-02 | 中山大学 | Application of antivirus compound in preparation of anti-HIV-1 virus medicament |
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