CN106279062A - A kind of benzothiazole 2 acetonitrile derivative and application thereof - Google Patents
A kind of benzothiazole 2 acetonitrile derivative and application thereof Download PDFInfo
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- CN106279062A CN106279062A CN201610575769.5A CN201610575769A CN106279062A CN 106279062 A CN106279062 A CN 106279062A CN 201610575769 A CN201610575769 A CN 201610575769A CN 106279062 A CN106279062 A CN 106279062A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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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- 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 present invention relates to a kind of benzothiazole 2 acetonitrile derivative, shown in its chemical constitution such as following formula (I).Benzothiazole 2 acetonitrile derivative of the present invention is to be obtained with benzothiazole 2 acetonitrile reaction by 4 (diethylamide) benzaldehyde.Benzothiazole 2 acetonitrile derivative of the present invention can carry out fluorescent labeling to intracellular mitochondrial.
Description
Technical field
The invention belongs to organic chemistry filed, relate to benzothiazole 2-cyanide compound, further relate to benzothiazole 2-acetonitrile
Purposes.
Background technology
Mitochondrion is the position that eukaryote carries out oxidative metabolism, is that saccharide, fat and aminoacid finally aoxidize release energy
The place of amount.Mitochondrion can provide place for the vital movement of cell, is Cellular Oxidation phosphorylation and the main field forming ATP
Institute.It addition, mitochondrion has DNA and the genetic system of self, but the gene dosage of mitochondrial genome is limited, therefore, and mitochondrion
Simply a kind of semi-autonomous organelle.In addition to for cell energy supply, mitochondrion also participates in such as cell differentiation, cellular informatics passes
Pass and the process such as apoptosis, and have regulating cell growth and the ability of cell cycle.Therefore, design synthesizing new is intracellular
Mitochondrion fluorescent 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 design has synthesized the asymmetric trimethine cyanine of benzothiazoles, is applied as nucleic acid fluorescent
Detection probe (Chen Xiuying, Guo Lin, Zheng Changge, high Potiria pectinifera (Mukller et Tro Sehel), An Wen, the synthesis of benzothiazoles nucleic acid molecules fluorescent probe and light
Spectral property, " applied chemistry ", 2012,29 (8): 892-897).But the easily cancellation in the solution of benzothiazole fluorochrome,
Do not possess and there is aggregation-induced emission effect.
Summary of the invention
It is an object of the invention to provide a kind of benzothiazole 2-acetonitrile derivative, this benzothiazole 2-acetonitrile derivative has
Aggregation-induced emission effect, can carry out fluorescence imaging to intracellular mitochondrion.
The present invention solves the technical scheme of the problems referred to above:
A kind of benzothiazole 2-acetonitrile derivative, its structural formula is shown in lower formula (I):
Benzothiazole 2-acetonitrile derivative of the present invention uses method commonly used in the art synthesis, e.g., by 4-(diethyl
Amine) benzaldehyde obtains with benzothiazole-2-acetonitrile reaction, and the following formula II of reaction equation of the method represents:
Benzothiazole 2-acetonitrile derivative of the present invention there is aggregation-induced emission effect (Aggregation-
Induced emission, AIE), under solvent state, i.e. do not show fluorescence, but have strong under coherent condition or solid state
Fluorescence.Therefore, it can be entirely applied in water solution system, and fluorescence spectrum has a hyperfluorescence peak at 550nm, at 365nm light
Excite lower display yellow-green fluorescence.
Benzothiazole 2-acetonitrile derivative of the present invention can be used for labeled cell mitochondrial.
In above-mentioned application, the concrete grammar of described labeled cell mitochondrial comprises the following steps:
The cell of trophophase of taking the logarithm is inoculated in and is equipped with in culture plate, adds containing 5～10 μMs of benzos of the present invention
After the RPMI-1640 of thiazole 2-acetonitrile derivative continues to cultivate 30min, clean 6 orifice plate 3 times with phosphate buffer, removed
The benzothiazole 2-acetonitrile derivative of amount, is placed in cell under laser confocal microscope observation, demonstrates green fluorescence position
It is mitochondrion.
Experiment results proved benzothiazole of the present invention 2-acetonitrile derivative can be with in highly selective labeled cell
Mitochondrion.The advantage that benzothiazole 2-acetonitrile derivative of the present invention has good biocompatibility.
Accompanying drawing explanation
Fig. 1 is the benzothiazole 2-acetonitrile derivative of the present invention fluorescence at the water/acetonitrile mixed solution of different ratios,
Wherein benzothiazole 2-acetonitrile derivative concentration is 5 μMs.
Fig. 2 is that the benzothiazole 2-acetonitrile derivative of the present invention water/DMSO mixed solution at different ratios is at 550nm
The fluorescence intensity at place, wherein 2-hydroxy phenyl-benzothiazole derivant concentration is 5 μMs.
Fig. 3 is that 2-hydroxy phenyl-benzothiazole derivant water/alcohol mixed solution at different ratios is at 550nm
Fluorescence intensity, wherein 2-hydroxy phenyl-benzothiazole derivant concentration is 5 μMs.
The cytotoxicity of Fig. 4 benzothiazole 2-acetonitrile derivative.
Detailed description of the invention
Embodiment 1
1. the preparation of benzothiazole 2-acetonitrile derivative
1.77g (10mmol) 4-(diphenylamines) benzaldehyde, 1.74g (10mmol) benzothiazole 2-is added in 50mL flask
Acetonitrile-2-acetonitrile and 0.77g (10mmol) ammonium acetate, be subsequently adding 20mL dehydrated alcohol.After reacting overnight under room temperature, precipitated
Filter, is recrystallized to give orange/yellow solid 2.36g in ethanol.Productivity 71%.
2. the sign 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 of preparation is chemistry benzothiazole 2-acetonitrile derivative shown in formula (I).
Embodiment 2 (fluorescent characteristic of benzothiazole 2-acetonitrile derivative)
Compound concentration is the benzothiazole 2-acetonitrile derivative acetonitrile solution of 5mM, takes 10 μ L benzothiazole 2-acetonitriles and derives
Thing acetonitrile solution, adds the volumetric flask of 10mL, is separately added into 1,2,3,4,5,6,7,8,9mL distilled water, is subsequently adding acetonitrile and adjusts
Whole liquor capacity is 10mL, respectively obtains benzothiazole 2-acetonitrile derivative water/acetonitrile solution (9/1, v/v) that concentration is 5 μMs,
Concentration is the benzothiazole 2-acetonitrile derivative water/acetonitrile solution (8/2, v/v) of 5 μMs, and concentration is the benzothiazole 2-acetonitrile of 5 μMs
Derivant water/acetonitrile solution (7/3, v/v), concentration is the benzothiazole 2-acetonitrile derivative water/acetonitrile solution (6/4, v/ of 1 μM
V), concentration is the benzothiazole 2-acetonitrile derivative water/acetonitrile solution (5/5, v/v) of 5 μMs, and concentration is the benzothiazole 2-of 5 μMs
Acetonitrile derivative water/acetonitrile solution (4/6, v/v), concentration is the benzothiazole 2-acetonitrile derivative water/acetonitrile solution (3/ of 5 μMs
7, v/v), concentration is the benzothiazole 2-acetonitrile derivative water/acetonitrile solution (2/8, v/v) of 5 μMs, and concentration is the benzo thiophene of 5 μMs
Azoles 2-acetonitrile derivative water/acetonitrile solution (1/9, v/v),.
Compound concentration is the benzothiazole 2-acetonitrile derivative acetonitrile solution of 5mM, takes 10 μ L benzothiazole 2-acetonitriles and derives
Thing acetonitrile solution, adds the volumetric flask of 10mL, with distilled water diluting to 10mL, obtains benzothiazole 2-acetonitrile derivative water-soluble
Liquid.
Compound concentration is the benzothiazole 2-acetonitrile derivative acetonitrile solution of 5mM, takes 10 μ L benzothiazole 2-acetonitriles and derives
Thing acetonitrile solution, adds the volumetric flask of 10mL, by dilution in acetonitrile to 10mL, obtains benzothiazole 2-acetonitrile derivative acetonitrile molten
Liquid.
Detecting the fluorescence of above-mentioned benzothiazole 2-acetonitrile derivative solution, result is as depicted in figs. 1 and 2.Benzothiazole 2-
The acetonitrile solution of acetonitrile derivative does not has fluorescence, when the volume ratio of water is more than 70% in mixed solution, demonstrates one at 550nm
Fluorescence peak.The fluorescence intensity of benzothiazole 2-acetonitrile derivative aqueous solution is the acetonitrile solution of benzothiazole 2-acetonitrile derivative
43 times of fluorescence intensity.Therefore, benzothiazole 2-acetonitrile derivative has AIE characteristic.
Foundation document (Chen Xiuying, Guo Lin, Zheng Changge, high Potiria pectinifera (Mukller et Tro Sehel), An Wen, benzothiazoles nucleic acid molecules fluorescent probe
Synthesis and spectral quality, " applied chemistry ", 2012,29 (8): 892-897) to prepare benzothiazoles asymmetric for described method
Trimethine cyanine, its structural formula is lower shown.
Being dissolved in ethanol by the asymmetric trimethine cyanine of benzothiazoles shown in chemistry formula (III), preparation one is
Row concentration is the ethanol/H of 5 μMs2O mixed solution, detects benzothiazoles asymmetric cyanine dye ethanol/H2O mixed solution glimmering
Light spectrum.Its result is as shown in Figure 3.Result shows along with in mixed solution, water content increases, the asymmetric front three of benzothiazoles
The fluorescence of river cyanines declines.
Detectable concentration is that the asymmetric trimethine cyanine of the benzothiazoles shown in chemical formula (III) of 5 μMs is water-soluble respectively
Liquid and concentration are the fluorescence light of the asymmetric trimethine cyanine ethanol solution of the benzothiazoles shown in chemical formula (III) of 5 μMs
Spectrum.Found that chemistry the benzothiazoles asymmetric trimethine cyanine aqueous solution fluorescence shown in formula (III) almost without.Change
The fluorescence intensity of the benzothiazoles asymmetric trimethine cyanine aqueous solution shown in formula (III) is shown in chemistry formula (III)
0.028 times of fluorescence intensity of ethanol solution of the asymmetric trimethine cyanine of benzothiazoles.
Therefore, document (Chen Xiuying, Guo Lin, Zheng Changge, high Potiria pectinifera (Mukller et Tro Sehel), An Wen, benzothiazoles nucleic acid molecules fluorescent probe
Synthesis and spectral quality, " applied chemistry ", 2012,29 (8): 892-897) the benzothiazoles asymmetric cyanine dye dye described in
Material does not has AIE performance.
Embodiment 3 (cell is cultivated and fluorescence imaging)
The HeLa cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, and overnight incubation is used instead containing 10 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative obtained continues to cultivate 30min, clean 6 orifice plates with phosphate buffer
3 times, remove the benzothiazole 2-acetonitrile derivative of excess.Cell continuously adds the RPMI-1640 of DAPI (10g/ml) and continues training
After supporting 30min, clean 6 orifice plate 3 times with phosphate buffer, remove the DAPI of excess.Cell is placed in laser co-focusing micro-
Microscopic observation.Yellow-green fluorescence is demonstrated at Cytoplasm.
Embodiment 4 (mtt assay detection cells growth activity)
By HeLa cell with every hole 6x103Individual cell is inoculated in 96 orifice plates, overnight incubation, uses the benzene containing embodiment 1 instead
And thiazole 2-acetonitrile derivative concentration is respectively 5, the culture fluid of 10,15,20 μMs, after continuing to cultivate 24h, inhale and abandon supernatant, often
Hole adds 200 μ l MTT reagent (5mg/ml prepares) with PBS, continues to cultivate 4h, discards culture fluid, and every hole adds 150 μ
LDMSO, is placed in 10min in cell shaking table, is completely dissolved to blue particle.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, measure each hole absorbance, with the culture fluid containing cell
It is matched group with MTT, with the culture fluid only adding equivalent and MTT as blank well.Calculate cell survival rate according to the following equation: cell
Survival rate (%)=(experimental port absorbance-blank well absorbance)/(control wells absorbance-blank well absorbance) ×
100%.Each concentration arranges 5 parallel holes, and experiment is repeated 3 times.Result is as shown in Figure 2.Fig. 2 shows that benzothiazole 2-acetonitrile spreads out
Biological cytotoxicity is low.
Embodiment 5
The HeLa cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, overnight incubation, use instead containing 5 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative arrived continues to cultivate 30min, clean 6 orifice plates 3 with phosphate buffer
Secondary, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Observe under light Laser Scanning Confocal Microscope.Red and the of the present invention fluorescent probe of mitochondrion is similar in intracellular distributed areas.Altogether
Orientation factor is high, and Pearson ' s coefficient (Rr) is 0.8, and overlap coefficient is 0.94.Illustrate of the present invention
Fluorescent probe main accumulation is in living cells mitochondrion.
Embodiment 6
The HeLa cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, and overnight incubation is used instead containing 10 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative obtained continues to cultivate 30min, clean 6 orifice plates with phosphate buffer
3 times, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Under light Laser Scanning Confocal Microscope, continuous exposure is observed.Continuously after scanning 360s, under the fluorescence intensity of fluorescent probe of the present invention
Dropped～23% and MTR have dropped～32%.Therefore, fluorescent probe of the present invention has good light stability.
Embodiment 7
The LoVo cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, overnight incubation, use instead containing 8 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative arrived continues to cultivate 30min, clean 6 orifice plates 3 with phosphate buffer
Secondary, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Observe under light Laser Scanning Confocal Microscope.Red and the of the present invention fluorescent probe of mitochondrion is similar in intracellular distributed areas.
Pearson ' s coefficient (Rr) is 0.84, and overlap coefficient is 0.96.Fluorescent probe master of the present invention is described
In living cells mitochondrion to be accumulated on.
Embodiment 8
The A549 cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, overnight incubation, use instead containing 8 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative arrived continues to cultivate 30min, clean 6 orifice plates 3 with phosphate buffer
Secondary, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Observe under light Laser Scanning Confocal Microscope.Red and the of the present invention fluorescent probe of mitochondrion is similar in intracellular distributed areas.
Pearson ' s coefficient (Rr) is 0.83, and overlap coefficient is 0.92.Fluorescent probe master of the present invention is described
In living cells mitochondrion to be accumulated on.
Embodiment 9
The PC3 cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, overnight incubation, use instead containing 10 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative arrived continues to cultivate 30min, clean 6 orifice plates 3 with phosphate buffer
Secondary, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Observe under light Laser Scanning Confocal Microscope.Red and the of the present invention fluorescent probe of mitochondrion is similar in intracellular distributed areas.
Pearson ' s coefficient (Rr) is 0.86, and overlap coefficient is 0.94.Fluorescent probe master of the present invention is described
In living cells mitochondrion to be accumulated on.
Embodiment 10
The LNCaP cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, and overnight incubation is used instead containing 10 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative obtained continues to cultivate 30min, clean 6 orifice plates with phosphate buffer
3 times, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Observe under light Laser Scanning Confocal Microscope.Red and the of the present invention fluorescent probe of mitochondrion is similar in intracellular distributed areas.
Pearson ' s coefficient (Rr) is 0.84, and overlap coefficient is 0.95.Fluorescent probe master of the present invention is described
In living cells mitochondrion to be accumulated on.
Embodiment 11
D μ 145 cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, and overnight incubation is used instead containing 10 μMs of embodiments 1
After the RPMI-1640 of the benzothiazole 2-acetonitrile derivative obtained continues to cultivate 30min, clean 6 orifice plates with phosphate buffer
3 times, remove the benzothiazole 2-acetonitrile derivative of excess, then with mitochondrion red (MTR) solution of the 250nM of PBS dilution at CO2
Incubator is cultivated 30min.Cleaning 6 orifice plate 3 times with phosphate buffer, the mitochondrion removing excess is red.Cell is placed in sharp
Observe under light Laser Scanning Confocal Microscope.Red and the of the present invention fluorescent probe of mitochondrion is similar in intracellular distributed areas.
Pearson ' s coefficient (Rr) is 0.82, and overlap coefficient is 0.93.Fluorescent probe master of the present invention is described
In living cells mitochondrion to be accumulated on.
Embodiment 12 (comparative example)
The HeLa cell of trophophase of taking the logarithm is inoculated in and is equipped with in 6 orifice plates, and overnight incubation is used instead containing 10 μMs of documents (old
Elegant English, Guo Lin, Zheng Changge, high Potiria pectinifera (Mukller et Tro Sehel), An Wen, the synthesis of benzothiazoles nucleic acid molecules fluorescent probe and spectral quality, " application
Chemistry ", 2012,29 (8): 892-897) chemistry the asymmetric trimethine cyanine of benzothiazoles shown in formula (III) 1640
After culture fluid continues to cultivate 30min, clean 6 orifice plate 3 times with phosphate buffer, remove the benzothiazoles asymmetric three of excess
Methine cyanine dyes, then in CO2 incubator, cultivate 30min with the Mito-Tracker Green solution of the 250nM of PBS dilution.
Clean 6 orifice plate 3 times with phosphate buffer, remove the Mito-Tracker Green of excess.Cell is placed in laser co-focusing
Basis of microscopic observation.Mito-Tracker Green is similar in intracellular distributed areas with fluorescent probe of the present invention.
Pearson ' s coefficient (Rr) is 0.54, and overlap coefficient is 0.63.Benzothiazoles described in explanation is asymmetric
The dyeing site of trimethine cyanine and Mito-Tracker Green is different, does not have special to mitochondrial fluorescence staining
Property probe.
Claims (3)
1. a benzothiazole 2-acetonitrile derivative, its structural formula is shown in lower formula (I):
2. the application in labeled cell mitochondrial of the benzothiazole 2-acetonitrile derivative described in claim 1.
Application the most according to claim 2, it is characterised in that the method for described labeled cell mitochondrial includes following step
Rapid:
The cell of trophophase of taking the logarithm is inoculated in and is equipped with in culture plate, adds containing the benzo thiophene described in 5～10 μMs of claim 1
After the RPMI-1640 of azoles 2-acetonitrile derivative continues to cultivate 30min, clean 6 orifice plate 3 times with phosphate buffer, remove excess
Benzothiazole 2-acetonitrile derivative, cell is placed under laser confocal microscope observation, demonstrates that green fluorescence position is i.e.
For mitochondrion.
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| CN106279062B (en) | 2018-06-05 |
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