CN104777141A - Application of detecting pH of benzothiazole derivative in extremely acidic environment - Google Patents
Application of detecting pH of benzothiazole derivative in extremely acidic environment Download PDFInfo
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- CN104777141A CN104777141A CN201510212698.8A CN201510212698A CN104777141A CN 104777141 A CN104777141 A CN 104777141A CN 201510212698 A CN201510212698 A CN 201510212698A CN 104777141 A CN104777141 A CN 104777141A
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- PXQMKXMYODRGKG-ASVGJQBISA-N CN(C)c1ccc(/C=C/C=C/c2nc3ccccc3[s]2)cc1 Chemical compound CN(C)c1ccc(/C=C/C=C/c2nc3ccccc3[s]2)cc1 PXQMKXMYODRGKG-ASVGJQBISA-N 0.000 description 1
Abstract
The invention discloses an application of detecting the pH of a benzothiazole derivative in the extremely acidic environment. The structural formula of the benzothiazole derivative is shown in the specification. A probe has the good selectivity and high sensitivity for H+ on the strongly acidic condition, has the obvious ratio fluorescence emission characteristic and has the advantages of having the large Stocks displacement and the like. The probe can be used for detecting the pH changes of escherichia coli in the extremely acidic environment, and can also be used for detecting pH changes in the extremely acidic environment.
Description
Technical field
The present invention relates to the purposes of benzothiazole compound, the application of specifically a kind of benzothiazole derivant pH change in detection extreme acidic environment is as Escherichia coli.
Background technology
PH homeostasis is kept to be all very important (people such as a.Baker-Austin.C., TrendsMicrobiol.2007,15:165-171.b.Krulwich to all biosomes, T.A. people is waited, Nat.Rev.Microbiol., 2011,9:330-343).PH value in different prokaryotes and eukaryotic different subcellular organelle (people such as a.Baker-Austin, C., Trends Microbiol. such as not from alkalescence to highly acid, 2007,15:165-171.b.Krulwich, the people such as T.A., Nat.Rev.Microbiol., 2011,9:330-343.c.Foster, the people such as J.W., Nat.Rev.Microbiol., 2004,2:898-907).Although for most of biology, extreme acidic (pH<4) is fatal, but there are some researches show, a large amount of microorganisms (as helicobacter pylori and " acidophil " etc.) has evolved to (a.Krulwich of can surviving under this extreme acidic's condition, T.A. people is waited, Nat.Rev.Microbiol., 2011,9:330-343.b.Merrell, D.S. people is waited, Curr.Opin.Microbiol., 2002,5:51-55).In addition, pathogenic entero becteria can arrive small intestine through mammiferous highly acid gastric juice (pH 0.9-1.5), causes fatal infection.Therefore be necessary to carry out sensitive, Real-Time Monitoring accurately to its pH, this has great importance for the physiology and toxicity process studying cell from molecular level.
The method of current mensuration internal pH has microelectrode method,
31p nuclear magnetic resonance method, ultraviolet absorption method and fluorometry etc.In these methods, fluorometry has easy and simple to handle, highly sensitive, selectivity good, the advantage that fast response time and non-destructive etc. are unique.In addition, can for molecular level detecting in real time the means that internal pH provides important in conjunction with laser confocal microscope.
Up to now, the pH fluorescence probe great majority of bibliographical information are applicable to the pH detection of faintly acid organelle (as: lysosome and endosome, pH 4.0-5.5) and weakly acidic pH tenuigenin (pH 6.8-7.4).But, few people pay close attention to the research of extreme acidic (pH<4) environment pH fluorescence probe, cause the very limited (people such as a.Su, M.H. of the kind of this aspect pH fluorescence probe, Chem.Commun., the people such as 2001,960-961.b.Xu, Y., Analytica Chimica Acta, the people such as 2014,820:146-151.c.Yang, M.Y., Angew.Chem.Int.Ed., the people such as 2012,51:7674-7679.Niu, W.F., Analytical Chemistry, 2015,87:2788-2793), the pH fluorescence probe especially with ratio test performance is very few.Therefore, in the urgent need to developing effective ratio emission type pH fluorescence probe, Sensitive Detection is carried out to the pH fluctuation of extreme acid condition (as Escherichia coli).
Summary of the invention
The object of this invention is to provide a kind of novelty teabag of benzothiazole derivant, specifically detecting as fluorescence probe the application that in extreme acidic's water environment and Escherichia coli, pH changes.This probe is to H in extreme acidic's environment
+there are higher sensitivity and good selectivity, and present ratio fluorescent emission characteristics, and there is large Stocks displacement, effectively can reduce the interference of exciting light in imaging process.
A kind of benzothiazole derivant provided by the invention detects the application in Escherichia coli pH change as fluorescence probe under extreme acidic's environment;
Described benzothiazole derivant, its structural formula is:
Described benzothiazole derivant also can be used as fluorescence probe for detecting the change of pH in extreme acidic's water environment.
Benzothiazole derivant synthetic route, reference literature people such as (, J.Photochem.Photobiol.A, 2006,177,238-247) Etaiw, S.H. synthesis:
Concrete synthetic method: under inert gas shielding, by 2-methylbenzothiazole, 4-(dimethylamino) cinnamic acid in molar ratio 1:1.5 is dissolved in N; dinethylformamide; by 2-methylbenzothiazole, add the KOH solid of 5 times of molal quantitys, stirring at room temperature is reacted.Cross after reacting completely and filter KOH, in filtrate, add H
2o (10ml), mixed solution dichloromethane extraction.After organic phase anhydrous sodium sulfate drying, decompression distillation, except desolventizing, obtains thick product.Thick product take methylene chloride as eluant, eluent, and silica gel column chromatography obtains sterling after being separated.
The pH response process of this fluorescence probe is as follows:
Its concrete operation step is as follows: probe molecule is dissolved in make in ethanol 10
-3the mother liquor of M, then use water/ethanol (V/V=2/1) system that probe dilution is carried out fluorescence spectrum pH titration to 10 μMs, fixing excitation wavelength is 383nm, fluorescence emission spectrum change under record condition of different pH.Along with the reduction of pH, the emission peak at 595nm place reduces, and new emission peak appears in 425nm place, the launching site appearance such as to have at 522nm place simultaneously.
Probe of the present invention can mark Escherichia coli, is applicable to the detection that Escherichia coli extreme acidic environment pH changes.Benzothiazole derivant also can be used for the change detecting pH in extreme acidic's water environment.
Compared with existing extreme acidic pH fluorescence probe, the present invention adopts benzothiazole derivant to belong to ratio emission type pH fluorescence probe as pH fluorescence probe, effectively can eliminate the error because the factors such as probe negative staining and biological sample uneven thickness, temperature and solvent cause, thus obtain than single transmit fluorescence probe measurement result more accurately; To H
+response has higher sensitivity and good selectivity; Have large Stokes displacement (177nm, pH 7.0 and 71nm, pH 1.0) simultaneously concurrently, the interference of exciting light can be effectively reduced.Probe of the present invention is based on Intramolecular electron transfer (ICT) principle design, benzothiazole and dimethylamino are respectively to electron accepter (A) and electron donor (D), under strongly acidic conditions, dimethylamino atom N occurs protonated, electron donation weakens, thus weaken the ICT effect of D-A in whole molecule, make uv absorption and the fluorescence emission spectrum generation blue shift of this probe.By naked eye to this solution under natural light and uviol lamp color change.This probe adopts Laser scanning confocal microscopy technology can mark Escherichia coli, carries out Sensitive Detection to the pH fluctuation of extreme acid condition.In addition, the synthesis of this probe is easy, with low cost, has very important actual application value.
Accompanying drawing explanation
Fig. 1. the uv absorption spectra of the embodiment of the present invention 1 middle probe under different pH condition.
Fig. 2. the embodiment of the present invention 1 middle probe identifies H under natural light
+front and back color change, color from yellow becomes colorless.
Fig. 3. the fluorescence spectrum figure of the embodiment of the present invention 1 middle probe under different pH condition.
Fig. 4. the embodiment of the present invention 1 middle probe identifies H under uviol lamp
+front and back color change, color from yellow becomes blueness.
Fig. 5. the fluorescence intensity ratio (F of the embodiment of the present invention 1 middle probe
425nm/ F
595nm) with the change curve of pH value.
Fig. 6. the embodiment of the present invention 1 middle probe common metal ion exist under to H
+selectivity.
Fig. 7. the embodiment of the present invention 1 middle probe and Escherichia coli (E.coli) jointly hatch the laser confocal imaging figure of 2h under the condition of pH value 7.0, and left figure is faint blue channel image, and right figure is bright red channel image,
Fig. 8. the embodiment of the present invention 1 middle probe and Escherichia coli (E.coli) jointly hatch the laser confocal imaging figure of 2h under the condition of pH value 1.0, and compared with pH 7.0, the almost cancellation of red channel fluorescence, blue channel fluorescence significantly strengthens.
Embodiment
Embodiment 1
The synthesis of probe of the present invention (benzothiazole derivant):
(1) under inert gas shielding; by 2-methylbenzothiazole (0.126ml; 1mmol); 4-(dimethylamino) cinnamic acid (0.263g; 1.5mmol) in 7ml DMF, stirring at room temperature reacts 24h with KOH (0.354g, 6.25mmol).
(2) excessively filter KOH solid, in reactant liquor, add H
2o (10ml), mixed solution dichloromethane extraction.After organic phase anhydrous sodium sulfate drying, decompression distillation, except desolventizing, obtains thick product.
(3) thick product take methylene chloride as eluant, eluent, after silica gel column chromatography is separated, obtain Orange red solid.1H NMR(300MHz,DMSO-d6):δ2.961-2.976(s,6H),6.674-6.893(m,3H),6.893-7.032(d,2H),7.290-7.476(m,5H),7.890-8.022(d,1H),8.022-8.047(d,1H).MS:m/z 307.1264for M+.
Embodiment 2
Be the storing solution of 1mM by the probe ethanol compound concentration in embodiment 1.Use water/ethanol (V/V=2/1) system by probe dilution in experiment, make its final concentration 10 μMs.The pH value of this system is regulated, the uv absorption spectra (Fig. 1) under record condition of different pH with the HCl of 1mol/L.Along with pH value is declined gradually by the absorption peak that 7.0 are reduced to 1.2,418nm place, the absorption peak at 354nm place strengthens successively, simultaneously in 383nm place existence obvious isoelectric point.Fig. 2 shows solution colour and is become colorless by yellow.
Embodiment 3
Probe dilution is carried out fluorescence spectroscopic titration to 10 μMs by same water/ethanol (V/V=2/1) system, and fixing excitation wavelength is 383nm, the fluorescence emission spectrum under record condition of different pH.As shown in Figure 3, under the neutrallty condition of pH 7.0, the maximum fluorescence emission of this probe is positioned at 595nm place, along with the reduction of pH, the fluorescent emission at 595nm place reduces gradually, and the fluorescent emission new 425nm place appearance one also significantly strengthens, simultaneously in 522nm place appearance one isoelectric point clearly.Under ultra violet lamp, the color of solution is from being become blueness (Fig. 4) from yellow.
Embodiment 4
By calculating fluorescence intensity ratio (F
425nm/ F
595nm) calculate pK from the matched curve of different pH value
avalue is 2.34 (Fig. 5).
Embodiment 5
Investigate probe (10 μMs) in embodiment 1 when common metal ion exists, to H
+selectivity.As shown in Figure 5, respectively under different pH (pH 7.0 and pH 1.2) condition, common metal ion is not almost responded, prove that this probe is to H
+there is very high selectivity.In Fig. 5, the order of each material and concentration are followed successively by: 1. probe; 2, Na
+(150mM); 3, K
+(150mM); 4, Ca
2+(10mM); 5, Mg
2+(2mM); 6, Co
2+(0.2mM); 7, Mn
2+(0.2mM); 8, Cd
2+(0.2mM); 9, Fe
3+(0.2mM); 10, Fe
2+(0.2mM); 11, Zn
2+(0.2mM); 12, Cu
2+(0.2mM); 13, Hg
2+(0.2mM); 14, Ni
2+(0.2mM); 15, Al
3+(0.2mM); 16, Ba
2+(0.2mM); 17, Pb
2+(0.2mM).
Embodiment 6
Probe in embodiment 1 and Escherichia coli (E.coli) are jointly hatched 2h respectively under pH value 7.0 and 1.0 condition in shaking table, under laser confocal microscope, observes fluorescence imaging change.Fixing excitation wavelength is 405nm, and emission band collects blue channel (420-460nm) and red channel (580-620nm) respectively.As shown in Figure 7, during pH 7.0, Escherichia coli present bright red fluorescence and faint blue-fluorescence; When pH is down to extreme acidic 1.0, colibacillary red fluorescence is cancellation completely almost, and blue-fluorescence significantly strengthens (Fig. 8).
Claims (2)
1. benzothiazole derivant detects the application in Escherichia coli pH change under extreme acidic's environment as fluorescence probe; Described benzothiazole derivant, its structural formula is:
2. benzothiazole derivant as fluorescence probe for detecting the change of pH in extreme acidic's water environment; Described benzothiazole derivant, its structural formula is:
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496214A (en) * | 2016-10-19 | 2017-03-15 | 山西大学 | The lysosome targeting type pH fluorescent probes of benzothiazoles and its preparation and application |
| CN111393462A (en) * | 2020-04-10 | 2020-07-10 | 山西大学 | Dual-mechanism-based fluorescent probe for detecting ONOO-, and preparation method and application thereof |
| CN112480052A (en) * | 2020-12-08 | 2021-03-12 | 山西大学 | Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application |
| CN112812075A (en) * | 2020-12-30 | 2021-05-18 | 山西大学 | Preparation method and application of benzothiazole Schiff base-based fluorescent probe |
| CN113234040A (en) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | Fluorescent probe molecule for detecting pH and preparation method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496214A (en) * | 2016-10-19 | 2017-03-15 | 山西大学 | The lysosome targeting type pH fluorescent probes of benzothiazoles and its preparation and application |
| CN106496214B (en) * | 2016-10-19 | 2019-02-26 | 山西大学 | The lysosome targeting type pH fluorescence probe of benzothiazoles and its preparation and application |
| CN111393462A (en) * | 2020-04-10 | 2020-07-10 | 山西大学 | Dual-mechanism-based fluorescent probe for detecting ONOO-, and preparation method and application thereof |
| CN112480052A (en) * | 2020-12-08 | 2021-03-12 | 山西大学 | Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application |
| CN112480052B (en) * | 2020-12-08 | 2022-05-31 | 山西大学 | Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application |
| CN112812075A (en) * | 2020-12-30 | 2021-05-18 | 山西大学 | Preparation method and application of benzothiazole Schiff base-based fluorescent probe |
| CN112812075B (en) * | 2020-12-30 | 2022-05-31 | 山西大学 | Preparation method and application of benzothiazole Schiff base-based fluorescent probe |
| CN113234040A (en) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | Fluorescent probe molecule for detecting pH and preparation method thereof |
| CN113234040B (en) * | 2021-05-28 | 2022-03-25 | 中国科学院新疆理化技术研究所 | Fluorescent probe molecule for detecting pH and preparation method thereof |
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Application publication date: 20150715 |
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