CN108129500A - A kind of synthesis and application of the ratio type fluorescence probe for identifying hydrogen peroxide - Google Patents
A kind of synthesis and application of the ratio type fluorescence probe for identifying hydrogen peroxide Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 60
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 230000015572 biosynthetic process Effects 0.000 title claims description 4
- 238000003786 synthesis reaction Methods 0.000 title claims description 4
- 238000001514 detection method Methods 0.000 claims abstract description 16
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 1
- 210000003470 mitochondria Anatomy 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 8
- 210000004027 cell Anatomy 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 4
- 230000006378 damage Effects 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
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- 238000002360 preparation method Methods 0.000 abstract description 2
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- 230000002708 enhancing effect Effects 0.000 abstract 1
- 238000002189 fluorescence spectrum Methods 0.000 abstract 1
- 208000014674 injury Diseases 0.000 abstract 1
- 238000003384 imaging method Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 5
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 5
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
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- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
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- 208000037273 Pathologic Processes Diseases 0.000 description 1
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- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
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- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000008965 mitochondrial swelling Effects 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
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- 210000003463 organelle Anatomy 0.000 description 1
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- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation 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
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- 150000003613 toluenes Chemical class 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The present invention relates to one kind for detecting hydrogen peroxide (H2O2) fluorescence probe preparation method and application, belong to chemical analysis detection technique field.Its molecular structure is as follows：The probe molecule maximum emission wavelength is in 666nm, probe molecule and hydrogen peroxide (H2O2) after effect, fluorescence spectrum is blue shifted at 594nm, realizes ratio feux rouges detection hydrogen peroxide (H2O2), improve the sensitivity of detection；Launch wavelength can reduce the light injury of the background fluorescence and living cells during probe in detecting in red light district, and enhancing biology is to the penetration capacity of tissue.Probe molecule of the present invention has good linear in certain time and concentration range, to hydrogen peroxide (H2O2) recognition capability is strong, mitochondria can be accurately positioned in the good, strong antijamming capability of selectivity, realize detection hydrogen peroxide in mitochondria, such probe has important application value in fields such as biochemistries.
Description
The invention belongs to chemical analysis detection technique fields, and in particular to detection hydrogen peroxide is novel in a kind of mitochondria
The preparation method of ratio feux rouges type fluorescence probe and its in vitro with detection hydrogen peroxide (H inside living cells2O2) in terms of should
With.
Background technology
Active oxygen (ROS) class plays important role in the physiology of biosystem and pathologic process.As important
One of ROS, hydrogen peroxide generate in almost all of oxidation process, and take part in the regulation and control to bioactivity.Endogenous mistake
Hydrogen oxide (H2O2) as a kind of " signaling molecule ", it can be conducted with activation signal, stimulate the physiological activities such as proliferation, the differentiation of cell.
Excessive endogenous hydrogen peroxide (H2O2) generated by the catalysis stimulation of desmoenzyme, so as to generate oxidation pressure, destroy albumen
The lipid moleculars such as matter, nucleic acid, damage biomolecule, it is considered to be the trigger of many diseases, as diabetes, cancer and old age are crazy about
Slow-witted disease.In addition, it is a large amount of the experimental results showed that, the excessive endogenous hydrogen peroxide (H generated during mitochondrial respiratory2O2)
Strong oxygen may be caused to damage, further cause mitochondrial swelling and Apoptosis.Therefore, it is necessary to it designs a kind of effective
Method, realize subcellular organelle in hydrogen peroxide Cut-set power space detection, particularly in mitochondria.
Invention content
It is an object of the present invention to provide a kind of fluorescence probe synthetic methods of simple and effective；Another object of the present invention
It is good to be to provide a kind of selectivity, strong antijamming capability has ratio red light emission wavelength, can realize to external or living cells
Inside particularly mitochondria hydrogen peroxide (H2O2) detection fluorescence probe.
The technical solution of the present invention for solving the problems, such as to take is that a kind of ratio type identifies hydrogen peroxide novel fluorescence probe,
Molecular structural formula is as follows：
Synthetic route is as follows：
Specific synthetic method is as follows：(a) by compound 1 (200.0g, 1.4mmol) and 4- bromomethyl benzene boric acid pinacol esters
(620.0g, 2.1mmol) is dissolved in 2.0mL dry toluenes 110 DEG C of stirring 6h in closed reactor.It is precipitated in reaction process a large amount of
White solid stops reaction and is cooled to room temperature suction filtration, and filter cake is washed with toluene, obtains pure product 2 (180.0mg, 29.3%).
(b) compound 2 (160.0g, 0.46mmol) and compound 3 (100.0g, 0.4mmol) are dissolved in 2.0mL ethyl alcohol at room temperature
Solution in, add in piperidines (5.0 μ L, 0.05mmol).It is stirred at room temperature after five minutes, acquired solution is flowed back 6 hours.So
Reaction solution is concentrated under reduced pressure afterwards, column chromatography for separation obtains the compound 4 (60.0mg, 22.6% yield) of Orange red solid.
The mechanism of action of the fluorescence probe of the present invention is as follows, and probe molecule enhances intramolecular by the introducing of positive charge
ICT effects and emit near infrared light (666nm).Hydrogen peroxide can be released in oxidised probe molecule in bromo borinate ester moiety, hydrolysis
Dyestuff 6 is released, intramolecular ICT processes is reduced and leads to launch wavelength blue shift and emit feux rouges (594nm), so as to fulfill special
Property detection hydrogen peroxide (H2O2) purpose.The response process of probe molecule is as follows：
The fluorescence probe of the present invention has ratio red emission, with hydrogen peroxide (H2O2) effect before transmitting near-infrared it is glimmering
Light is at 666nm, and fluorescence emission peak is at 594nm after effect.
The fluorescence probe of the present invention has Mitochondrially targeted site, shows that the probe can realize mitochondria hydrogen peroxide
(H2O2) detection.
The fluorescence probe of the present invention is selectively good.The PBS bufferings that the test system of probe molecule is the 10mM that pH is 7.0 are molten
Liquid includes 30% acetonitrile, measures at room temperature.Probe molecule emits near-infrared fluorescent in itself, is adding in 40 times of equivalents of hydrogen peroxide
(H2O2) after, fluorescence intensity, which has occurred, at maximum emission wavelength 666nm is blue shifted at 594nm.And adding in other active oxygens
Substance (ROO.,.OH,NO.,TBHP,NaClO,.O2,KO2,.OtBu,ONOO-) after, fluorescence emission peak does not change.
The fluorescence probe strong antijamming capability of the present invention, other reactive oxygen species (ROO.,.OH,NO.,TBHP,NaClO,.O2,KO2,.OtBu,ONOO-) presence do not influence probe molecule and hydrogen peroxide (H2O2) effect.
The fluorescence probe of the present invention is in the hydrogen peroxide (H for adding in 40 times of equivalents2O2) after effect, fluorescence emission peak is blue shifted to
594nm, in 2h, fluorescence reaches maximum value at 594nm, and the emission peak at 666nm disappears.
The fluorescence probe of the present invention shows good dynamic phenomena, probe molecule and hydrogen peroxide (H2O2) selectivity
After identification good linear relationship is shown with the time in, fluorescence intensity ratio.
The fluorescence probe of the present invention can realize intraor extracellular detection hydrogen peroxide (H2O2), pass through common location reagent Luo Dan
The Mitochondrially targeted performance that bright 123 confirmation probe has had, overlap coefficient have reached 94%, have shown that the probe can realize mitochondria
Interior detection hydrogen peroxide (H2O2)。
Probe molecule of the present invention is to hydrogen peroxide (H2O2) launch wavelength before response sends out after near-infrared, response
It penetrates blue shift and red fluorescence is presented and active oxygen is had good selectivity and antijamming capability, and the spirit having had
Sensitivity has wider application range.Long ratio transmitted wave with strong tissue penetration, the fluorescence probe biology with
The fields such as chemistry have practical application value.
Description of the drawings
Fig. 1 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in PBS (10mM, pH=7.0)/CH3CN30% is molten
In liquid, with hydrogen peroxide (H2O2) the front and rear spectrum change figure of effect, abscissa is wavelength, and ordinate is respectively that absorbing/fluorescent is strong
Degree.
Fig. 2 is the fluorescence probe (10.0 × 10-6mol/L) of the present invention in PBS (10mM, pH=7.0)/CH3In CN30%
Fluorescent emission spectrogram with add in hydrogen peroxide (H2O2) amount variation.
Fig. 3 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in PBS (10mM, pH=7.0)/CH3In CN30%
The fluorogram variation of selectivity test, abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 4 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in PBS (10mM, pH=7.0)/CH3In CN30%
The bar chart of selectivity, ordinate I594/I666The variation of intensity.
Fig. 5 is the fluorescence probe (10.0 × 10-6mol/L) of the present invention in PBS (10mM, pH=7.0)/CH3In CN30%
Detect hydrogen peroxide (H2O2) there is (ROO.,.OH,NO.,TBHP,NaClO,.O2,KO2,.OtBu,ONOO-) isoreactivity oxygen species
When anti-interference bar chart, ordinate I594/I666The variation of intensity.
Fig. 6 is the fluorescence probe (10.0 × 10-6mol/L) of the present invention in PBS (10mM, pH=7.0)/CH3CN30%
In, with hydrogen peroxide (H2O2) the linear relationship research of ratio fluorescence intensity and time in mechanism.
Fig. 7 is the fluorescence probe (10.0 × 10-6mol/L) of the present invention in PBS (10mM, pH=7.0)/CH3CN30%
In, with hydrogen peroxide (H2O2) before and after effect, I594/I666The curve that intensity changes over time.
Fig. 8 is the toxicity research experiment of the fluorescence probe cell applicability of the present invention, and abscissa is concentration and probe concentration, ordinate
For cell survival rate.
The fluorescence probe that Fig. 9 is the present invention detects endogenous, exogenous hydrogen peroxide (H in the cell2O2) imaging it is real
It tests, first is horizontally-arranged for the probe imaging contexts of (10 μM) in the cell in itself；Second is horizontally-arranged to add in probe (10 μM) and external source
Property hydrogen peroxide (H2O2) imaging contexts after (0.5mM)；Third it is horizontally-arranged for add in endogenous stimulus drug PMA (1 μ g/mL) and
Probe (10 μM) detects hydrogen peroxide (H afterwards2O2) imaging contexts, the experimental result obtained using 488nm as excitation wavelength.
Figure 10 is the common location imaging experiment of the fluorescence probe of the present invention, and b is common location reagent Rhodamine 123 (2.0 μM)
Image, c be probe (10 μM) imaging experiment figure, a be b and c Overlay figure, d be light field design sketch, use
The experimental result that 488nm is obtained as excitation wavelength.
Figure 11 is the overlap coefficient that the fluorescence probe common location of the present invention is tested, and illustrates probe and common location reagent energy
Overlapping very well, so as to verify that mitochondria can be accurately positioned in probe.
Example is embodied
Embodiment 1：The synthesis of probe molecule
Compound 2 (160.0g, 0.46mmol) and compound 3 (100.0g, 0.4mmol) are dissolved in 2.0mL ethyl alcohol at room temperature
In solution in, add in piperidines (5.0 μ L, 0.05mmol).It is stirred at room temperature after five minutes, acquired solution is flowed back 6 hours.
Then reaction solution is concentrated under reduced pressure, with methylene chloride/methanol (v/v=15/1) for eluant, eluent carry out column chromatography for separation obtain it is orange red
The compound 4 (60.0mg, 22.6% yield) of color solid.HRMS(EI)m/z:calcd for C37H34BN2O3S+(M+H)+
597.23777,found597.23822.1H NMR(400MHz,DMSO)δH 12.35(s,1H),9.61(s,1H),9.12(s,
1H), 8.77 (s, 1H), 8.68 (s, 1H), 8.46 (d, J=15.1Hz, 2H), 8.33 (s, 3H), 8.19 (s, 2H), 8.12 (s,
2H), 8.00 (s, 1H), 7.79 (d, J=6.2Hz, 1H), 7.59 (s, 1H), 7.49 (s, 1H), 7.33 (s, 2H), 6.28 (s,
2H),1.23(s,13H).13C NMR(101MHz,DMSO)δc 163.98,160.12,159.12,154.31,151.85,
149.78,148.53,144.18,138.51,137.27,136.62,135.61,135.14,132.04,131.72,130.05,
129.72,127.97,127.58,127.25,127.01,126.36,125.68,123.47,122.60,120.00,118.45,
116.54,60.00,59.43,31.59,30.26,29.36,20.37.
Embodiment 2：The application of the fluorescence probe of the present invention
Probe molecule is dissolved in PBS (10mM, pH=7.0)/CH310.0 × 10 are configured in CN30%-6The solution of mol/L, to
Various reactive oxygen species (ROO are added in solution.,.OH,NO.,TBHP,NaClO,.O2,KO2,.OtBu,ONOO-) after do not cause
The significant change of probe itself emission peak, as hydrogen peroxide (H2O2) and interfering substance (ROO.,.OH,NO.,TBHP,NaClO,.O2,KO2,.OtBu,ONOO-) when coexisting, can occur to respond and the transmitting of itself is caused to disappear and blue shift occurs and generates new hair
Peak is penetrated, the influence of the interference-free factor of probe shows very strong antijamming capability.The probe molecule and hydrogen peroxide
(H2O2) response all have good linear relationship within certain time and concentration range.Show probe energy by Cellular imaging assays
For endogenous, exogenous hydrogen peroxide (H2O2) detection, common location experimental result confirm probe mitochondria can be accurately positioned,
Good biological adaptation ability is shown, so as to fulfill mitochondria hydrogen peroxide (H2O2) detection.
Claims (2)
1. a kind of identification hydrogen peroxide (H2O2) fluorescence probe synthesis, structure is：
2. a kind of detection hydrogen peroxide (H in the cell2O2) ratio type red light emitting phosphor probe application.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108727362A (en) * | 2018-08-01 | 2018-11-02 | 中南大学 | The synthesis and application of a kind of solid fluorescence small molecule |
| CN108752371A (en) * | 2018-07-13 | 2018-11-06 | 济南大学 | A kind of two-photon hydrogen peroxide fluorescence probe based on quinoline |
| CN110172070A (en) * | 2019-06-05 | 2019-08-27 | 商丘师范学院 | A kind of fluorescence probe and its synthetic method and application detecting viscosity and hydrogen peroxide |
| CN110894201A (en) * | 2019-12-13 | 2020-03-20 | 安徽大学 | Single-molecule fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid and preparation and application thereof |
| CN112358508A (en) * | 2020-12-01 | 2021-02-12 | 南京工业大学 | Accurate detection of H in vivo through light control2O2Fluorescent probe and preparation method and application thereof |
| CN113387973A (en) * | 2021-05-24 | 2021-09-14 | 云南师范大学 | Double-recognition fluorescent probe molecule and preparation method and application thereof |
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| CN108752371A (en) * | 2018-07-13 | 2018-11-06 | 济南大学 | A kind of two-photon hydrogen peroxide fluorescence probe based on quinoline |
| CN108752371B (en) * | 2018-07-13 | 2020-06-30 | 济南大学 | Two-photon hydrogen peroxide fluorescent probe based on quinoline |
| CN108727362A (en) * | 2018-08-01 | 2018-11-02 | 中南大学 | The synthesis and application of a kind of solid fluorescence small molecule |
| CN110172070A (en) * | 2019-06-05 | 2019-08-27 | 商丘师范学院 | A kind of fluorescence probe and its synthetic method and application detecting viscosity and hydrogen peroxide |
| CN110172070B (en) * | 2019-06-05 | 2021-11-02 | 商丘师范学院 | Fluorescent probe for detecting viscosity and hydrogen peroxide as well as synthesis method and application thereof |
| CN110894201A (en) * | 2019-12-13 | 2020-03-20 | 安徽大学 | Single-molecule fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid and preparation and application thereof |
| CN110894201B (en) * | 2019-12-13 | 2023-02-03 | 安徽大学 | Single-molecule fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid and preparation and application thereof |
| CN112358508A (en) * | 2020-12-01 | 2021-02-12 | 南京工业大学 | Accurate detection of H in vivo through light control2O2Fluorescent probe and preparation method and application thereof |
| CN114736223A (en) * | 2021-01-07 | 2022-07-12 | 湖南超亟检测技术有限责任公司 | Preparation of novel near-infrared fluorescence detection reagent and in-vitro diagnosis application thereof |
| CN114736223B (en) * | 2021-01-07 | 2024-05-10 | 湖南超亟检测技术有限责任公司 | Preparation of near infrared fluorescence detection reagent and in-vitro diagnosis application thereof |
| CN113387973A (en) * | 2021-05-24 | 2021-09-14 | 云南师范大学 | Double-recognition fluorescent probe molecule and preparation method and application thereof |
| CN115112656A (en) * | 2022-07-08 | 2022-09-27 | 四川大学华西医院 | Method for integral imaging of mucosa lymphatic vessel by combining immunofluorescence staining |
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