CN107603603A - A kind of fluorescence probe for identifying hydrogen peroxide - Google Patents
A kind of fluorescence probe for identifying hydrogen peroxide Download PDFInfo
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- CN107603603A CN107603603A CN201711060748.0A CN201711060748A CN107603603A CN 107603603 A CN107603603 A CN 107603603A CN 201711060748 A CN201711060748 A CN 201711060748A CN 107603603 A CN107603603 A CN 107603603A
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- hydrogen peroxide
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Abstract
It is used to detect hydrogen peroxide (H the present invention relates to one kind2O2) fluorescence probe preparation method and application, belong to chemical analysis detection technique field.Its molecular structure is as follows：The probe molecule maximum absorption wavelength is in 480nm, probe molecule and hydrogen peroxide (H2O2) after effect, fluorescence spectrum at 619nm intensity by without to having and constantly strengthening, fluorescence self-absorption can be reduced by showing larger Stokes shift (Stokes shifts), improve the sensitivity of detection；Launch wavelength can reduce the light injury of the background fluorescence and living cells during probe in detecting, penetration capacity of the enhancing biology to tissue in red light district.Probe molecule of the present invention has good linear in certain time and concentration range, to hydrogen peroxide (H2O2) recognition capability is strong, selectivity, strong antijamming capability, such probe have important application value in fields such as biochemistries.
Description
Technical field
The invention belongs to chemical analysis detection technique field, and in particular to one kind has big Stokes shift detection peroxide
Change hydrogen new feux rouges-near infrared fluorescent probe preparation method and its in vitro with living cells inside detection hydrogen peroxide side
The application in face.
Background technology
Hydrogen peroxide (H2O2) it is one of most important active oxygen (ROS), crucial work is played in many physiology and pathologic process
With.Hydrogen peroxide (H2O2) can be by hormone, cell factor, lead to during the cytositimulation of neurotransmitter and peptide growth factor
Cross activation endogenous generation in cell of nadph oxidase compound (NOX).Endogenous hydrogen peroxide (H2O2) be not only to make
For the main oxidation metabolin of the oxidative stress index of biosystem, and it is many bioprocess such as wound healings of regulation, exempts from
The signal of interest molecule of epidemic disease, cell propagation, differentiation and migration.However, hydrogen peroxide (H2O2) abnormal level and many diseases it is close
Cut is closed, including nerve degenerative diseases, diabetes, cancer and aging.Due to hydrogen peroxide (H2O2) influence it is very big, design
Effective method is qualitative and quantitatively detects hydrogen peroxide (H2O2) it is significant and necessary.At present, it has been reported that a variety of to have
High-sensitivity detection hydrogen peroxide (H2O2) fluorescence probe.But this kind of probe emission wavelength of major part is short, has small Si Tuo
The shortcomings that gram this displacement (Stokes shifts).But growing launch wavelength can be reduced during probe in detecting to living cells
Light injury, strengthen penetration into tissue, big Stokes shift can also reduce self-absorption improve detection sensitivity.Report at present
Road one kind has big Stokes shift, for detecting the also fewer of hydrogen peroxide near infrared fluorescent probe.
The content of the invention
It is an object of the present invention to provide a kind of fluorescence probe synthetic method of simple and effective；Another object of the present invention
It is to provide a kind of good selectivity, strong anti-interference ability, big Stokes shift (Stokes shifts), launch wavelength is near red
Outside, can be to the fluorescence probe of progress hydrogen peroxide detection inside external or living cells.
The technical scheme of the present invention for solving the problems, such as to take is that a kind of fluorescent switch (off-on) method identification hydrogen peroxide is new
Fluorescence probe, its molecular structural formula are as follows：
Synthetic route is as follows：
Specific synthetic method is as follows：(a) it is compound 1 (234.0mg, 1.0mmol) and potassium carbonate (300.1mg, 2.2mmol) is molten
Stirred at room temperature in 8.0mL acetone 10 minutes.Then 2- (4- (bromomethyl) phenyl) -4,4,5,5- four is added into mixture
Methyl isophthalic acid, 3,2- dioxaborolan alkane (356.2mg, 1.2mmol), react 3h at 70 DEG C.After being cooled to room temperature, it will react
Mixture removal of solvent under reduced pressure, column chromatography for separation obtain compound 2 (197.2mg, 43.7%), are yellow solid.(b) in room temperature
It is lower compound 2 (90.3mg, 0.2mmol) and cyan-acetic ester (48.2mg, 0.4mmol) are dissolved in it is molten in 5.0mL ethanol
In liquid, piperidines (5.0 μ L, 0.05mmol) is added.After being stirred at room temperature 5 minutes, resulting solution is flowed back 1 hour.Then, will
Reaction solution is concentrated under reduced pressure, and column chromatography for separation obtains the compound 3 (46.0mg, 39.4% yield) of red solid.
The mechanism of action of the fluorescence probe of the present invention is as follows, and probe molecule fluorescence itself is in cancellation state.Hydrogen peroxide
Can fragrant borinate ester moiety in oxidised probe molecule, hydrolysis obtains phenolic hydroxyl group, and then leaving away of proton translocation occurs for intramolecular
The part of compound 4 obtains intermediate 5, and nucleophilic substitution intramolecular cyclization finally occurs and obtains compound 6.So as to realize specificity
Detect hydrogen peroxide (H2O2) purpose.The response process of probe molecule is as follows：
The fluorescence probe of the present invention is near infrared emission, itself and hydrogen peroxide (H2O2) unstressed configuration before effect, fluorescence after effect
Emission peak is at 619nm.
The fluorescence probe of the present invention has a big Stokes shift (Stokes shifts), absorption maximum in 480nm, with
Hydrogen peroxide (H2O2) emission maximum in 619nm, Stokes shift (Stokes shifts) is 139nm after effect.
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 7.4 are molten
Liquid includes 30% ethanol, measures at room temperature.Probe molecule does not have fluorescence in itself, is adding 40 times of equivalents of hydrogen peroxide (H2O2) it
Afterwards, fluorescence intensity increases 22 times at maximum emission wavelength 619nm.And add other reactive oxygen species and some negative and positive from
Son (ROO., NO., TBHP, ClO-, KO2, ONOO-, OH-, I-, F-, PO4 3-, NO2-, NO3 -, SO3 2-, S2O3 2-, SCN-, CN-,
CO3 2-, Fe2+, Fe3+) after, fluorescence only has almost negligible increase.
The fluorescence probe strong antijamming capability of the present invention, other reactive oxygen species and some zwitterions (ROO., NO.,
TBHP, ClO-, KO2, ONOO-, OH-, I-, F-, PO4 3-, NO2-, NO3 -, SO3 2-, S2O3 2-, SCN-, CN-, CO3 2-, Fe2+, Fe3+)
In the presence of not influenceing probe molecule and hydrogen peroxide (H2O2) effect.
The fluorescence probe of the present invention is adding the hydrogen peroxide (H of 40 times of equivalents2O2) after effect, fluorescence quickly strengthens, 40
Minute reaches maximum, and the significant change of fluorescence can be observed.
The fluorescence probe of the present invention shows wider application, and probe molecule all may be used in the range of being 7 to 11 in pH
With to hydrogen peroxide (H2O2) Selective recognition.
Probe molecule of the present invention is to hydrogen peroxide (H2O2) launch wavelength after response is in near-infrared, stoke position
Move greatly, there is good selectivity and antijamming capability, and the sensitivity having had, there is wider application, this is glimmering
Light probe has actual application value in biology and the fields such as chemistry.
Brief description of the drawings
Fig. 1 be fluorescence probe of the present invention selectivity, fluorescence probe (10.0 × 10-6Mol/L) in PBS (10mM, pH=
7.4) in/EtOH30% solution, with hydrogen peroxide (H2O2) fluorescence spectrum after effect, abscissa is wavelength, and ordinate is fluorescence
Intensity.
Fig. 2 be fluorescence probe of the present invention antijamming capability, hydrogen peroxide (H2O2) be total to other oxides and zwitterion
When depositing, with fluorescence probe (10.0 × 10-6Mol/L) in PBS (10mM, pH=7.4)/EtOH30% with hydrogen peroxide (H2O2)
Fluorescence intensity ratio (I/I after effect0) block diagram.
Fig. 3 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in PBS (10mM, pH=7.4)/EtOH30%,
With various concentrations hydrogen peroxide (H2O2) fluorescence spectrum change after effect, 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=7.4)/EtOH30%,
With hydrogen peroxide (H2O2) concentration linear relationship, abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 5 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in PBS (10mM, pH=7.4)/EtOH30%,
With hydrogen peroxide (H2O2) fluorescence intensity changes with time at 619nm in mechanism, abscissa is the time, and ordinate is glimmering
Luminous intensity.
Fig. 6 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in PBS (10mM, pH=7.4)/EtOH30%,
With hydrogen peroxide (H2O2) fluorescence intensity is with the linear relationship of time at 619nm in mechanism, abscissa is the time, ordinate
For fluorescence intensity.
Fig. 7 is the fluorescence probe (10.0 × 10 of the present invention-6Mol/L) in different pH value cushioning liquid, with hydrogen peroxide
(H2O2) the front and rear fluorescence intensity of effect, abscissa pH, ordinate is fluorescence intensity.
The fluorescence probe that Fig. 8 is the present invention detects Hela intracellular hydrogen peroxides (H2O2) activity application, A1-A3 for visit
Pin (10.0 × 10-6Mol/L) 37 DEG C when cultivate 30min imaging effect in the cell.B1-B3 is that cell is first used at 37 DEG C
Hydrogen oxide (0.4 × 10-3Mol/L 30min) is cultivated, then with probe (10.0 × 10-6Mol/L 30min imaging effect) is cultivated
Fruit.
Example is embodied
Embodiment 1：The synthesis of intermediate product 2
By compound 1 (234.0mg, 1.0mmol) and potassium carbonate (300.1mg, 2.2mmol) in 8.0mL acetone at room temperature
Stirring 10 minutes.Then 2- (4- (bromomethyl) phenyl) -4,4,5,5- tetramethyl -1,3,2- dioxa boron is added into mixture
Heterocycle pentane (356.2mg, 1.2mmol), obtained mixture is reacted into 3h at 70 DEG C.After reaction is cooled to room temperature after terminating,
Reactant mixture is concentrated under reduced pressure, obtains crude product.Finally, entered using petroleum ether/dichloromethane (5/1, v/v) as eluent
Row column chromatography, compound 3 (197.2mg, 43.7%) is obtained, be yellow solid.1H NMR (500MHz, CDCl3)δH 10.26
(s, 1H), 7.83 (d, J=7.7Hz, 2H), 7.45 (d, J=7.7Hz, 2H), 7.01 (s, 1H), 6.04,1.55 (s, 2H),
1.36 (s, 12H), 1.16 (t, J=7.0Hz, 3H), 1.12 (t, J=7.1Hz, 3H)
Embodiment 2：The synthesis of probe molecule
Compound 2 (90.3mg, 0.2mmol) and cyan-acetic ester (48.2mg, 0.4mmol) are dissolved in 5.0mL at room temperature
Ethanol, piperidines (5.0 μ L, 0.05mmol) is added, after being stirred at room temperature 5 minutes, resulting solution is flowed back 1 hour.Reaction knot
Shu Hou, reaction solution is concentrated under reduced pressure, obtains crude product.Finally, using petrol ether/ethyl acetate (4/1, v/v) as elution
Agent carries out column chromatography, obtains the Probe1 (46.0mg, 39.4% yield) of red solid.HRMS(ESI)m/z：C22H 21N 6O5[M+1]+, 449.1573；1H NMR (400MHz, CDCl3)δH8.72 (s, 1H), 7.82 (d, J=8.0Hz, 2H), 7.73
(s, 1H) 7.42 (d, J=8.0Hz, 2H), 5.98 (s, 1H), 5.15 (s, 2H), 4.31 (q, J=7.1Hz, 2H), 3.31 (m,
8H), 1.35 (t, J=15.5,3H), 1.34 (s, 12H), 1.21 (t, J=7.0Hz, 3H), 1.07 (t, J=7.1Hz, 3H).13C
NMR (100MHz, CDCl 3)δC165.2,155.9,147.1,143.4,140.0,135.1,128.8,126.1,118.8,
109.6,94.4,90.3,83.8,71.1,61.3,48.0,45.8,45.5,44.8,24.6,14.3,10.7,9.8.
Embodiment 3：The application of the fluorescence probe of the present invention
Probe molecule is dissolved in PBS (10mM, pH=7.4)/EtOH30% and is configured to 10.0 × 10-6Mol/L solution, to
Various reactive oxygen species and zwitterion (ROO., NO., TBHP, ClO are added in solution-, KO2, ONOO-, OH-, I-, F-, PO4 3-,
NO2-, NO3 -, SO3 2-, S2O3 2-, SCN-, CN-, CO3 2-, Fe2+, Fe3+) after do not cause the significant change of fluorescence, work as hydrogen peroxide
(H2O2) and interfering material (ROO., NO., TBHP, ClO-, KO2, ONOO-, OH-, I-, F-, PO4 3-, NO2-, NO3 -, SO3 2-,
S2O3 2-, SCN-, CN-, CO3 2-, Fe2+, Fe3+) when coexisting, the influence of the interference-free factor of probe, show very strong anti-dry
Disturb ability.The probe molecule and hydrogen peroxide (H2O2) response all have good linear relationship within certain time and concentration range.
Probe molecule can be to hydrogen peroxide (H in the range of being 7 to 11 in pH2O2) Selective recognition, show good biology
Adaptability.
Claims (1)
1. a kind of fluorescence probe for identifying hydrogen peroxide, its structure are：
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109180716A (en) * | 2018-09-19 | 2019-01-11 | 中南大学 | A kind of multi signal Ratio-type differentiation detection H2O2And H2Design, synthesis and the application of the fluorescence probe of S |
| 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 |
| CN110818734A (en) * | 2019-10-23 | 2020-02-21 | 中南大学 | Fluorescent probe with double-ratio recognition function for hydrogen peroxide and hypochlorous acid |
| 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 |
| 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 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109180716A (en) * | 2018-09-19 | 2019-01-11 | 中南大学 | A kind of multi signal Ratio-type differentiation detection H2O2And H2Design, synthesis and the application of the fluorescence probe of S |
| CN109180716B (en) * | 2018-09-19 | 2020-11-13 | 中南大学 | Multi-signal ratio type distinguishing detection H2O2And H2Design, synthesis and application of fluorescent probe of S |
| 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 |
| CN110818734A (en) * | 2019-10-23 | 2020-02-21 | 中南大学 | Fluorescent probe with double-ratio recognition function for hydrogen peroxide and hypochlorous acid |
| 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 |
| 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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