CN107417638B - A kind of glutathione and cysteine fluorescence probe and preparation method thereof based on 7- nitrobenzofurazan - Google Patents
A kind of glutathione and cysteine fluorescence probe and preparation method thereof based on 7- nitrobenzofurazan Download PDFInfo
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Abstract
The invention discloses a kind of glutathione based on 7- nitrobenzofurazan and cysteine fluorescence probes and preparation method thereof, intermediate 4- amino -7- nitro benzofuraxan and triethylamine are mixed in dichloromethane solution, the dichloromethane solution containing acryloyl chloride is added dropwise under condition of ice bath, it is reacted later, reaction product obtains glutathione and cysteine fluorescence probe by subsequent processing.The fluorescence probe is for the analysis of the fluorescence sense of environment or glutathione and cysteine in biological sample.Evident characteristics of the probe MP1 in DMSO/PBS (10 mM, pH=7.4,1/9, v/v) solution system with each amino acid are had studied by sepectrophotofluorometer.The result shows that: probe MP1 is good to the selectivity of glutathione and cysteine, strong antijamming capability, quickly can distinguish glutathione and cysteine from a variety of amino acid with sensitive, have a good application prospect.
Description
Technical field
The present invention relates to a kind of novel detection agents for glutathione and cysteine fluorescence identifying, and in particular to a kind of
The preparation method and application of glutathione and cysteine fluorescent probe molecule based on 7- nitrobenzofurazan.
Background technique
In organism, biological thiol such as glutathione (GSH), cysteine (Cys) etc. is in physiology course such as cell generation
It plays an important role during journey of apologizing for having done sth. wrong and maintenance redox reaction balance etc..The variation of such intracellular mercaptans content
It can cause a series of disease, such as AIDS, oedema, coronary heart disease, parkinsonism, cancer and psoriasis.It is included as cell
Highest nonprotein mercaptan is measured, glutathione (GSH) plays extremely important in keeping redox homeostasis process
Effect, content it is excessively high or too low, have direct relationship with many diseases such as cancer.Cysteine in organism
(Cys) shortage can cause a series of illnesss, such as skin injury, the illnesss such as liver oedema.Therefore, such sulphur of fast real-time monitoring
The content of alcohol in vivo has great significance in clinical medicine and biochemistry.
Fluorescence probe have many advantages, such as the good, high sensitivity of selectivity, it is easy to operate quickly, it is few to detectable substance damage wide
The general metal cation being applied in detection environmental and biological materials system, anion, active small molecular etc. in organism.Fluorescence
The development and utilization research of probe molecule is the crossing domain of the science such as chemistry and biology, medicine, agricultural.Fluorescent probe technique is
The analysis method that research object is studied on a molecular scale.Fluorescence probe has been widely used in environment and life at present
The detection of the substances such as heavy metal, bioactive small molecule in object.
Because it is with easy to operate, single-minded efficient selectivity has been widely used in detecting environment and life fluorescence probe method
Active small molecular etc. in metal cation, anion, organism in objects system.Compared to common other in organism
Amino acid, biological thiol such as cysteine (Cys) all contain sulfydryl in glutathione (GSH) molecular structure, therefore to biological sulphur
The detection of alcohol can be realized by the identification to sulfydryl.Sulfydryl has stronger nucleophilicity, many mercaptan reported at present
Fluorescence probe mainly realizes the efficient identification to biological thiol using its stronger nucleophilicity.
Summary of the invention
The invention proposes a kind of glutathione based on 7- nitrobenzofurazan and cysteine fluorescence probe and its preparations
The fluorescence probe of method, synthesis sensitive quickly can distinguish glutathione and cysteine from a variety of amino acid.
It realizes the technical scheme is that a kind of glutathione and cysteine fluorescence based on 7- nitrobenzofurazan is visited
Needle (is denoted as MP1), and the structural formula of the fluorescent probe molecule is as follows:
。
The preparation method of the glutathione and cysteine fluorescence probe based on 7- nitrobenzofurazan, step is such as
Under:
Under the conditions of being protected from light, by intermediate 4- amino -7- nitro benzofuraxan (NBD-NH2) with triethylamine be mixed in dichloromethane
In alkane solution, the dichloromethane solution containing acryloyl chloride, after reaction, reaction solution saturated sodium chloride solution are added dropwise under condition of ice bath
Washing, organic phase are dried, filtered with anhydrous sodium sulfate, solvent are removed under reduced pressure, is eluant, eluent with ethyl acetate and n-hexane, through silicon
The isolated light yellow solid of plastic column chromatography, obtains glutathione and cysteine fluorescence probe.
In the step (1), the condition that is protected from light is using masking foil encapsulation reaction container.
Triethylamine is acid binding agent, the molar ratio of intermediate 4- amino -7- nitro benzofuraxan and triethylamine in the step
For 1:(2-10), methylene chloride used need to be 0 DEG C through Non-aqueous processing, the ice bath reaction temperature, and the reaction time is 3-12 hours.
The molar ratio of the intermediate 4- amino -7- nitro benzofuraxan and acryloyl chloride is 1:(2-5).
The eluent of the silica gel post separation is ethyl acetate and n-hexane, the volume ratio of the ethyl acetate and n-hexane
For 1:(5-20).
The intermediate 4- amino -7- nitro benzofuraxan the preparation method comprises the following steps: being protected from light under the conditions of, dropwise by ammonia spirit
It is added drop-wise in the methanol solution containing the chloro- 7- nitrobenzofurazan of 4-, the concentration of the methanol solution of the chloro- 7- nitrobenzofurazan of 4-
It for 40-70%, is stirred to react at room temperature 20-30 hours, vacuum distillation obtains solid, and the solid is dissolved with ethyl acetate, with second
Acetoacetic ester and petroleum ether are as eluant, eluent, and the isolated Tan solid of silica gel column chromatography, the Tan solid is intermediate
4- amino -7- nitro benzofuraxan.
The ratio between the chloro- 7- nitrobenzofurazan of the 4- and the amount of substance of ammonia spirit are 1:(15-30).
Glutathione and cysteine fluorescence probe based on 7- nitrobenzofurazan are in detection glutathione and cysteine
In application.
The method of the methylene chloride Non-aqueous processing are as follows: it is distilled after being added calcium hydride reflux 2-4 hours into methylene chloride,
Up to anhydrous methylene chloride.
Used chemical reagent, solvent, metal salt etc. during fluorescence probe MP1 are prepared in the present invention is purchased from state
Chemical reagent Co., Ltd, medicine group.Bruke company DTX-400 is used in the confirmation and performance test process of fluorescence probe MP1
Type nuclear magnetic resonance spectrometer, solvent are deuterated chloroform, record nuclear magnetic resonance spectroscopy as internal standard using TMS and carbon is composed.Using Thermo public affairs
The Q-Exactive HR-MS mass spectrograph of department records high resolution mass spectrum data.Using Hitachi, Japan F-7000 Fluorescence Spectrometer
Record fluorescence spectrum.
Specific synthetic route of the invention is as follows:
The beneficial effects of the present invention are: obtaining fluorescence probe by simple single step reaction, synthetic route is short, method letter
Just, yield is high.Fluorescence probe in DMSO/PBS (10 mM, pH=7.4,1/9, v/v) solution system to glutathione and
The selectivity of cysteine is good, strong antijamming capability, can with it is sensitive quickly distinguished from a variety of amino acid glutathione and
Cysteine.Fluorogen 4- amino -7- nitro benzofuraxan in fluorescence probe structure has longer launch wavelength and good
Cell permeability, be conducive to such probe and identify glutathione and cysteine in vivo, therefore have and preferably answer
Use prospect.
Detailed description of the invention
Fig. 1 is fluorescence probe MP1 fluorescence selectivity figure of the invention, 380 nm of excitation wavelength.
Fig. 2 is the competitive lab diagram that fluorescence probe MP1 of the invention identifies GSH, 380 nm of excitation wavelength, launch wavelength
500 nm。
Fig. 3 is the competitive lab diagram that fluorescence probe MP1 of the invention identifies Cys, 380 nm of excitation wavelength, launch wavelength
500 nm。
Fig. 4 is fluorescence probe MP1 fluorescence kinetics lab diagram of the invention, 380 nm of excitation wavelength, launch wavelength 500
nm。
Fig. 5 is the fluorescence titration figure that fluorescence probe MP1 of the invention identifies GSH, and illustration is minimum detection limit figure, excitation wave
Long 380 nm, 500 nm of launch wavelength.
Fig. 6 is the fluorescence titration figure that fluorescence probe MP1 of the invention identifies Cys, and illustration is minimum detection limit figure, excitation wave
Long 380 nm, 500 nm of launch wavelength.
Fig. 7 is the pH value lab diagram that fluorescence probe MP1 of the invention identifies GSH and Cys, 380 nm of excitation wavelength, hair
Ejected wave grows 500 nm.
Specific embodiment
Embodiment 1
The synthesis of fluorescence probe:
(1) under the conditions of being protected from light, ammonia spirit is added drop-wise to dropwise in the methanol solution containing the chloro- 7- nitrobenzofurazan of 4-,
The ratio between the chloro- 7- nitrobenzofurazan of the 4- and the amount of substance of ammonia spirit are 1:15, the first of the chloro- 7- nitrobenzofurazan of 4-
The concentration of alcoholic solution is 40%, is stirred to react at room temperature 20 hours, and vacuum distillation obtains solid, and the solid ethyl acetate is molten
Solution, using ethyl acetate and petroleum ether as eluant, eluent, the isolated Tan solid of silica gel column chromatography, the Tan solid is
Intermediate 4- amino -7- nitro benzofuraxan；
(2) under the conditions of being protected from light, by intermediate NBD-NH2(180 mg, 1 mmol) and triethylamine (270 μ L, 2
Mmol it) is mixed in 15 mL dichloromethane solutions, the dichloro of acryloyl chloride (162 μ L, 2 mmol) is added dropwise under condition of ice bath
Methane (10 mL) solution, under condition of ice bath after fully reacting (3 hours), reaction solution is washed with saturated sodium chloride solution (15 mL*3)
It washs, organic phase is dried, filtered with anhydrous sodium sulfate, and solvent is removed under reduced pressure, is eluant, eluent (volume ratio with ethyl acetate and n-hexane
For 1:6), the light yellow solid through isolated 105.3 mg of silica gel column chromatography, as fluorescent probe molecule, yield 45%.
Nuclear magnetic resonance hydrogen spectruming determining:1H NMR (400 MHz, DMSO, ppm) δ: 5.97 (dd, 1 H, J =
1.6 Hz), 6.46 (dd, 1 H, J = 1.6 Hz), 6.93 (t, 1 H, J = 9.0 Hz), 8.51 (d, 1 H,J = 8.0 Hz), 8.79 (d, 1 H, J = 8.4 Hz), 11.62 (s, 1 H)。
Carbon-13 nmr spectra measurement:13C NMR (100 MHz, DMSO, ppm) δ: 114.7, 130.6, 130.8,
131.0, 135.1, 136.2, 143.9, 146.1, 165.5。
High resolution mass spectrum measurement: HR-MS m/z: theoretical value: C9H7N4O4([M+H+]+) 235.0647, detection value:
235.0649。
Embodiment 2
In the present embodiment fluorescence probe the preparation method is as follows:
(1) under the conditions of being protected from light, ammonia spirit is added drop-wise to dropwise in the methanol solution containing the chloro- 7- nitrobenzofurazan of 4-,
The ratio between the chloro- 7- nitrobenzofurazan of the 4- and the amount of substance of ammonia spirit are 1:20, the first of the chloro- 7- nitrobenzofurazan of 4-
The concentration of alcoholic solution is 60%, is stirred to react at room temperature 25 hours, and vacuum distillation obtains solid, and the solid ethyl acetate is molten
Solution, using ethyl acetate and petroleum ether as eluant, eluent, the isolated Tan solid of silica gel column chromatography, the Tan solid is
Intermediate 4- amino -7- nitro benzofuraxan；
(2) under the conditions of being protected from light, by intermediate NBD-NH2(180 mg, 1 mmol) and triethylamine (810 μ L, 6
Mmol it) is mixed in 15 mL dichloromethane solutions, the dichloro of acryloyl chloride (324 μ L, 4 mmol) is added dropwise under condition of ice bath
Methane (10 mL) solution, under condition of ice bath after fully reacting (6 hours), reaction solution is washed with saturated sodium chloride solution (15 mL*3)
It washs, organic phase is dried, filtered with anhydrous sodium sulfate, and solvent is removed under reduced pressure, is eluant, eluent (volume ratio with ethyl acetate and n-hexane
For 1:8), the light yellow solid through isolated 128 mg of silica gel column chromatography, as fluorescent probe molecule, yield 55%.
Nuclear magnetic resonance hydrogen spectruming determining:1H NMR (400 MHz, DMSO, ppm) δ: 5.97 (dd, 1 H, J =
1.6 Hz), 6.46 (dd, 1 H, J = 1.6 Hz), 6.93 (t, 1 H, J = 9.0 Hz), 8.51 (d, 1 H,J = 8.0 Hz), 8.79 (d, 1 H, J = 8.4 Hz), 11.62 (s, 1 H);
Carbon-13 nmr spectra measurement:13C NMR (100 MHz, DMSO, ppm) δ: 114.7, 130.6, 130.8,
131.0, 135.1, 136.2, 143.9, 146.1, 165.5；
High resolution mass spectrum measurement: HR-MS m/z: theoretical value: C9H7N4O4 ([M+H+]+) 235.0647, detection value:
235.0649。
Embodiment 3
In the present embodiment fluorescence probe the preparation method is as follows:
(1) under the conditions of being protected from light, ammonia spirit is added drop-wise to dropwise in the methanol solution containing the chloro- 7- nitrobenzofurazan of 4-,
The ratio between the chloro- 7- nitrobenzofurazan of the 4- and the amount of substance of ammonia spirit are 1:30, the first of the chloro- 7- nitrobenzofurazan of 4-
The concentration of alcoholic solution is 70%, is stirred to react at room temperature 30 hours, and vacuum distillation obtains solid, and the solid ethyl acetate is molten
Solution, using ethyl acetate and petroleum ether as eluant, eluent, the isolated Tan solid of silica gel column chromatography, the Tan solid is
Intermediate 4- amino -7- nitro benzofuraxan；
(2) under the conditions of being protected from light, by intermediate NBD-NH2(180 mg, 1 mmol) and triethylamine (1350 μ L, 6
Mmol it) is mixed in 15 mL dichloromethane solutions, the dichloro of acryloyl chloride (405 μ L, 5 mmol) is added dropwise under condition of ice bath
Methane (10 mL) solution, under condition of ice bath after fully reacting (12 hours), reaction solution is with saturated sodium chloride solution (15 mL*3)
Washing, organic phase are dried, filtered with anhydrous sodium sulfate, solvent are removed under reduced pressure, is eluant, eluent (volume with ethyl acetate and n-hexane
Than for 1:8), the light yellow solid through isolated 128 mg of silica gel column chromatography, as fluorescent probe molecule, yield 55%.
The application examples of probe MP1 prepared by embodiment 1
(1) preparation of solution:
Calcium chloride, potassium chloride, sodium chloride, sodium nitrite, hydrogen peroxide and each amino acid are purchased from the examination of Chinese medicines group chemistry
Agent Co., Ltd.Precise respective metal salt, amino acid, are dissolved in the solution for standby that 10 mM are prepared in high purity water.Purchase
Hydrogen peroxide is diluted with deionized water, according to Beer law, the concentration of hydrogen peroxide by 240 nm of ultraviolet spectra extinction scale
It is fixed, according to formula c=A/Kb(b=1 cm, K=43.6 M-1 cm-1) calculate.
The probe solution of 1 mM is prepared: fluorescence probe is dissolved in DMSO solution and prepares 1 by precise correspondent probe
The solution for standby of mM.
Test system preparation method: in test bottle, being added the 10 mM PBS buffer (pH 7.4) of 2.5 mL,
The test sample solution of respective volume is added, after mixing, the 1 mM probe solution of 30 μ L is added, corresponding body is finally added dropwise
Long-pending DMSO and 10 mM PBS buffer (pH 7.4) solution makes the 10 mM PBS buffer (pH of 3 mL of total system
7.4) system containing 10 % DMSO.
(2) selectivity experiment:
Good selectivity and stronger anti-interference are the major criterions for examining a fluorescent probe molecule whether excellent.
Probe has been primarily looked to the selectivity of common active sulfur, amino acid, metal ion.All of above test substances, which are added, not to be had
Apparent variation is generated to the fluorescent emission intensity of test system.As a comparison, corresponding glutathione is added under the same terms
(GSH) and when cysteine (Cys), system fluorescent emission intensity, which has, apparent reduces (Fig. 1).It should be the experimental results showed that probe
Has preferable selectivity to glutathione (GSH) and cysteine (Cys).In order to test probe to glutathione (GSH) and
The anti-interference ability of cysteine (Cys) identification, tests the anti-interference of the probe respectively in fluorescence emission spectrum.Such as figure
Shown in 2 and Fig. 3, test is separately added into DMSO/PBS (10 mM, pH=7.4,1/9, v/v) solution at (10 μM)
Various common amino acids (100 μM) test its fluorescent emission intensity (500 nm), then again to molten containing various amino acid
100 μM of glutathione (GSH) and cysteine (Cys) solution are separately added into liquid, by Fig. 2 and Fig. 3 it is found that in common ammonia
Glutathione (GSH) and cysteine (Cys) are added in the presence of base acid and is individually added into glutathione (GSH) and cysteine
(Cys) obtained fluorescent emission intensity (500 nm) is essentially identical when, is somebody's turn to do the result shows that probe is to glutathione (GSH) and half
The detection of cystine (Cys) has strong anti-interference ability.
(3) dynamic experiment is identified:
Quick response, real-time monitoring are one of the important features of fluorescence probe detection.It has been investigated instead by Fluorescence Spectrometer
The influence of probe in detecting glutathione (GSH) and cysteine (Cys) between seasonable.As shown in figure 4, DMSO/PBS (10 mM,
PH=7.4,1/9, v/v) in solution system, probe is 45 to glutathione (GSH) and cysteine (Cys) recognition time
Minute, and reach balance after 45 mins.The result shows that probe is to glutathione (GSH) and cysteine (Cys) recognition time
It is 45 minutes, reaction product has stronger stability.
(4) fluorescence titration and detection limit test:
In order to further study the sensitivity that probe identifies glutathione (GSH) and cysteine (Cys), pass through fluorescence
Titration experiments test its minimum detection limit.As shown in figure 5, molten in DMSO/ PBS (10 mM, pH=7.4,1/9, v/v)
In liquid system, control concentration and probe concentration be 10 μM, be continuously increased GSH concentration (0,1,2,3,5,7,10,15,
20,30,40,50,60,70,80,100 μM) its fluorescence emission spectrum is tested, with the increase of GSH concentration, body
Be that fluorescent emission intensity constantly reduces, meanwhile, the reduction of system fluorescent emission intensity be 1-30 μM in GSH concentration between be in
Linearly (R 2 =0.991, Fig. 5), according to IUPAC rule, measures probe and 106 nM are limited to the detection of GSH.Same method, is examined
The fluorescence titration for having examined probe and Cys is tested, and is measured and is limited to 103 nM (Fig. 6) to the detection of Cys.
(5) influence of the pH value to probe identification glutathione (GSH) and cysteine (Cys):
Glutathione (GSH) and cysteine (Cys) can be identified under different physiological conditions in order to test probe, examined
Its influence for identifying glutathione (GSH) and cysteine (Cys) to it in different pH values is examined.It is as shown in Figure 7: In
In DMSO/PBS (10 mM, pH=7.4,1/9, v/v) solution system, change the pH value (4-9) of buffer system, individually visits
Less, but (concentration is respectively in glutathione (GSH) and cysteine (Cys) for the fluorescence emission spectrum variation of needle (10 μ L)
100 μ L) it is existing under the conditions of, system fluorescent emission intensity has for 7-9 between in pH to be significantly quenched, this is the result shows that spy
Needle can be applied to identify glutathione (GSH) and cysteine (Cys) under physiological condition.
Claims (8)
1. a kind of glutathione and cysteine fluorescence probe based on 7- nitrobenzofurazan, which is characterized in that the fluorescence is visited
The structural formula of needle molecule is as follows:
。
2. the preparation side of glutathione and cysteine fluorescence probe as described in claim 1 based on 7- nitrobenzofurazan
Method, it is characterised in that steps are as follows:
Under the conditions of being protected from light, intermediate 4- amino -7- nitro benzofuraxan and triethylamine are mixed in dichloromethane solution, ice bath
Under the conditions of be added dropwise acryloyl chloride dichloromethane solution, after reaction, reaction solution is washed with saturated sodium chloride solution, organic phase nothing
Aqueous sodium persulfate dries, filters, and solvent is removed under reduced pressure, and is eluant, eluent with ethyl acetate and n-hexane, separates through silica gel column chromatography
To light yellow solid, glutathione and cysteine fluorescence probe are obtained.
3. the preparation side of glutathione and cysteine fluorescence probe as claimed in claim 2 based on 7- nitrobenzofurazan
Method, it is characterised in that: the condition that is protected from light is using masking foil encapsulation reaction container.
4. the preparation side of glutathione and cysteine fluorescence probe as claimed in claim 2 based on 7- nitrobenzofurazan
Method, it is characterised in that: the molar ratio of the intermediate 4- amino -7- nitro benzofuraxan and triethylamine is 1:(2-10), it is used
Methylene chloride need to be 0 DEG C through Non-aqueous processing, the ice bath reaction temperature, and the reaction time is 3-12 hours.
5. the preparation side of glutathione and cysteine fluorescence probe as claimed in claim 2 based on 7- nitrobenzofurazan
Method, it is characterised in that: the molar ratio of the intermediate 4- amino -7- nitro benzofuraxan and acryloyl chloride is 1:(2-5).
6. the preparation side of glutathione and cysteine fluorescence probe as claimed in claim 2 based on 7- nitrobenzofurazan
Method, it is characterised in that: the eluent of silica gel column chromatography separation is ethyl acetate and n-hexane, the ethyl acetate with just oneself
The volume ratio of alkane is 1:(5-20).
7. such as the described in any item glutathione and cysteine fluorescence probe based on 7- nitrobenzofurazan of claim 2-6
Preparation method, which is characterized in that the intermediate 4- amino -7- nitro benzofuraxan the preparation method comprises the following steps: being protected from light under the conditions of, will
Ammonia spirit is added drop-wise to dropwise in the methanol solution containing the chloro- 7- nitrobenzofurazan of 4-, the first of the chloro- 7- nitrobenzofurazan of 4-
The concentration of alcoholic solution is 40-70%, is stirred to react at room temperature 20-30 hours, and vacuum distillation obtains solid, the solid acetic acid
Ethyl ester dissolution, using ethyl acetate and petroleum ether as eluant, eluent, the isolated Tan solid of silica gel column chromatography, the yellowish-brown
Solid is intermediate 4- amino -7- nitro benzofuraxan.
8. the preparation side of glutathione and cysteine fluorescence probe as claimed in claim 7 based on 7- nitrobenzofurazan
Method, it is characterised in that: the ratio between the chloro- 7- nitrobenzofurazan of the 4- and the amount of substance of ammonia spirit are 1:15-30.
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| 一种用于活细胞中检测还原性谷胱甘肽的试卤灵类荧光探针;赵灵芝 等;《分析试验室》;20160131;第35卷(第1期);第7-11页 * |
| 基于硝基苯并呋咱的荧光探针;古振远 等;《化学进展》;20130831;第25卷(第8期);第1302-1311页 * |
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