CN105694857B - A kind of Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe and its preparation method and application - Google Patents
A kind of Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe and its preparation method and application Download PDFInfo
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- 0 *c1ccc(C(c(cccc2)c2C(O)=O)O)c(O)c1 Chemical compound *c1ccc(C(c(cccc2)c2C(O)=O)O)c(O)c1 0.000 description 1
- VILGTSHLXGZPEL-UHFFFAOYSA-N OC(C1)=CC=CC1N1CCNCC1 Chemical compound OC(C1)=CC=CC1N1CCNCC1 VILGTSHLXGZPEL-UHFFFAOYSA-N 0.000 description 1
- KUJBJYHESZLJNB-UHFFFAOYSA-N OC(c1ccccc1C(C(C=C1)C(Oc2c3)=CC1=O)c2ccc3N1CCNCC1)=O Chemical compound OC(c1ccccc1C(C(C=C1)C(Oc2c3)=CC1=O)c2ccc3N1CCNCC1)=O KUJBJYHESZLJNB-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a kind of Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe and its preparation method and application, belong to technical field of analytical chemistry.Shown in the structural formula of the probe molecule such as formula (I)：The fluorescence probe that the HNO molecules being directed in cell mitochondrial that the probe molecule system synthesizes first are detected, its synthetic method is relatively easy, and yield is higher, and last handling process is relatively easy.The present invention realizes the selective quick detection of HNO molecular probes, and selective good, resists other molecule interference performances strong.The present invention is a kind of simple, quickly, sensitive HNO molecular specificities detection reagent, is had broad application prospects in biomolecule detection field.
Description
Technical field
The present invention relates to a kind of Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe and its preparation method and application, belong to
Technical field of analytical chemistry.
Background technology
Research shows, nitrosyl hydrogen (HNO) plays an important role in many physiology courses in vivo.Such as HNO
The contraction of heart cell can be strengthened, can increase the expansion of blood vessel in musculature and reduce hematoblastic cohesion
And other effects.In non-vascular cell, HNO be it is known can prevent lipid peroxidation, so as to promote the table of heme oxidase
Reach.In addition research shows, HNO can be by triggering the K with voltage-dependent+Passage, promote the relaxation of artery.Pass through animal
Experiment finds that HNO can not only strengthen the flexible energy of mouse cardiac muscle myofilament, while can increase Ca2+In mouse cardiac muscle
Absorb.But the exception of HNO contents in vivo may have a larger infringement to body, for example, HNO oxidation product have compared with
Strong oxidation energy, can destroy intracellular DNA.In addition, substantial amounts of HNO may cause the apoptosis of nerve cell.
In intracellular organelle level, mitochondria and HNO have it is close contact, have a document report, the cytochrome c in mitochondria
NO can be changed into HNO, HNO generation there may be important contact with the effect of mitochondria.In addition, one in mitochondria
The effect of a little enzymes, may be suppressed by HNO, and therefore, the detection to the HNO of cell mitochondrial is very necessary and with important
Meaning.
The content of the invention
To be set for there is presently no the fluorescence probe detected to the HNO in cell mitochondrial, the present invention by molecule
Meter, synthesize a kind of HNO fluorescence probes with good selectivity and mitochondria body targeting.
Present invention also offers its preparation method of above-mentioned Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe and application.
The present invention uses following technical scheme：
A kind of Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe, it is characterised in that the structural formula of the fluorescence probe
As shown in formula (I), compound Mito-HN is abbreviated as：
A kind of preparation method of above-mentioned nitrosyl hydrogen molecule fluorescence probe, it is characterised in that it comprises the following steps：
1) by 2 ', 4 '-dihydroxy -2- benzophenones benzoic acid and a hydroxy phenyl piperazine in trifluoroacetic acid, nitrogen protection, 90
DEG C be heated to reflux, react 24h, after reaction terminates decompression be spin-dried for solvent and obtain crude product, then with the isolated compound of silicagel column
MHN-1, structural formula are as follows：
2) compound MHN-1 obtained by step 1) is dissolved in acetonitrile, adds Fmoc-Cl after adding sodium acid carbonate, nitrogen is protected
Shield, 3h is reacted at room temperature, extraction after reacting completely, dry, be spin-dried for and with the isolated compound MHN-2 of silicagel column, the MHN-2
It is as follows with Fmoc-Cl structural formula difference：
3) compound diphenylphosphine benzoic acid is dissolved in dichloromethane, adds dicyclohexylcarbodiimide and 4- diformazan ammonia
Compound MHN-2 is added after 20min is stirred at room temperature after yl pyridines, 8h is stirred at room temperature, is extracted after reacting completely, is dry
It is dry, be spin-dried for and as follows with the isolated compound MHN-3 of silicagel column, structural formula：
4) compound MHN-3 is dissolved in acetonitrile, 15% piperidines is then added dropwise, 30min is stirred at room temperature, reacted
After completely, add water quenching and go out reaction, then extracted at twice with dichloromethane, successively with drying after water, saturated common salt water washing,
Solvent is spin-dried for, and isolated compound MHN-4 is carried out with silicagel column, structural formula is as follows：
5) compound MHN-4 is dissolved in DMF, adds K2CO3After be stirred at room temperature 10min, add triphenylphosphine butyl
Reaction overnight, is detected with TLC plates and reacted at room temperature after bromine, and separating-purifying obtains target-probe compound after reacting completely
Mito-HN。
The synthetic route of fluorescence probe of the present invention is as follows：
Silica gel post separation eluant, eluent proportioning is methanol in the step 1)：Dichloromethane=1:5.
Extractant is ethyl acetate in the step 2), and drier is anhydrous sodium sulfate, and silicagel column elution agent proportioning is first
Alcohol/dichloromethane=1:30.
Extractant is ethyl acetate in the step 3), and drier is anhydrous sodium sulfate, and silicagel column elution agent proportioning is first
Alcohol：Dichloromethane=1:50.
Drier is anhydrous sodium sulfate in the step 4), and silicagel column elution agent proportioning is ethanol/methylene=1:30.
The specific method of separating-purifying is in the step 5)：Reaction product is diluted with ethyl acetate, washed 2 times, is satisfied
With brine It 2 times, anhydrous sodium sulfate drying, decompression is spin-dried for solvent and obtains crude product, and is separated with silicagel column, eluant, eluent
Match as methanol：Dichloromethane=1:20.
The purposes of HNO probes of the present invention：The fluorescence probe can apply to the biography of HNO in water environment and biological cell system
Sense detection；Described sensing detection includes fluoroscopic examination, visual qualitative detection, cell imaging detection.
The beneficial effects of the invention are as follows：
(1) fluorescence probe that the HNO molecules that synthesis is directed in cell mitochondrial first are detected, the synthesis phase of the probe
To being easier, yield is higher, and last handling process is relatively easy.
(2) present invention realizes the selective quick detection of HNO molecular probes, and selective good, resists other molecules to do
It is strong to disturb ability.
(3) this probe can apply to detect the detection of the HNO in intracellular mitochondrial, by being contaminated with commercialization mitochondria
Material is compared, it is found that the coincidence factor that this fluorescence probe is imaged to HNO in mitochondria is high, the common location coefficient of two kinds of dyestuffs is
0.93, illustrate that this probe can be used as HNO detection probes in intracellular mitochondrial.Specific and significant face based on this probe
Color change, the reagent can also be used as selectivity indicator existing for HNO molecules in the display aqueous solution, can carry out real-time qualitative and determine
The optical colorimetry detection of amount.So the present invention is a kind of simple, quickly, sensitive HNO molecular specificities detection reagent,
Biomolecule detection field has broad application prospects.
Brief description of the drawings
Fig. 1 is the middle probe Mito-HN of embodiment 11H NMR spectras.
Fig. 2 is situations of change of the probe Mito-HN with HNO addition fluorogram.
Fig. 3 is probe Mito-HN to different ions and the selective fluorogram of molecule.
Fig. 4 be probe Mito-HN to different ions and the selective histogram data of molecule, wherein 1, blank；2、Cu2+；
3、Al3+；4、Ca2+；5.Fe3+；6、Mg2+；7、Cys；8、GHS；9、HS-；10、S2-；11、HClO；12、H2O2；13rd, the tertiary fourth of peroxide
Alcohol；14th, the tertiary butyl ether of peroxide；15、NO；16、NO2 -；17、；NO3 -；18、HNO.
Fig. 5 is that probe Mito-HN carries out fluorescence imaging using with exogenous HNO in cell, wherein a) there was only fluorescence spy
Pin Mito-HN (5 μM) fluorescence imaging light field figure；B) there was only fluorescence probe Mito-HN (5 μM) green passage image；C) light field
With fluorescence imaging figure overlay chart；D) probe Mito-HN (5 μM) is added to after cultivating 30min in HeLa cells, adds 20 μM
HNO is further cultured for light field figure after 20min, e) add green channel fluorescence image after HNO；F) light field d is overlapping with fluorescence imaging e figures
Figure.
Fig. 6 is Mito-HN fluorescence probes HNO is carried out the fluorescence imaging figure common location red with being commercialized dyestuff mitochondria into
As figure.Wherein a) concentration and probe concentration is 5 μM and cultivates light field figure after 30min, b with red be added in HeLa cells of mitochondria), probe point
Son is to the HNO red fluorescence imaging figure to intracellular mitochondrial of fluorescence imaging figure c) mitochondrias, d) light field, green passage and red passage
Stacking chart, e) green passage and red passage stacking chart, f) green passage is compared with red passage superposition arrow head part fluorescence intensity, g) green lead to
Road and red passage Overlapping intensities scatter diagram.
Embodiment
With reference to embodiment and accompanying drawing, the present invention will be further described, but the present invention is not limited by following embodiments
Make, the number of compound corresponds to the number of compound in such scheme in embodiment.
Embodiment 1
Target-probe Mito-HN synthesis
1) compound MHN-1 synthesis：
Compound 2 ', 4 '-dihydroxy -2- benzophenones benzoic acid (1.0g, 3.88mmol, 1eq) and a hydroxy phenyl piperazine
(758.3mg, 4.26mmol, 1.1eq) is dissolved in 20mL trifluoroacetic acids, and 90 DEG C are heated to reflux, and reaction is overnight.Detected with TLC plates
Reaction, after reaction completely, decompression is spin-dried for solvent trifluoroacetic acid, is dissolved with 1ml ethyl acetate, and separated with silicagel column, silicon
Glue granular size is 200-300 mesh, and eluant, eluent proportioning is ethanol/methylene=1:5.Yield is 81%.
2) compound MHN-2 synthesis：
By compound MHN-2 (1.1g, 2.74mmol, 1eq), NaHCO3(691mg, 8.22mmol, 3eq) is dissolved in 20mL second
Fmoc-Cl (850mg, 3.29mmol, 1.2eq) is added after 10min is reacted at room temperature in nitrile, under nitrogen protection, 3h is reacted at room temperature, uses
The detection reaction of TCL plates, the quenching reaction of 100mL water is added after reacting completely, is extracted 2 times with ethyl acetate 200mL, saturated aqueous common salt
Washing 2 times, anhydrous sodium sulfate drying.Decompression is spin-dried for solvent and obtains crude product, and is separated with silicagel column, and silica gel particle size is
200-300 mesh, eluant, eluent proportioning is ethanol/methylene=1:30.Yield is 72%.
3) compound MHN-3 synthesis：
By compound, diphenylphosphine benzoic acid (235mg, 0.77mmol, 1.2eq), DMAP (586mg, 4.8mmol,
7.5eq), DCC (192mg, 1.92mmol, 3eq) is dissolved in react 20min at room temperature in 20mL dichloromethane after add MHN-2
(400mg, 0.64mmol, 1eq) and under nitrogen protection, normal-temperature reaction 8h, detected and reacted with TLC plates, after reaction completely, added
The quenching reaction of 50mL water, is extracted 2 times, saturated common salt water washing 2 times, anhydrous sodium sulfate drying with ethyl acetate 100mL.Decompression rotation
Dry solvent obtains crude product, and is separated with silicagel column, and silica gel particle size is 200-300 mesh, and eluant, eluent proportioning is methanol/bis-
Chloromethanes=1:50.Yield is 56%.
4) compound MHN-4 synthesis：
Compound MHN-3 (240mg, 0.264mmol, 1eq) is dissolved in 5mL acetonitriles, then adds 15% piperidines, and normal temperature stirs
Mix 30min, TLC detection reactions are complete, add 10mL water quenchings and go out reaction, are extracted at twice with 50mL dichloromethane, with 20mL water
20mL saturated common salt water washing is used after washing, anhydrous sodium sulfate drying, decompression is spin-dried for solvent and obtains crude product, and is entered with silicagel column
Row separation, silica gel particle size are 200-300 mesh, and eluant, eluent proportioning is ethanol/methylene=1:30, compound MHN-4 is obtained,
Yield 35%.
5) compound Mito-HN synthesis：
Compound MHN-4 (60mg, 0.087mmol, 1eq), potassium carbonate (60.2mg, 0.435mmol, 5eq) are dissolved in
Reaction 10min is stirred at room temperature in 5mLDMF, adds triphenylphosphine butyl bromide (69.3mg, 0.174mmol, 2eq), nitrogen is protected
Shield, room temperature reaction overnight, are detected with TCL plates and reacted, after reaction completely, diluted with ethyl acetate, washed 2 times, saturated aqueous common salt
Washing 2 times, anhydrous sodium sulfate drying, decompression is spin-dried for solvent and obtains crude product, and is separated with silicagel column, and silica gel particle size is
200-300 mesh, eluant, eluent proportioning is ethanol/methylene=1:20.Yield is 64%.1H NMR(400MHz,DMSO-d6)δ
8.25 (s, 1H), 8.05 (d, J=7.5Hz, 1H), 7.89 (d, J=6.6Hz, 3H), 7.86-7.70 (m, 14H), 7.67-7.58
(m, 2H), 7.41 (s, 5H), 7.33 (d, J=7.3Hz, 1H), 7.21 (s, 4H), 7.09 (d, J=8.1Hz, 1H), 6.82 (dd,
J=17.4,8.6Hz, 4H), 6.63 (d, J=8.8Hz, 1H), 4.07 (s, 2H), 3.63 (s, 4H), 3.20 (s, 4H), 1.80
(s,2H),1.62(s,2H),1.23(s,2H).
Embodiment 2
Fluorescence probe Mito-HN is with the change of the increase fluorogram of HNO additions
Mito-HN nitrosyl hydrogen fluorescence probes prepared by Example 1 are dissolved in DMF (DMF), are made
Into 1mmol/L storing solutions.Take out 30 μ L from storing solution to be added among 5mL centrifuge tube, with PBS 2mL (0.1mol/L, pH
=7.4) it is diluted, AS salt (HNO donors) standard liquid of different equivalents (0-10eq) is then added, with PBS cushioning liquid
Solution is diluted to 3mL, using 450nm to excite its photoluminescent property of photo measure.Fluorescence spectrum is as shown in Figure 2.From Figure 2 it can be seen that with
Fluorescence of the increase of HNO additions at 545nm gradually strengthens.
Embodiment 3
Selectivity of the compound Mito-HN nitrosyl hydrogen fluorescence probes to different molecular or ion
30 μ L are taken out in fluorescence probe storing solution from embodiment 2 to be added separately among 18 5mL centrifuge tube, are used
After 2mLPBS is diluted, then the competition molecular criteria solution of equimolar amounts is added into 16 centrifuge tubes respectively, one of them
Any ion is also not added with, and as blank sample, another adds the HNO standard liquids of equimolar amounts, solution is detected after 15min
Fluorescence emission spectrum changes, using 450nm as exciting light, as a result as shown in Figure 3 and Figure 4.By Fig. 3 and Fig. 4 it can be found that other are golden
Category ion, reproducibility compound, oxidative compound etc. have little to no effect to fluorescence of the compound Mito-HN at 545nm,
And the addition of nitrosyl hydrogen solution significantly increases fluorescence of the compound Mito-HN at 545nm.
Embodiment 4
Compound Mito-HN fluorescence probes are in the cell to nitrosyl hydrogen fluorescence imaging
Fluorescence probe Mito-HN obtained by the present invention is applied in HeLa cells and carries out fluorescence to nitrosyl hydrogen by we
Imaging applications (Fig. 5) concrete operation step is as follows：The DMSO solution of 5 μM of Mito-HN fluorescence probes is added into two to give birth to
In the culture dish of HeLa cells, and culture dish is numbered.What is all not added with No.1 culture dish, adds in No. two culture dishes
Enter 20 μM of nitrosyl hydrogen donors.After cultivating 30min in CO2gas incubator, it is imaged with Laser Scanning Confocal Microscope.First
No.1 culture dish is imaged.Excited with 488nm laser, the profile diagram of cell can be watched in light field passage,
And when green channel (500nm-550nm) carries out fluorescence imaging to cell, fluorescent emission is not observed now.No. two are cultivated
Ware carries out imaging research and finds that what can be regained consciousness under green channel sees fluorescence imaging figure of the probe to HNO.This experiment can be with
Illustrate that probe Mito-HN can carry out fluorescence imaging to HNO in the cell.
Embodiment 5
Compound Mito-HN fluorescence probes are in the cell to nitrosyl hydrogen fluorescence imaging and fixed altogether with business mitochondrial dye
Bit comparison we the fluorescence probe Mito-HN obtained by the present invention is applied in HeLa cells and commercialized mitochondrial dye
Common location experiment is carried out, illustrates that this probe can be navigated in mitochondria, and fluorescence imaging application is carried out to HNO in mitochondria
(Fig. 6).Concrete operation step is as follows：5 μM of Mito-HN probes DMSO solutions and commercialization mitochondrial dye-mitochondria are red
10nM is added in the culture dish for giving birth to HeLa cells, and after cultivating 30min in CO2gas incubator, 20 are added into system
μM HNO donor solutions after, then be imaged with Laser Scanning Confocal Microscope after waiting 20min, now swashed with (Ex=488nm)
Hair can be by green channel to being imaged, and this is Mito-HN probes and the light launched after HNO responses, uses up (Ex=
561nm) carry out exciting and cell can be imaged by red channel, this is red to intracellular for commercialization dyestuff mitochondria
The fluorescence imaging that mitochondria is carried out, carrying out processing with software can show that the common location coefficient of two kinds of dyestuffs is 0.96.
Claims (7)
1. a kind of Mitochondrially targeted nitrosyl hydrogen molecule fluorescence probe, it is characterised in that the structural formula of the fluorescence probe is such as
Shown in formula (I)：
The preparation method of described nitrosyl hydrogen molecule fluorescence probe comprises the following steps：
1) by 2 ', 4 '-dihydroxy -2- benzophenones benzoic acid and a hydroxy phenyl piperazine in trifluoroacetic acid, nitrogen protection, 90 DEG C add
Heat backflow, react 24h, after reaction terminates decompression be spin-dried for solvent and obtain crude product, then with the isolated compound MHN- of silicagel column
1, structural formula is as follows：
2) compound MHN-1 obtained by step 1) is dissolved in acetonitrile, Fmoc-Cl, nitrogen protection, room is added after adding sodium acid carbonate
Temperature reaction 3h, extraction after reacting completely, dry, be spin-dried for and with the isolated compound MHN-2 of silicagel column, the MHN-2 with
Fmoc-Cl structural formula difference is as follows：
3) compound diphenylphosphine benzoic acid is dissolved in dichloromethane, adds dicyclohexylcarbodiimide and 4- dimethylamino pyrroles
Compound MHN-2 is added after 20min is stirred at room temperature after pyridine, 8h is stirred at room temperature, extracts, dry after reacting completely, rotation
The isolated compound MHN-3 of dry doubling silicagel column, structural formula are as follows：
4) compound MHN-3 is dissolved in acetonitrile, 15% piperidines is then added dropwise, 30min is stirred at room temperature, reaction is complete
Afterwards, add water quenching to go out reaction, then extracted at twice with dichloromethane, successively with drying after water, saturated common salt water washing, be spin-dried for
Solvent, and isolated compound MHN-4 is carried out with silicagel column, structural formula is as follows：
5) compound MHN-4 is dissolved in DMF, adds K2CO3After be stirred at room temperature 10min, after adding triphenylphosphine butyl bromide
Reaction overnight, is detected with TLC plates and reacted at room temperature, and separating-purifying obtains target-probe compound Mito-HN after reacting completely.
2. nitrosyl hydrogen molecule fluorescence probe according to claim 1, it is characterised in that silicagel column point in the step 1)
It is methanol from eluant, eluent proportioning：Dichloromethane=1:5.
3. nitrosyl hydrogen molecule fluorescence probe according to claim 1, it is characterised in that extractant is in the step 2)
Ethyl acetate, drier are anhydrous sodium sulfate, and silicagel column elution agent proportioning is ethanol/methylene=1:30.
4. nitrosyl hydrogen molecule fluorescence probe according to claim 1, it is characterised in that extractant is in the step 3)
Ethyl acetate, drier are anhydrous sodium sulfate, and silicagel column elution agent proportioning is methanol：Dichloromethane=1:50.
5. nitrosyl hydrogen molecule fluorescence probe according to claim 1, it is characterised in that drier is in the step 4)
Anhydrous sodium sulfate, silicagel column elution agent proportioning is ethanol/methylene=1:30.
6. nitrosyl hydrogen molecule fluorescence probe according to claim 1, it is characterised in that separating-purifying in the step 5)
Specific method be：Reaction product is diluted with ethyl acetate, washed 2 times, saturated common salt water washing 2 times, anhydrous sodium sulfate is done
Dry, decompression is spin-dried for solvent and obtains crude product, and is separated with silicagel column, and eluant, eluent proportioning is methanol：Dichloromethane=1:20.
7. the application of the nitrosyl hydrogen molecule fluorescence probe described in a kind of claim 1, it is characterised in that the probe can be applied
The HNO sensing detection in water environment and biological cell system；Described sensing detection includes fluoroscopic examination, visual qualitative inspection
Survey, cell imaging detection.
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| CN106928276A (en) * | 2017-03-07 | 2017-07-07 | 济南大学 | A kind of fluorescence probe of the intracellular nitrosyl hydrogen of detection based on Rhodamine Derivatives |
| CN108129403B (en) * | 2018-01-23 | 2020-05-29 | 陕西师范大学 | Application of 6-fluoro-7-piperazinyl-4-arylaminoquinazoline derivative as mitochondrial fluorescent probe |
| CN108358966B (en) * | 2018-03-05 | 2020-04-28 | 南京工业大学 | Targeting-mitochondrial function enhancing drug Mito-VB3 and preparation method and application thereof |
| CN110724520B (en) * | 2019-09-23 | 2020-11-17 | 济南大学 | Fluorescent probe for detecting water content in heavy water and application thereof |
| CN110885675B (en) * | 2019-11-22 | 2022-07-12 | 山东师范大学 | Nano fluorescent probe, preparation method and application thereof in detection of HNO in Golgi apparatus |
| CN114573635B (en) * | 2020-11-30 | 2024-01-30 | 中国科学院大连化学物理研究所 | Photocrosslinking probe and mitochondrial protein enrichment method based on photocrosslinking |
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| US8791258B2 (en) * | 2008-06-10 | 2014-07-29 | The Regents Of The University Of California | Pro-fluorescent probes |
| US20100081159A1 (en) * | 2008-09-26 | 2010-04-01 | Lebedeva Irina V | Profiling reactive oxygen, nitrogen and halogen species |
| CN104237503A (en) * | 2014-09-01 | 2014-12-24 | 上海师范大学 | Mitochondria targeted fluorescent labeling magnetic nano material as well as preparation method and application of mitochondria targeted fluorescent labeling magnetic nano material |
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