CN108864161A - It is a kind of to detect the compound and its preparation method and purposes that cell autophagy enhances - Google Patents
It is a kind of to detect the compound and its preparation method and purposes that cell autophagy enhances Download PDFInfo
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- CN108864161A CN108864161A CN201810563989.5A CN201810563989A CN108864161A CN 108864161 A CN108864161 A CN 108864161A CN 201810563989 A CN201810563989 A CN 201810563989A CN 108864161 A CN108864161 A CN 108864161A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- General Health & Medical Sciences (AREA)
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Abstract
A kind of compound of detection cell autophagy enhancing, it is just like flowering structure：
Description
Technical field
The present invention relates to field of biological detection, are a kind of materials that detection autophagy enhancing is acidified by real-time detection organelle
Material.
Background technique
Autophagy is a kind of intracellular degradation process, cell by the process control Cell Homeostasis and survival [referring to：
Kroemer,G.； Marino,G.；Levine,B.Molecular cell 2010,40,280.].In aging and various nerves
Autophagy increases in degenerative disease model such as Alzheimer disease, Parkinson's disease, Huntington's chorea and amyotrophic lateral sclerosis
Be by force it is beneficial [referring to：Tan,C.C.；Yu,J.T.；Tan,M.S.；Jiang,T.；Zhu,X.C.；Tan,
L.Neurobiology of aging 2014,35,941；Wolfe,D.M.；Lee,J.H.；Kumar,A.；Lee,S.；
Orenstein,S. J.；Nixon,R.A.The European journal of neuroscience 2013,37,1949；
Menzies,F. M.；Fleming,A.；Rubinsztein,D.C.Nature reviews.Neuroscience 2015,16,
345.].In the disease, the removing of the aggregation as caused by disease is can be enhanced in the enhancing of cell autophagy, to facilitate
Treatment and mitigation the severity of disease.Treatment for cancer, the enhancing of autophagy play double-edged sword but very important work
With：It activates autophagy to protect cancer cell in response to radiotherapy or chemotherapy, and has been demonstrated to can promote tumor cell survival and generation
Drug resistance；On the other hand, when cancer cell is when excessive autophagy causes autophagy cell death, autophagy is as tumor suppression mechanism
Work [referring to：Chen, N.；Debnath,J.FEBS letters 2010,584,1427].There is a large amount of evidence to prove certainly
Biting enhancing is to treat the effective ways of a wide range of disease and disorder, therefore exploitation has wide city based on the drug that autophagy enhances
Field prospect, this also proposes demand to the detection method developed and optimization autophagy enhances.
In the detection method of autophagy enhancing, what is be widely used is to mark autophagosome using autophagy marker GFP-LC3, it
It is the fusions of a kind of GFP fluorescin and LC3 marker.GFP-LC3 is as autophagosome fluorescent marker, in label autophagy
Be very effective in terms of body, but due to its using complex steps and it is expensive the problems such as, greatly limit in drug high pass
The application of amount screening etc..For new drug development, fundamental way is to improve efficiency, and reduces cost, in the shorter time
Upper market.Therefore, developing one kind, quickly and effectively autophagy enhances detection probe for improving drug screening efficiency --- especially
Drug primary dcreening operation --- it is very important.
The acidification of organelle and autophagy process it is closely related [referring to：Wijdeven,R.H.；Janssen,H.；
Nahidiazar, L.；Janssen,L.；Jalink,K.；Berlin,I.；Neefjes,J.Nature communications
2016,7, 11808.Mehrpour,M.；Esclatine,A.；Beau,I.；Codogno,P.Cell research 2010,
20, 748.Lamb,C.A.；Yoshimori,T.；Tooze,S.A.Nature reviews.Molecular cell
biology 2013,14,759.Eskelinen,E.-L.；Reggiori,F.；Baba,M.；Kovács,A.L.；Seglen,P.
O.Autophagy 2011,7,935.], for example, autophagosome merges (early endosome, late endosome and more with endocytic pathway
Foam (MVBs)), and enhanced by the fusion that these fusion process generate than autophagosome acidity；Meanwhile in endocytic pathway
Late endosomeCompare early endosomeAcidity enhancing [referring to：Mellman,
I.Annual Review of Cell and Developmental Biology 1996,12,575].It is acidified organelle quantity
Sharply increase and reflect the enhancing of autophagy indirectly.Therefore, the formation of real time imagery acidic organelles for further understand with
The enhancing of aging neurodegenerative disease, drug resistance in the treatment of cancer autophagy closely related with tumor suppression has weight
Want significance and importance.Many molecular probes such as lysosome for long-life acidic organelles has been reported, but has no and is used for
The organelle being newly acidified.The identification of new acidification organelle and characterization rely primarily on electron microscope and fluorescence microscope, however,
The dead cell that electron microscope needs chemistry fixed may draw the wrong conclusion during fixed cell because of pollution.
The living cells imaging of late endosome can be realized by transfection endosome specific protein gene, however, these methods
Take a long time and complicated operating procedure, and inefficiency.Nano-probe, especially overdelicate pH nano-probe,
Research late endosome plays an important role in terms of the mechanism understanding in encytosis and intracellular transport nano particle,
But intracellular invalid the problem of delivering, hinders them in autophagy approach for marking late endosome and other acidifications
Organelle [referring to：Liu,M.；Li,Q.；Liang, L.；Li,J.；Wang,K.；Li,J.；Lv,M.；Chen,N.；Song,H.；
Lee,J.；Shi,J.；Wang, L.；Lal,R.；Fan,C.Nature communications 2017,8,
15646.Richardson,D.S.；Gregor, C.；Winter,F.R.；Urban,N.T.；Sahl,S.J.；Willig,
K.I.；Hell,S.W.Nature communications 2017,8,577.Wang,C.；Zhao,T.；Li,Y.；Huang,
G.；White,M.A.； Gao,J.Advanced drug delivery reviews 2017,113,87.Zhou,K.；Wang,
Y.；Huang,X.； Luby-Phelps,K.；Sumer,B.D.；Gao,J.Angewandte Chemie-International
Edition 2011, 50,6109.].Molecular probe is a kind of more promising substitute, because they usually have biofacies
Capacitive and usually have membrane permeability.Proper probes for acidic organelles are usually by fluorogen and lipophilic alkalescent base
Group composition so that probe in acidophilia and after protonation in acid vesica accumulation [referring to：Xu,W.；Zeng,Z.B.；
Jiang,J.H.；Chang, Y.T.；Yuan,L.Angewandte Chemie-International Edition 2016,
55,13658.].However, this skill is not particularly suited for the organelle being newly acidified.When cell and molecular probe are incubated with,
The lysosome of long-life is labeled, and newly-generated acidic organelles are not labeled but, because once being protonated in lysosome,
Net positive charge probe cannot readily pass through organelle film and return in cytosol, and probe is enriched in lysosome.Therefore,
Low 1 to 2 order of magnitude of concentration and probe concentration concentration and probe concentration usually more intracorporal than lyase outside lysosome is produced in the organelle being newly acidified
Have little time to dye it when raw.There is patent report metal complex to be used as the label of autophagy lysosome, but metal complex
Object membrane permeability itself is poor, and being entered after cell by endocytic pathway can not be discharged into the cell by endosome retention, therefore only
It can be transported in autophagy lysosome and finally be degraded, and pH is changed without response, therefore also can not be newly acidified by detection
Organelle quickly indicate autophagocytosis enhancing [referring to：CN 104152529A].Success detects the key of new acidification organelle
It is molecular probe to be entered into cell with a kind of invisible, non-fluorescence " sleep state ", and make them in cytoplasm and cell
It is uniformly distributed in device, to " can wake up " when generating acidic organelles, that is, be transformed into another visible fluorescent form.
Summary of the invention
The content of present invention is to design and provide a kind of material that detection autophagy enhancing is acidified by real-time detection organelle
Preparation method and use.
In the present invention, we cleverly devise the non-conjugated systems compound of fluorine boron, they are all neutral
Small-molecule probe therefore can be evenly distributed in cell, and cytoplasm, it is neutral in alkaline cell device almost without
Any fluorescence.However strong fluorescence can be emitted under conditions of organelle acidification.When with cell autophagy enhance drug
When (rapamycin) stimulates cell, observes that acidic organelles acutely increase, indirectly indicate the enhancing of cell autophagy.It visits
Needle not only shows good cell permeability, almost without any cytotoxicity, and almost without background fluorescence, is not necessarily to
Any washing step.
Summary of the invention：
A kind of compound of detection cell autophagy enhancing, it is just like flowering structure：
SN x (x representation compound number)
Wherein R1、R2、R3、R4、R5、R6, it is independently selected from H, halogen, nitro, (C1-C6) alkyl, (C3-C6) cycloalkanes
Base, (C6-C10) aryl, (C3-C10) heteroaryl, (C3-C6) alkenyl, CH3COO-、CCl3COO-、CF3COO-、ClO4-、BF4-、
BPh4-、-CN、-N3Or-OCH3。
The compound of above-mentioned detection cell autophagy enhancing, the R1Group is preferably H or CH3。
The compound of above-mentioned detection cell autophagy enhancing, the R2Group is preferably H, NO2Or N (CH3)2。
The compound of above-mentioned detection cell autophagy enhancing, the R3Group is preferably OCnH2n+1, wherein n=1-8.
The compound of above-mentioned detection cell autophagy enhancing, the R4Group is preferably CN or COOC2H5。
The compound of above-mentioned detection cell autophagy enhancing, the R5Group is preferably COOC2H5。
The compound of above-mentioned detection cell autophagy enhancing, the R6Group is preferably H or CH3。
A method of above compound being prepared, it is shown below：
A method of the compound of above-mentioned detection cell autophagy enhancing being prepared, it includes the following steps:
(the R containing substituent group is added in the reaction vessel1、R2) benzaldehyde and the (R containing substituent group4-R6) pyrroles, substituted benzene
The ratio between amount of substance of formaldehyde and substituted azole is 1:2, it is dissolved in anhydrous methylene chloride, catalyst trifluoro second is added
Sour (TFA), catalyst amount are 1-5 times of the amount of substituted benzaldehyde substance, stir, return under conditions of completely cutting off air and being protected from light
Stream 4-7 days, contact plate detection is completely depleted until substituted azole, then will be with equimolar 2, the 3- bis- chloro- 5,6- of substituted benzaldehyde
Dicyano-Isosorbide-5-Nitrae-benzoquinones (DDQ) is added in reaction solution, stirs 30 minutes at room temperature, excessive triethylamine is added later
(TEA) and boron trifluoride-diethyl ether solution (BF3Et2O), continue to be stirred at reflux reaction 1 hour, reaction is quenched with water, reaction
Mixture distilled water, saturated sodium bicarbonate solution, saturated salt solution successively wash, and are then concentrated with Rotary Evaporators, use
100-200 mesh silica gel column chromatography separates and collects component of the maximum absorption wavelength at 600-700 nanometers, and eluant, eluent is dichloromethane
Alkane after solvent evaporated, is added methanol and potassium carbonate, is stirred at room temperature 5 minutes, is filtered to remove potassium carbonate, and rotation solvent evaporated obtains mesh
Mark compound.
It is a kind of to pass through real-time detection organelle acidification detection autophagy using the above-mentioned compound for detecting cell autophagy enhancing
The method of enhancing, this method comprise the following steps：
The first step：Obtain cell needed for testing；
Second step：The compound that will test cell autophagy enhancing is incubated for altogether with cell.
Third step：Emit the acidification that fluorescence determines organelle by the compound of detection cell autophagy enhancing.
4th step：Pass through the enhancing of the acidification instruction autophagy of organelle.
The compound of above-mentioned detection cell autophagy enhancing drug screening, cell experiment, histological examination, fluorescence experiments,
Application in high flux screening, preparation detection reagent or kit.
Beneficial effects of the present invention
Compared with prior art, remarkable advantage is the present invention：Invented for the first time one kind can real-time detection be newly acidified carefully
The non-conjugated material of two pyrroles of fluorine-containing boron of born of the same parents' device, this material itself do not have fluorescence, can but send out at once when organelle is acidified
Strong fluorescence is penetrated, the enhancing of autophagy is detected by detecting the organelle being newly acidified.For the first time using the side of detection organelle acidification
Method detects cell autophagy enhancing, proposes completely new detection idea and method.Autophagosome is marked compared to classical GFP-LC3
Method, the method for the present invention operation is simpler, quickly, more conducively high flux screening.Other than the above advantage, probe has excellent
Different cell permeability, insignificant toxicity are interfered during fluorescence imaging without background fluorescence, and any washing step is not necessarily to,
This is the outstanding advantages of quick detection and high flux screening.
Detailed description of the invention
Fig. 1 is the compound SN1 of detection cell autophagy enhancing of the invention1H NMR spectra；
Fig. 2 is the compound SN1 of detection cell autophagy enhancing of the invention13C NMR spectra；
Fig. 3 is that the pH of the compound SN1 of detection cell autophagy enhancing of the invention titrates UV absorption and fluorescence spectra；
Fig. 4 is newly acidified organelle lab diagram for the detection for the compound SN1 that detection cell autophagy of the invention enhances, wherein
The enlarged drawing in region 1a, 2a, 3a, 4a selected (at i.e. 0 minute) before representing addition autophagy agonist, 1b, 2b, 3b, 4b
The enlarged drawing that autophagy agonist selection area after ten minutes is added is represented, the arrow in figure, which indicates, is added autophagy agonist 10
The acidic organelles newly increased before comparing 10 minutes after minute, Fig. 4 clearly illustrate that material of the present invention being capable of real-time detection autophagy
Adjoint organelle acidification during enhancing；
Fig. 5 is the compound SN2 of detection cell autophagy enhancing of the invention1H NMR spectra；
Fig. 6 is the compound SN2 of detection cell autophagy enhancing of the invention13C NMR spectra；
Fig. 7 is the compound SN3 of detection cell autophagy enhancing of the invention1H NMR spectra；
Fig. 8 is the compound SN3 of detection cell autophagy enhancing of the invention13C NMR spectra；
Fig. 9 is the compound SN4 of detection cell autophagy enhancing of the invention1H NMR spectra；
Figure 10 is the compound SN4 of detection cell autophagy enhancing of the invention13C NMR spectra；
Figure 11 is the compound SN5 of detection cell autophagy enhancing of the invention1H NMR spectra；
Figure 12 is the compound SN5 of detection cell autophagy enhancing of the invention13C NMR spectra；
Figure 13 is the compound SN6 of detection cell autophagy enhancing of the invention1H NMR spectra；
Figure 14 is the compound SN6 of detection cell autophagy enhancing of the invention13C NMR spectra.
Specific embodiment
Detecting instrument is：(TMS is internal standard, CD to BrukerARX500 type Nuclear Magnetic Resonance2Cl2And CD3OD is molten
Agent), Shimadzu UV-4500 type ultraviolet-visible spectrophotometer (300-900 nanometers of scanning range, 2.0 nanometers of slit width), day
Vertical F4600 Fluorescence Scanner, U.S. BrukerDaltonicsautoflexII mass spectrum work station.
Embodiment 1：The synthesis of compound SN1
(R1=R2=H) benzaldehyde (0.106g, 1mmol) and 2- cyano -2- (carbethoxyl group second are added in the reaction vessel
Alkenyl) -1-H- pyrroles (0.380g, 2mmol), it is dissolved in 50ml anhydrous methylene chloride.Trifluoroacetic acid is extracted with syringe
(TFA) catalyst 0.5ml injection system, it is anhydrous be protected from light under the conditions of be put into magnetic stir bar stirring, reflux 7 days after use thin layer color
Plate monitoring is composed until 2- cyano -2- (the ethoxycarbonyl-ethenyl) -1-H- pyrroles is completely depleted.Then 2,3- bis- is chloro-
5,6- dicyano -1,4- benzoquinones (DDQ：0.227g, 1mmol) it is added in reaction solution, it stirs 30 minutes at room temperature, Zhi Houjia
Enter triethylamine (TEA) 3ml after mixing evenly, Eorontrifluoride etherate solution (BF is added in three times3·Et2O) 6ml (quality percentage
Than 48%), continuing to be stirred at reflux reaction 1 hour, obtain the solution that blue has red fluorescence, contact plate is monitored to not being coordinated
Product exhausts, and reaction is quenched with water, and reaction mixture distilled water, saturated sodium bicarbonate solution, saturated salt solution are successively washed
It washs, is then concentrated with Rotary Evaporators, separated with 100-200 mesh silica gel column chromatography and collect 630 nanometers of maximum absorption wavelength
Component, eluant, eluent are methylene chloride, after solvent evaporated, methanol, potassium carbonate are added, is stirred at room temperature 5 minutes, is filtered to remove carbonic acid
Potassium, rotation solvent evaporated obtain the two azole compounds (R of different fluorine boron containing 2- cyanacrylate group1=H；R2=H；R3
=OCH3；R4=CN；R5=COOCH2CH3；R6=H).Product is pale yellow powder, 298mg, yield 58%.MALDI TOF
MS:calculated for[C28H25BF2N4O5+H]+m/z 547.18,found 547.50.1H NMR (see Fig. 1)
δ=8.83 (600MHz, MeOD) (s, 2H), 7.77 (d, J=4.2,2H), 7.38-7.33 (m, 2H), 7.28-7.22 (m,
2H), 7.21-7.17 (m, 1H), 6.18 (d, J=4.1,2H), 3.68 (ddd, J=5.9,4.6,1.0,1H), 3.65-3.62
(m, 2H), 3.57 (ddd, J=17.3,8.9,4.4,2H), 3.33 (dt, J=3.3,1.6,4H), 1.19 (dd, J=8.4,
5.7,4H).13C NMR (see Fig. 2) (151MHz, MeOD) δ=165.92,145.85,145.26,128.69,127.72,
126.82,125.17,121.34,118.42,112.04,87.96,76.80, 72.38,63.16,57.15,53.47,
53.29,53.11,52.92,52.74,48.43,48.29,48.14,48.00, 47.86,47.72,47.58,17.23.。
Embodiment 2：The pH titration experiments of SN1
Colourless to flaxen solution is presented in compound SN1 in methyl alcohol, and ultra-violet absorption spectrum is shown in Fig. 3, at 374 nanometers
There are two absorption peaks with 413 nanometers.TFA is added to in the methanol solution of iso-BODIPY progress pH titration, pH value from
It is observed in 7.0 to 2.12 titration process from light yellow to red obvious color change, and along with strong red
The generation of fluorescence.It is 6.0 hereinafter, being initially observed the generation of fluorescence in pH value, when pH value becomes 4.1 from 7.0, fluorescence hair
It penetrates intensity (λ em=635nm) and increases 17 times or more.Illustration in Fig. 3 shows the absorption in pH titration process at 618nm
Variation.Obtaining pKa value by using Henderson-Hasselbach equation is 4.71.Ultra-violet absorption spectrum and fluorescence spectrum
Variation show iso-BODIPY be suitable for pH fluorescence probe, potential acidic organelles fluorescent marker function may be possessed.
Embodiment 3：The lysosome fluorescent marker of SN1 and real-time monitoring application
KB cell strain is provided by Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences.
KB cell is cultivated in the serum-free cell freezing media (RPMI) 1640 mixed with 10% fetal calf serum (FBS).It places it in
In carbon dioxide incubator, 37 DEG C incubator 4 hours.Cell need not clean direct scanning imagery.Skill is imaged by 3D living cells
Art has done further research to the iso-BODIPY and label lysosome fluorescence probe of synthesis.Hela cell is loaded into 1 (15 μM)
30 minutes, and confocal microscopy is carried out, rapamycin (20 μM) then are added under the microscope.For each field, note
A series of 10-20 continuous image focal planes are recorded, continuous focal plane is at a distance of 0.8 μm, for the upright projection of each focal plane
Mapping.Fluorescent image is recorded at selected time point, calculates the increased percentage of pixel.10 μm of image scale (Fig. 4).
Embodiment 4：The synthesis of compound SN2
Paranitrobenzaldehyde (0.128g) and 2- cyano -2- (ethoxycarbonyl-ethenyl) -1-H- is added in the reaction vessel
Pyrroles (0.380g), is dissolved in 70ml anhydrous methylene chloride.Trifluoroacetic acid (TFA) catalyst 0.5ml note is extracted with syringe
Enter system, it is anhydrous be protected from light under the conditions of be put into magnetic stir bar stirring, with chromatographic sheet monitoring until pyrroles is complete after reflux 5 days
Fully- depleted.Then by 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ：0.227g, 1mmol) it is added in reaction solution, room
Temperature lower stirring 30 minutes, triethylamine (TEA) 3ml is added after mixing evenly later, Eorontrifluoride etherate solution is added in three times
(BF3·Et2O) 6ml (mass percent 48%) continues to be stirred at reflux reaction 1 hour, obtains blue with the molten of red fluorescence
Liquid, contact plate, which is monitored to the product not being coordinated, to be exhausted, and reaction is quenched with water, and reaction mixture distilled water, saturated sodium bicarbonate are molten
Liquid, saturated salt solution successively wash, and are then concentrated with Rotary Evaporators, are separated with 100-200 mesh silica gel column chromatography and collect tool
There is a component of red fluorescence, eluant, eluent is methylene chloride, after solvent evaporated, methanol, potassium carbonate is added, is stirred at room temperature 5 minutes,
It is filtered to remove potassium carbonate, rotation solvent evaporated obtains different two azole compounds of fluorine boron containing 2- cyanacrylate group
(R1=H；R2=NO2；R3=OCH3；R4=CN；R5=COOCH2CH3；R6=H).Product is pale yellow powder, yield 55%.Change
Object is closed by nucleus magnetic hydrogen spectrum and carbon stave sign (see Fig. 5, Fig. 6).
Embodiment 5：The synthesis of compound SN3
It is added in the reaction vessel to dimethylamino benzaldehyde (0.150g) and 2- cyano -2- (ethoxycarbonyl-ethenyl) -
1-H- pyrroles (0.380g), is dissolved in 50ml anhydrous methylene chloride.Trifluoroacetic acid (TFA) catalyst is extracted with syringe
0.5ml injection system, it is anhydrous be protected from light under the conditions of be put into magnetic stir bar stirring, reflux 7 days after with chromatographic sheet monitoring until
Pyrroles is completely depleted.Then by 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ：0.227g, 1mmol) it is added and reacts molten
In liquid, stirs at room temperature 30 minutes, triethylamine (TEA) 3ml is added after mixing evenly later, boron trifluoride-second is added in three times
Ethereal solution (BF3·Et2O) 6ml (mass percent 48%) continues to be stirred at reflux reaction 1 hour, obtains the solution of blue, point
Plate, which is monitored to the product not being coordinated, to be exhausted, and reaction is quenched with water, and reaction mixture distilled water, is satisfied at saturated sodium bicarbonate solution
It successively washs with saline solution, is then concentrated with Rotary Evaporators, separated with 100-200 mesh silica gel column chromatography and collect maximum suction
It receives wavelength and is greater than 600 nanometers of component, eluant, eluent is methylene chloride, after solvent evaporated, methanol, potassium carbonate is added, is stirred at room temperature
5 minutes, it is filtered to remove potassium carbonate, rotation solvent evaporated obtains different two pyrrolesization of fluorine boron containing 2- cyanacrylate group
Close object (R1=H；R2=N (CH3)2； R3=OCH3；R4=CN；R5=COOCH2CH3；R6=H).Product is pale yellow powder, is produced
Rate 57%.Compound is by nucleus magnetic hydrogen spectrum and carbon stave sign (see Fig. 7, Fig. 8).
Embodiment 6：The synthesis of compound SN4
2,6- dimethylbenzaldehyde (0.140g) and 2- cyano -2- (ethoxycarbonyl-ethenyl)-are added in the reaction vessel
1-H- pyrroles (0.380g), is dissolved in 50ml anhydrous methylene chloride.Trifluoroacetic acid (TFA) catalyst is extracted with syringe
0.5ml injection system, it is anhydrous be protected from light under the conditions of be put into magnetic stir bar stirring, reflux 6 days after with chromatographic sheet monitoring until
Pyrroles is completely depleted.Then by 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ：It 0.227g) is added in reaction solution, room
Temperature lower stirring 30 minutes, triethylamine (TEA) 3ml is added after mixing evenly later, Eorontrifluoride etherate solution is added in three times
(BF3·Et2O) 6ml (mass percent 48%) continues to be stirred at reflux reaction 1 hour, obtain molten with intense red fluorescence
Liquid, contact plate, which is monitored to the product not being coordinated, to be exhausted, and reaction is quenched with water, and reaction mixture distilled water, saturated sodium bicarbonate are molten
Liquid, saturated salt solution successively wash, and are then concentrated with Rotary Evaporators, are separated and are collected most with 100-200 mesh silica gel column chromatography
Big absorbing wavelength is greater than 600 nanometers of component, and eluant, eluent is methylene chloride, and after solvent evaporated, ethyl alcohol, potassium carbonate, room temperature is added
Stirring 5 minutes, is filtered to remove potassium carbonate, and rotation solvent evaporated obtains different two pyrrole of fluorine boron containing 2- cyanacrylate group
Cough up compound (R1=CH3；R2=H；R3=OCH2CH3；R4=CN；R5=COOCH2CH3；R6=H).Product is pale yellow powder,
Yield 68%.Compound is by nucleus magnetic hydrogen spectrum and carbon stave sign (see Fig. 9, Figure 10).
Embodiment 7：The synthesis of compound SN5
Benzaldehyde (0.106g) and 2 is added in the reaction vessel, 2- bis--(ethoxycarbonyl-ethenyl) -1-H- pyrroles
(0.410g) is dissolved in 50ml anhydrous methylene chloride.Trifluoroacetic acid (TFA) catalyst 0.4ml, which is extracted, with syringe injects body
System, it is anhydrous be protected from light under the conditions of be put into magnetic stir bar stirring, consumed completely with chromatographic sheet monitoring until pyrroles after reflux 4 days
To the greatest extent.Then by 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ：It 0.227g) is added in reaction solution, stirs 30 at room temperature
Minute, triethylamine (TEA) 3ml is added after mixing evenly later, Eorontrifluoride etherate solution (BF is added in three times3·Et2O)
6ml (mass percent 48%) continues to be stirred at reflux reaction 1 hour, obtains with rose fluorescent solutions, contact plate monitoring
It is exhausted to the product not being coordinated, reaction, reaction mixture distilled water, saturated sodium bicarbonate solution, saturated common salt is quenched with water
Water successively washs, and is then concentrated with Rotary Evaporators, is separated with 100-200 mesh silica gel column chromatography and collects maximum absorption wavelength
Component greater than 600 nanometers, eluant, eluent are methylene chloride, after solvent evaporated, octanol, potassium carbonate are added, is stirred at room temperature 5 minutes,
It is filtered to remove potassium carbonate, rotation solvent evaporated obtains different two azole compounds of fluorine boron containing 2- cyanacrylate group.
(R1=H；R2=H； R3=OCH2CH2CH2CH2CH2CH2CH2CH3；R4=COOCH2CH3；R5=COOCH2CH3；R6=H).Product
For pale yellow powder, yield 65%.Compound is by nucleus magnetic hydrogen spectrum and carbon stave sign (see Figure 11, Figure 12).
Embodiment 8：The synthesis of compound SN6
2,6- dimethylbenzaldehyde (0.135g) and 2,2- bis--(ethoxycarbonyl-ethenyl) -1- are added in the reaction vessel
Methyl-pyrroles (0.460g), is dissolved in 50ml anhydrous methylene chloride.Trifluoroacetic acid (TFA) catalyst is extracted with syringe
0.5ml injection system, it is anhydrous be protected from light under the conditions of be put into magnetic stir bar stirring, reflux 7 days after with chromatographic sheet monitoring until
Pyrroles is completely depleted.Then by 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ：It 0.227g) is added in reaction solution, room
Temperature lower stirring 30 minutes, triethylamine (TEA) 3ml is added after mixing evenly later, Eorontrifluoride etherate solution is added in three times
(BF3·Et2O) 6ml (mass percent 48%) continues to be stirred at reflux reaction 1 hour, obtain with red fluorescence solution, point
Plate, which is monitored to the product not being coordinated, to be exhausted, and reaction is quenched with water, and reaction mixture distilled water, is satisfied at saturated sodium bicarbonate solution
It successively washs with saline solution, is then concentrated with Rotary Evaporators, separated with 100-200 mesh silica gel column chromatography and collect maximum suction
It receives wavelength and is greater than 600 nanometers of component, eluant, eluent is methylene chloride, after solvent evaporated, propyl alcohol, potassium carbonate is added, is stirred at room temperature
5 minutes, it is filtered to remove potassium carbonate, rotation solvent evaporated obtains different two pyrrolesization of fluorine boron containing 2- cyanacrylate group
Close object (R1=CH3；R2=H； R3=OCH2CH2CH3；R4=COOCH2CH3；R5=COOCH2CH3；R6=CH3).Product is yellowish
Color powder, yield 51%.
Embodiment 9：The pH titration experiments of compound SN2
Colourless to flaxen solution is presented in compound SN2 in methyl alcohol, and in 375 nanometers and 415 nanometers, there are two inhale
Receive peak.TFA is added to progress pH titration in the methanol solution of iso-BODIPY, in titration process of the pH value from 7.0 to 2.12
In observe from light yellow to red obvious color change, and along with the generation of strong red fluorescence.It is in pH value
5.5 hereinafter, be initially observed the generation of fluorescence, when pH value becomes 2.0 from 7.0, fluorescent emission intensity (λ em=635nm)
Increase 20 times or more.Obtaining pKa value by using Henderson-Hasselbach equation is 2.0.Ultra-violet absorption spectrum and
The variation of fluorescence spectrum shows that iso-BODIPY is suitable for pH fluorescence probe, may possess potential acidic organelles fluorescence mark
It cites sb. for meritorious service energy.
Embodiment 10：The pH titration experiments of compound SN4
Colourless to flaxen solution is presented in compound SN2 in methyl alcohol, and in 375 nanometers and 415 nanometers, there are two inhale
Receive peak.TFA is added to progress pH titration in the methanol solution of iso-BODIPY, in titration process of the pH value from 7.0 to 2.00
In observe from light yellow to red obvious color change, and along with the generation of strong red fluorescence.It is in pH value
7.0 hereinafter, be initially observed the generation of fluorescence, when pH value becomes 2.0 from 7.0, fluorescent emission intensity (λ em=630nm)
Increase 30 times or more.Obtaining pKa value by using Henderson-Hasselbach equation is 5.5.0.Ultra-violet absorption spectrum
Variation with fluorescence spectrum shows that iso-BODIPY is suitable for pH fluorescence probe, may possess potential acidic organelles fluorescence
Mark function.
Embodiment 11：The lysosome fluorescent marker of SN2 and real-time monitoring application
KB cell strain is provided by Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences.
KB cell is cultivated in the serum-free cell freezing media (RPMI) 1640 mixed with 10% fetal calf serum (FBS).It places it in
In carbon dioxide incubator, 37 DEG C incubator 4 hours.Cell need not clean direct scanning imagery.Skill is imaged by 3D living cells
Art has done further research to the iso-BODIPY and label lysosome fluorescence probe of synthesis.Hela cell is loaded into 1 (15 μM)
30 minutes, and confocal microscopy is carried out, rapamycin (20 μM) then are added under the microscope.For each field, note
A series of 10-20 continuous image focal planes are recorded, continuous focal plane is at a distance of 0.8 μm, for the upright projection of each focal plane
Mapping.Fluorescent image is recorded at selected time point, calculates the increased percentage of pixel.Observe acidic organelles pixel
Increasing for point, shows that compound can detecte the enhancing of cell autophagy.
Embodiment 12：The lysosome fluorescent marker of SN4 and real-time monitoring application
KB cell strain is provided by Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences.
KB cell is cultivated in the serum-free cell freezing media (RPMI) 1640 mixed with 10% fetal calf serum (FBS).It places it in
In carbon dioxide incubator, 37 DEG C incubator 4 hours.Cell need not clean direct scanning imagery.Skill is imaged by 3D living cells
Art has done further research to the iso-BODIPY and label lysosome fluorescence probe of synthesis.Hela cell is loaded into 1 (15 μM)
30 minutes, and confocal microscopy is carried out, rapamycin (20 μM) then are added under the microscope.For each field, note
A series of 10-20 continuous image focal planes are recorded, continuous focal plane is at a distance of 0.8 μm, for the upright projection of each focal plane
Mapping.Fluorescent image is recorded at selected time point, calculates the increased percentage of pixel.Observe acidic organelles pixel
Increasing for point, shows that compound can detecte the enhancing of cell autophagy.
Claims (10)
1. a kind of compound of detection cell autophagy enhancing, it is characterized in that it is just like flowering structure：
Wherein R1、R2、R3、R4、R5、R6, it is independently selected from H, halogen, nitro, (C1-C6) alkyl, (C3-C6) naphthenic base,
(C6-C10) aryl, (C3-C10) heteroaryl, (C3-C6) alkenyl, CH3COO-、CCl3COO-、CF3COO-、ClO4-、BF4-、
BPh4-、-CN、-N3Or-OCH3。
2. the compound of detection cell autophagy enhancing according to claim 1, it is characterized in that：The R1Group be H or
CH3。
3. the compound of detection cell autophagy enhancing according to claim 1, it is characterized in that：The R2Group is H, NO2
Or N (CH3)2。
4. the compound of detection cell autophagy enhancing according to claim 1, it is characterized in that：The R3Group is
OCnH2n+1, wherein n=1-8.
5. the compound of detection cell autophagy enhancing according to claim 1, it is characterized in that：The R4Group be CN or
COOC2H5。
6. the compound of detection cell autophagy enhancing according to claim 1, it is characterized in that：The R5Group is
COOC2H5。
7. the compound of detection cell autophagy enhancing according to claim 1, it is characterized in that：The R6Group be H or
CH3。
8. a kind of method for the compound for preparing detection cell autophagy enhancing described in claim 1, it is characterized in that under it includes
Column step:
(the R containing substituent group is added in the reaction vessel1、R2) benzaldehyde and the (R containing substituent group4-R6) pyrroles, substituted benzaldehyde and
The ratio between amount of substance of substituted azole is 1:2, it is dissolved in anhydrous methylene chloride, is added catalyst trifluoroacetic acid (TFA),
Catalyst amount is 1-5 times of the amount of substituted benzaldehyde substance, stirs, flows back 4-7 days under conditions of completely cutting off air and being protected from light, point
Plate detection is completely depleted until substituted azole, then will be with chloro- 5, the 6- dicyano-Isosorbide-5-Nitrae-of equimolar 2, the 3- bis- of substituted benzaldehyde
Benzoquinones (DDQ) is added in reaction solution, stirs 30 minutes at room temperature, excessive triethylamine (TEA) and boron trifluoride-are added later
Diethyl ether solution (BF3Et2O) continues to be stirred at reflux reaction 1 hour, and reaction is quenched with water, and reaction mixture distilled water is satisfied
It successively washs with sodium bicarbonate solution, saturated salt solution, is then concentrated with Rotary Evaporators, with 100-200 mesh silica gel column chromatography
Separate and collect component of the maximum absorption wavelength at 600-700 nanometers, eluant, eluent is methylene chloride, and after solvent evaporated, first is added
Pure and mild potassium carbonate is stirred at room temperature 5 minutes, is filtered to remove potassium carbonate, and rotation solvent evaporated obtains target compound.
9. a kind of compound using detection cell autophagy enhancing described in claim 1 passes through the acidification inspection of real-time detection organelle
The method for surveying autophagy enhancing, it is characterized in that this method comprises the following steps：
The first step：Obtain cell needed for testing；
Second step：The compound that will test cell autophagy enhancing is incubated for altogether with cell；
Third step：Emit the acidizing degree that fluorescence determines organelle by the compound of detection cell autophagy enhancing；
4th step：Pass through the enhancing degree of the acidizing degree indicating cell autophagy of organelle.
10. the compound of detection cell autophagy enhancing described in claim 1 is in drug screening, cell experiment, histological examination, glimmering
Application in light experiment, high flux screening and preparation detection reagent or kit.
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