CN106810561A - A kind of lysosome targeting hypochlorous acid ratio fluorescent probe and preparation method and application - Google Patents
A kind of lysosome targeting hypochlorous acid ratio fluorescent probe and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of lysosome targeting hypochlorous acid ratio fluorescent probe and preparation method and application, the molecular formula of the probe is：C49H55N7O5, structural formula is as follows：The probe increases lysosome seeking group by reaction site, fluorescence is caused to change using the energy transmission between two fluorophors, design Ratio-type and recognize hypochlorous probe and response time non-constant velocity, can apply to hypochlorous sensing detection in biological cell system；Described sensing detection includes fluoroscopic examination, visual qualitative detection, cell imaging detection.
Description
Technical field
The present invention relates to a kind of lysosome targeting hypochlorous acid ratio fluorescent probe and preparation method and application, belong to analysis
Technical field of chemistry.
Background technology
Hypochlorous acid (HClO) is the various biological natural bactericidal agents for defending in nature.It is secondary in biological cell
Chloric acid is produced by hydrogen peroxide and chlorion under the catalytic action of MPer (MPO).As a kind of important active oxygen
Species, hypochlorous acid plays very important effect in the intracellular redox equilibrium state of maintenance, but once secondary in cell
There is exception in the concentration of chloric acid, will cause including the various diseases including rheumatic arthritis, angiocardiopathy and cancer, inspection
Surveying hypochlorous concentration in living things system has turned into an important problem.
20-400 μM of hypochlorous acid can be per hour produced in the case of document report, neutral grain (white) inflammation cells, by
In hypochlorous pKaIt is 7.46, in vivo, part hypochlorous acid (HClO) is with (ClO with hypochlorous acid-) form exist.
Compared with traditional detection method, fluorescent spectrometry as a kind of Noninvasive detection technique, with its numerous advantage.It is such as sensitive
Degree is high, selectivity is good, respond rapid, in situ detection, real-time monitoring etc..
Many endogeneous activity species are widely present in organism, active oxygen species are a very important classes, and secondary chlorine
Acid is a member of active oxygen family, and under conditions of the presence of other interference active oxygen species, selectivity is to internal secondary chlorine
Acid is identified, it is necessary to probe molecule has preferable anti-interference.At the same time, for concentration and probe concentration, detection temperature, response
Time and instrument error all produce influence to identification.At present, the hypochlorous acid probe of most reports is the change of single fluorescence intensity
Change type probe, Ratio-type probe is less.This patent, using FRET principles, increases molten by Molecular Design in reaction site
Enzyme body seeking group, designs Ratio-type and recognizes hypochlorous probe and response time non-constant velocity.
The content of the invention
The present situation of problem encountered is detected for current hypochlorous acid fluorescence probe, the present invention passes through MOLECULE DESIGN,
Synthesize a kind of Ratio-type hypochlorous acid fluorescence probe fast with the response time.
The present invention uses following technical scheme：
A kind of lysosome targets hypochlorous acid ratio fluorescent probe, and the molecular formula of the probe molecule is：C49H55N7O5, with such as
Structure shown in lower：
The probe is gradually reduced with the increase of NaClO equivalents, the fluorescence probe in the absorption of 385nm, and in 560nm
Absorption gradually strengthen, ultraviolet spectra is into rate of change.
The probe is excited under the conditions of hypochlorous without addition with 380nm excitation wavelengths, is only had at 455nm
Very strong fluorescent emission, is excited with 500nm excitation wavelengths, does not have fluorescent emission；The increase of equivalent, 455nm are added with NaClO
The fluorescence intensity at place gradually weakens, and the fluorescence intensity at 584nm gradually strengthens, and fluorescence spectrum is into rate of change.
Above-mentioned lysosome targets the preparation method of hypochlorous acid ratio fluorescent probe, and it is comprised the following steps：
1) hydroxy phenyl piperazine between the 4- diethylin ketone acid and 1eq of 1eq is dissolved in the 3ml concentrated sulfuric acids, 90 DEG C of heating 3
Hour, room temperature is cooled to, under ice bath, 4ml perchloric acid is added, crude product is obtained, column chromatography for separation obtains compound 1；
2) compound 1 of 1eq is dissolved in ethanol, 80% hydrazine hydrate of 10eq is added dropwise, flowed back 3 hours, after the completion of reaction, subtracted
Pressure is spin-dried for solvent and obtains crude product and obtain compound 2 after using column chromatography；
3) EDCI of the 6- diethylin naphthalene-carboxylic acid of 1eq compounds 2 and 1eq, HOBt, 1eq of 1eq is dissolved in DMF, room
Temperature reaction 6h, after reaction completely, is added water, and ethyl acetate is extracted 2 times, is washed 2-3 times, anhydrous sodium sulfate drying, and decompression is spin-dried for molten
After agent crude product, and with separating to obtain compound after column chromatography；
4) compound 3 of 1eq is dissolved in chloroform, 3-4 drop triethylamines is added dropwise thereto, it is then that 20 μ l bromoacetyl bromides are molten
In 1ml chloroforms, it is slowly added dropwise in mixed solution under the conditions of 0 DEG C, room temperature reaction 3-4 hours after completion of dropping, reaction
After completely, extracted with dichloromethane, then washing, salt washing, anhydrous sodium sulfate drying successively, decompression is spin-dried for after solvent slightly producing
Product, and use column chromatography and obtain compound 4；
5) by the compound 4 of 1eq, the potassium carbonate of 3eq, the morpholine of micro KI and 3eq is dissolved in DMF, room temperature
6h is reacted, after reaction completely, ethyl acetate extraction is washed 2-3 times, anhydrous sodium sulfate drying, after back-out solvent obtains crude product,
Target-probe compound is obtained with column chromatography, Lyso-HClO is abbreviated as.
The eluant, eluent proportioning of column chromatography is in the step (1)：Methylene chloride/methanol=20:1.
The eluant, eluent proportioning of column chromatography is in the step (2)：Methylene chloride/methanol=20:1.
The eluant, eluent proportioning of column chromatography is in the step (3)：Methylene chloride/methanol=30:1.
The eluant, eluent proportioning of column chromatography is in the step (4)：Methylene chloride/methanol=20:1.
The eluant, eluent proportioning of column chromatography is in the step (5)：Dichloromethane/petroleum ether=1:1.
The synthetic route of above-mentioned hypochlorous acid fluorescence probe is as follows：
Lysosome of the present invention targets the application of hypochlorous acid ratio fluorescent probe, and the fluorescence probe can apply to life
Hypochlorous sensing detection in thing cell system；Described sensing detection includes fluoroscopic examination, visual qualitative detection, cell imaging
Detection.
The fluorescent molecular structure increases morpholine as lysosome seeking group, can be effectively single-minded in cell imaging process
Navigate to lyase body region.Based on FRET mechanism, under the conditions of hypochlorous without addition, the fluorescence probe is only in 385nm
There is UV absorption in place, and with the increase of NaClO equivalents, the fluorescence probe is gradually reduced in the absorption of 385nm, and in 560nm
Absorption gradually strengthen, ultraviolet spectra is into rate of change.Under the conditions of hypochlorous without addition, the fluorescence probe is in excitation wave
Under the conditions of (380nm) long, the fluorescence probe only has very strong fluorescent emission at 455nm, in excitation wavelength (500nm) condition
Under, the fluorescence probe does not have fluorescent emission.The increase of equivalent is added with NaClO, fluorescence intensity gradually weakens (455nm)；
The increase of equivalent is added under the conditions of excitation wavelength (500nm) with NaClO, fluorescence intensity gradually strengthens (584nm), fluorescence spectrum
Equally into rate of change, the change of fluorescence intensity ratio can effectively weaken the certain error that single fluorescence intensity change is caused.Utilize
Laser Scanning Confocal Microscope is imaged, without in the cell for adding sodium hypochlorite, by 405nm for the blue channel of exciting light has fluorescence to send out
Go out, and the red passage that 561nm is excited is barely perceivable fluorescence and sends.However, can be in the cell of addition sodium hypochlorite simultaneously
Fluorescence imaging is observed in blue channel and red passage.
Advantages of the present invention：(1) the synthesis cost of probe is than relatively low, and last handling process is relatively easy；(2) present invention is real
Show the selective quick detection of hypochlorous acid molecular probe, and it is selective good, resist other molecule interference performances strong.Additionally, with
Naked eyes are just it is observed that the change of solution colour.Based on its specificity and significant color change, the reagent can be used as display
The selectivity indicator existed with hypochlorous acid molecule in biological cell in the aqueous solution, can carry out real-time qualitative and quantitative visual ratio
Color method is detected.So, the present invention is simple one kind, quickly, sensitive hypochlorous acid molecular specificity detection reagent, in biomolecule
Detection field has broad application prospects.Its performance will give with reference to accompanying drawing in embodiment and describe in detail.
Brief description of the drawings
Fig. 1 is the middle probe Lyso-HClO of embodiment 11H NMR spectras；
Fig. 2 is situations of change of the probe Lyso-HClO with the addition fluorogram of sodium hypochlorite；
Fig. 3 is the situation of change of fluorescence intensity after probe Lyso-HClO is added to different analytes, wherein (1) blank；
(2)t-butylhydroperoxide；(3)H2O2；(4)NO；(5)peroxide tert-butyl ether；(6)OH—；(7)
NO2 —；(8)NO3 2—；(9)I—；(10)S2—；(11)Cys；(12)GHS；(13)Hcy；(14)Cu2+；(15)；Co2+(16)Ca2+；
(17)Mg2+；(18)K+,(19)ClO—；
Fig. 4 is the change of probe Lyso-HClO solution solution colour before and after hypochlorous acid is added；
Fig. 5 is that exogenous hypochlorous acid carries out fluorescence imaging during probe Lyso-HClO is applied to cell.
Specific 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
System, number of the number of compound for compound in such scheme in embodiment.
Embodiment 1
The synthesis of compound L yso-HClO hypochlorous acid fluorescence probes
The synthesis of compound 1：
4- diethylin ketone acid (10mmol, 3g) and a hydroxy phenyl piperazine (10mmol, 1.77g) are dissolved in the 3ml concentrated sulfuric acids
In, 90 DEG C of heating 3h are cooled to room temperature, and 4ml perchloric acid is added under ice bath, obtain crude product, and column chromatography for separation, silica gel particle is big
Small is 200-300 mesh, and eluant, eluent proportioning is methylene chloride/methanol=20:1, yield is 80%.
The synthesis of compound 2：
Compound 1 (2.5g, 5mmol, 1eq) is dissolved in 20mL ethanol, 80% hydrazine hydrate 5ml heating reflux reactions are added dropwise
3h, is reacted with the detection of TCL plates, and after reaction completely, decompression obtains crude product after being spin-dried for solvent, and is separated with silicagel column, silica gel
Granular size is 200-300 mesh, and eluant, eluent proportioning is methylene chloride/methanol=20:1, yield is 85%.
The synthesis of compound 3：
By compound 2 (0.23g, 5mmol, 1eq) and 6- diethylin naphthalene-carboxylic acid (1.2g, 5mmol, 1eq), HOBt
(0.675g, 5mmol, 1eq), EDCI (0.96g, 5mmol, 1eq) are dissolved in DMF, room temperature reaction 6h, after reaction completely, are added water,
Ethyl acetate is extracted 2 times, is washed 2-3 times, anhydrous sodium sulfate drying, and decompression obtains crude product after being spin-dried for solvent, and is entered with silicagel column
Row is separated, and silica gel particle size is 200-300 mesh, and eluant, eluent proportioning is methylene chloride/methanol=30:1, yield is 50%.
The synthesis of compound 4：
Compound 3 (100mg, 1mmol, 1eq) is dissolved in 2ml CHCl3In, 3-4 drop triethylamines, the μ of bromoacetyl bromide 20 is added dropwise
L is dissolved in 1ml chloroforms, is slowly added dropwise under the conditions of 0 DEG C.Detected with TCL plates and reacted, after reaction completely, 2 are extracted with dichloromethane
It is secondary, to wash 2-3 times, salt is washed 1 time, anhydrous sodium sulfate drying, and decompression obtains crude product after being spin-dried for solvent, and is divided with silicagel column
From silica gel particle size is 200-300 mesh, and eluant, eluent proportioning is methylene chloride/methanol=20:1, yield is 86%.1H NMR
(400MHz,DMSO-d6) δ 9.41 (s, 1H), 7.85 (d, J=6.8Hz, 1H), 7.78 (d, J=8.5Hz, 2H), 7.66 (d, J
=8.6Hz, 1H), 7.59 (p, J=7.2Hz, 2H), 7.36 (d, J=8.4Hz, 1H), 7.20 (d, J=9.1Hz, 1H), 7.06
(d, J=7.1Hz, 1H), 6.92 (s, 1H), 6.66 (s, 2H), 6.55 (d, J=9.3Hz, 1H), 6.46 (d, J=8.8Hz,
1H), 6.45-6.26 (m, 2H), 3.67 (s, 4H), 3.47 (d, J=6.9Hz, 4H), 3.32 (d, J=6.9Hz, 4H), 3.25
(s, 4H), 2.96-2.76 (m, 2H), 2.12 (s, 4H), 1.16 (t, J=6.9Hz, 6H), 1.08 (t, J=6.8Hz, 6H)
The synthesis of compound L yso-HClO：
By compound 4 (50mg, 0.1mmol, 1eq), K2CO3(45mg, 0.3mmol, 3eq), KI is micro, is dissolved in 2mLDMF
In, morpholine (26mg, 3eq) is added in being dissolved in DMF, and room temperature reaction 6 hours is detected with TCL plates and reacted, after reaction completely, acetic acid
Ethyl ester is extracted, and is washed 2-3 times, anhydrous sodium sulfate drying, screws out solvent.Silica gel particle size is 200-300 mesh, eluant, eluent proportioning
It is dichloromethane/petroleum ether=1:1, yield is 81%.
Embodiment 2
Compound L yso-HClO hypochlorous acid fluorescence probe adds the change of the increase fluorogram of equivalent with NaClO
Lyso-HClO hypochlorous acid fluorescence probes prepared by Example 1 are dissolved in DMF (DMF), are made
Into 1mmol/L storing solutions.Take out 40 μ L from storing solution to be added in the middle of the centrifuge tube of 5mL, with PBS cushioning liquid (0.1mol/
L, pH=7.4) with DMF volume ratios be 1:1 solution is diluted to 4mL, adds the NaClO standards of different equivalents (0-12eq) molten
Liquid, measures its photoluminescent property.Fluorescence spectrum is as shown in Figure 2.Left figure be the fluorescence probe under the conditions of excitation wavelength (380nm) with
The increase that NaClO adds equivalent, fluorescence intensity gradually weakens (455nm), right figure is the fluorescence probe in excitation wavelength
The increase of equivalent is added under the conditions of (500nm) with NaClO, fluorescence intensity gradually strengthens (584nm).The two is in finite concentration model
Enclose interior into linetype scale relation, can be with quantitative determination low concentration hypochlorous acid.
Embodiment 3
Compound L yso-HClO hypochlorous acid fluorescence probe is to different molecular or the selectivity of ion
30 μ L are taken out in fluorescence probe storing solution from embodiment 2 to be added in the middle of the centrifuge tube of 5mL, is separately added into etc. and to be rubbed
The competition molecular criteria solution of your amount, hypochlorous acid standard liquid [(1) blank of one of addition equimolar amounts；(2)t-
butylhydroperoxide；(3)H2O2；(4)NO；(5)peroxide tert-butyl ether；(6)OH—；(7)NO2 —；
(8)NO3 2—；(9)I—；(10)S2—；(11)Cys；(12)GHS；(13)Hcy；(14)Cu2+；(15)；Co2+(16)Ca2+；(17)
Mg2+；(18)K+,(19)OCl—], with PBS after 30min:DMF(1:1) it is solvent, 365nm is exciting light, detects the fluorescence of solution
Emission spectrum changes, as a result as shown in Figure 3.It is investigated shadow of other metal ions to fluorescence probe intensity of the present invention simultaneously
Ring, with PBS:DMF(1:1) it is solvent, 380nm is exciting light.Seen by Fig. 3 it can be found that other metal ions are to compound
Fluorescence of the Lyso-HClO at 584nm has little to no effect, and the addition of hypochlorite solution makes compound L yso-HClO exist
Fluorescence at 584nm is significantly increased.
Embodiment 4
Compound L yso-HClO fluorescence probes are to hypochlorous Visual retrieval
30 μ L are taken out in fluorescence probe storing solution from embodiment 2 to be added in the middle of the sample cell of 5mL, 80 moles are added
Hypochlorous acid standard liquid, hypochlorous acid can make the PBS of compound Lyso-HClO fluorescence probes：DMF volume ratios are 1:1 buffering
There is obvious color change in solution, solution, solution colour becomes red from colourless, as shown in Figure 4.This explanation is that one kind has
Add lustre to the fluorescence probe of sensing function.
Embodiment 5
Compound L yso-HClO hypochlorous acid fluorescence probe is to the exogenous hypochlorous acid ratio fluorescent imaging of cell
The hypochlorous acid of probe application of the present invention exogenous in HeLa cells is carried out fluorescence imaging by us.Concrete operations
Step is as follows：5 μM of Lyso-HClO hypochlorous acid probe DMSO solutions are added in two parts of nutrient solutions for giving birth to HeLa cells
After the secondary chlorine sodium water solution of 30min and then a 20 μM of addition thereto is cultivated in CO2gas incubator, another addition
The distilled water of same volume, cultivates in the incubator after 20min carries out washing three times with PBS, is carried out into Laser Scanning Confocal Microscope
Picture.Imaging results are as shown in Figure 5.It is the indigo plant of exciting light by 405nm without in the cell for adding sodium hypochlorite as reference
Passage has fluorescence to send, and the red passage that 561nm is excited is barely perceivable fluorescence and sends.However, adding the thin of sodium hypochlorite
Fluorescence imaging can be observed in blue channel and red passage simultaneously in born of the same parents, be imaged by two ratios of passage, it can be found that two
There is obvious difference between person, illustrate that this fluorescence probe can carry out ratio fluorescent imaging with the hypochlorous acid of exogenous.
Claims (10)
1. a kind of lysosome targeting hypochlorous acid ratio fluorescent probe, it is characterised in that the molecular formula of the probe molecule is：
C49H55N7O5, with structure as shown below：
2. fluorescence probe according to claim 1, it is characterised in that the probe is with the increase of NaClO equivalents, and this is glimmering
Light probe is gradually reduced in the absorption of 385nm, and the absorption in 560nm gradually strengthens, and ultraviolet spectra is into rate of change.
3. fluorescence probe according to claim 1, it is characterised in that the probe is without adding hypochlorous condition
Under, excited with 380nm excitation wavelengths, only there is very strong fluorescent emission at 455nm, excited with 500nm excitation wavelengths, do not have
Fluorescent emission；The increase of equivalent is added with NaClO, the fluorescence intensity at 455nm gradually weakens, the fluorescence intensity at 584nm
Gradually strengthen, fluorescence spectrum is into rate of change.
4. the lysosome described in a kind of claim 1,2 or 3 targets the preparation method of hypochlorous acid ratio fluorescent probe, and its feature exists
In it is comprised the following steps：
1) hydroxy phenyl piperazine between the 4- diethylin ketone acid and 1eq of 1eq is dissolved in the 3ml concentrated sulfuric acids, 90 DEG C of heating 3 are small
When, room temperature is cooled to, under ice bath, 4ml perchloric acid is added, crude product is obtained, column chromatography for separation obtains compound 1, the compound 1
Structural formula is as follows：
2) compound 1 of 1eq is dissolved in ethanol, 80% hydrazine hydrate of 10eq is added dropwise, flowed back 3 hours, after the completion of reaction, decompression rotation
Dry solvent obtains crude product and obtains compound 2 after using column chromatography, and the structural formula of the compound 2 is as follows：
3) EDCI of the 6- diethylin naphthalene-carboxylic acid of 1eq compounds 2 and 1eq, HOBt, 1eq of 1eq is dissolved in DMF, room temperature is anti-
6h is answered, after reaction completely, is added water, ethyl acetate is extracted 2 times, washed 2-3 times, anhydrous sodium sulfate drying, after decompression is spin-dried for solvent
Crude product is obtained, and with compound 3 is separated to obtain after column chromatography, the structural formula of the compound 3 is as follows：
4) compound 3 of 1eq is dissolved in chloroform, 3-4 drop triethylamines is added dropwise thereto, be then dissolved in 20 μ l bromoacetyl bromides
In 1ml chloroforms, it is slowly added dropwise in mixed solution under the conditions of 0 DEG C, room temperature reaction 3-4 hours after completion of dropping, has been reacted
Quan Hou, is extracted with dichloromethane, then washing, salt washing, anhydrous sodium sulfate drying successively, and decompression is spin-dried for after solvent slightly producing
Product, and use column chromatography and obtain compound 4, the structural formula of the compound 4 is as follows：
5) by the compound 4 of 1eq, the potassium carbonate of 3eq, the morpholine of micro KI and 3eq is dissolved in DMF, room temperature reaction
6h, after reaction completely, ethyl acetate extraction is washed 2-3 times, anhydrous sodium sulfate drying, after back-out solvent obtains crude product, uses post
Chromatography obtains target-probe compound, is abbreviated as Lyso-HClO.
5. preparation method according to claim 4, it is characterised in that the eluant, eluent proportioning of column chromatography in the step (1)
For：Methylene chloride/methanol=20:1.
6. preparation method according to claim 4, it is characterised in that the eluant, eluent proportioning of column chromatography in the step (2)
For：Methylene chloride/methanol=20:1.
7. preparation method according to claim 4, it is characterised in that the eluant, eluent proportioning of column chromatography in the step (3)
For：Methylene chloride/methanol=30:1.
8. preparation method according to claim 4, it is characterised in that the eluant, eluent proportioning of column chromatography in the step (4)
For：Methylene chloride/methanol=20:1.
9. preparation method according to claim 4, it is characterised in that the eluant, eluent proportioning of column chromatography in the step (5)
For：Dichloromethane/petroleum ether=1:1.
10. the lysosome described in a kind of claim 1,2 or 3 targets the application of hypochlorous acid ratio fluorescent probe, it is characterised in that
The fluorescence probe can apply to hypochlorous sensing detection in biological cell system；Described sensing detection is examined comprising fluorescence
Survey, visual qualitative detection, cell imaging detection.
Priority Applications (1)
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