CN105524079A - Ratio-type pH fluorescence probe for water-soluble locating lysosome as well as preparation method, application and test method of ratio-type pH fluorescence probe - Google Patents
Ratio-type pH fluorescence probe for water-soluble locating lysosome as well as preparation method, application and test method of ratio-type pH fluorescence probe Download PDFInfo
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Abstract
The invention provides a ratio-type pH fluorescence probe for water-soluble locating lysosome as well as a preparation method, an application and a test method of the ratio-type pH fluorescence probe. The chemical structural formula of the ratio-type pH fluorescence probe is shown in the specification. The probe can detect changes of strength of fluorescence with two emission wavelengths simultaneously, external influences are eliminated through self-calibration, the detection accuracy is high, the speed of response to pH changes is high, and the probe has excellent water solubility and has very good anti-interference capacity in the existence of various metal ions. The fluorescence strength changes remarkably in the pH range from 5.8 to 6.8, and the probe is suitable for pH detection under a weak acidic condition. Experiments prove that the probe can selectively dye lysosome in cells, the light stability in the cells is good, and the probe is non-toxic to the cells and can be used for pH detection of live cells. The probe has very important application value in cell imaging and detection of changes of the pH value in lysosome.
Description
One, technical field
The present invention relates to a kind of Ratio-type pH fluorescent probe, its preparation method, application and testing method, especially a kind of for water-soluble location lysosomal Ratio-type pH fluorescent probe, its preparation method, application and testing method.
Two, background technology
Intracellular ph value plays a significant role in many cell behaviors, as Growth of Cells, and apoptosis, signal transduction and cell proliferation etc.As an important indicator of cell health, the subtle change of internal pH can cause cellular abnormality, even can cause cell dysfunction and serious disease, as cancer and Alzheimer.Intracellular pH value is not identical, and tenuigenin is weakly alkaline.And lysosome internal pH-values is about 4.0-6.0, in slightly acidic.Weak acid environment in lysosome can activate the function of inner lytic enzyme, decomposing protein in cellular metabolism.Therefore, for water-soluble location lysosomal Ratio-type pH fluorescent probe, its preparation method, application and testing method are to the method for lysosomal pH value detection by quantitative to cell analysis or diagnosis, in existing Ratio-type pH fluorescent probe and its preparation method and application, the method detecting intracellular ph value mainly contains nucleus magnetic resonance, Absorption and fluorescence spectrum etc., detection technique of fluorescence is due to highly sensitive, easy to operate, can the advantage such as Real-Time Monitoring and being widely applied, but most of fluorescent probe is all the change detecting single wavelength fluorescent intensity, such probe output signal can be subject to temperature, solvent polarity, the impact of concentration and probe concentration, the accuracy of detected result can be affected.
Three, summary of the invention
Object first one of the present invention is used for water-soluble location lysosomal Ratio-type pH fluorescent probe,
Object of the present invention its two are a kind of its preparation methods for water-soluble location lysosomal Ratio-type pH fluorescent probe,
Object of the present invention its three are a kind of application for water-soluble location lysosomal Ratio-type pH fluorescent probe,
Object of the present invention its four are a kind of testing method for water-soluble location lysosomal Ratio-type pH fluorescent probe.
In order to overcome above-mentioned technical disadvantages, the object of this invention is to provide a kind of for water-soluble location lysosomal Ratio-type pH fluorescent probe, its preparation method, application and testing method, therefore detect the fluorescence intensity change of two emission wavelengths simultaneously, eliminate ectocine by self calibration, improve detection accuracy.
For achieving the above object, the technical scheme that the present invention takes is:
A kind of for water-soluble location lysosomal Ratio-type pH fluorescent probe, its chemical structural formula is shown below:
。
Owing to devising Ratio-type pH fluorescent probe, by showing for the FRET (fluorescence resonance energy transfer) FRET mechanism of water-soluble location lysosomal Ratio-type pH fluorescent probe, therefore detect the fluorescence intensity change of two emission wavelengths simultaneously, eliminate ectocine by self calibration, improve detection accuracy.
The present invention devises, a kind of for water-soluble location lysosomal Ratio-type pH fluorescent probe, has:
Nuclear magnetic resonance hydrogen spectruming determining result:
1hNMR (DMSO-d
6, 300MHz), δ (ppm)=8.71-8.75 (m, 1H), 8.67 (d, J=7.5Hz, 1H), 8.14 (s, 1H), 7.80 (t, J=4.5Hz, 1H), 7.51 (t, J=4.5Hz, 2H), 7.39 (dd, J=1.5Hz, J=7.5Hz, 1H), 7.00 (t, J=7.5Hz, 1H), 6.30-6.43 (m, 6H), 4.03 (s, 3H), 3.39-3.48 (m, 4H), 3.18-3.32 (m, 10H), 1.56-1.64 (m, 2H), (1.39 q, J=7.5Hz, 2H), 0.97-1.09 (m, 12H), 0.91 (t, J=7.5Hz, 3H)
Mr carbon spectrum measurement result:
13cNMR (DMSO-d
6, 75MHz), δ (ppm)=12.31 (4C), 13.48 (2C), 21.66 (2C), 25.12,28.57,29.96,43.51 (3C), 64.31,64.31,97.27 (2C), 104.74 (2C), 108.07 (2C), 110.93 (2C), 116.01,119.24,121.51,122.22,123.60,124.20,128.25,128.60,130.65 (2C), 131.45,132.68,138.47 (2C), 152.51 (2C), 153.87,163.76,167.69
High resolution mass spectrum measurement result: Calcd. [M-I]
-: 733.3633; Foundvalue [M-I]
-: 733.3623,
Compound.
The present invention devises, a kind of its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe, its step:
Intermediate compound III, HOBT(1-hydroxybenzotriazole), EDC [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride], triethylamine be dissolved in DMF and obtain in intermediate solution, intermediate compound V is joined in intermediate solution, be obtained by reacting finalization compound RMPM
Wherein: intermediate compound III chemical structural formula is shown below:
；
Intermediate compound V chemical structural formula is shown below:
。
The present invention devises, a kind of its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe, its step: its step:
Intermediate compound III 1.0-1.4mmol, HOBT1.0-1.4mmol, EDC1.0-1.4mmol, triethylamine 1.8-2.2mmol is dissolved in DMF18-22mL, stirring at room temperature 0.8-1.2h under nitrogen protection, obtain intermediate solution, then intermediate compound V 0.8-1.2mmol is joined in intermediate solution, under nitrogen protection, stirring at room temperature 18-22h, silica-gel plate is followed the tracks of and is determined reaction end, reaction solution reduction vaporization is concentrated to obtain thick terminated compound, extract in thick terminated compound methylene dichloride and water, collect organic phase, dry, concentrated, then purification by column chromatography is passed through, the faint yellow solid obtained and terminated compound RMPM, yield 45.2%, fusing point: 197-201 DEG C.
The present invention devises, a kind of its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe, its step: its step:
3-butyl-1-chlorine imidazo (1,5-α) pyridine-7-carboxylic acid ethyl ester and intermediate compound I 8-12mmol and NaOH18-22mmol be dissolved in the mixing solutions of ethanol 18-22ml and water 13-17ml, after back flow reaction 1.8-2.2h, gained solution is poured in 180-220ml water, be neutralized to neutrality with dilute hydrochloric acid 1.8-2.2M, have yellow solid to separate out to filter, wash 2 × 5mL with water, in an oven after 50 DEG C of dryings, obtain yellow solid and intermediate compound II, yield is 90.8%
By intermediate compound II 8-12mmol and CH
3i48-52mmol is dissolved in tetrahydrofuran (THF) 28-32ml solution, heating reflux reaction 7.8-8.2h under nitrogen protection, cool to room temperature; separate out yellow solid, by vacuum filtration, filter cake ethyl acetate 8-12ml washs 3 times; obtain yellow solid and intermediate compound III, yield is 73.0%
Intermediate compound V can pass through to be synthesized by the intermediate compound IV of method in the literature and reacting ethylenediamine,
Wherein: intermediate compound IV chemical structural formula is shown below:
。
The present invention devises, and a kind of synthetic route of its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe is as follows:
The present invention devises, a kind of for the application of water-soluble location lysosomal Ratio-type pH fluorescent probe as Ratio-type pH fluorescent probe.
The present invention devises, and a kind of is the application of the Ratio-type pH fluorescent probe of 5.8 to 6.8 for water-soluble location lysosomal Ratio-type pH fluorescent probe as measuring pH.
The present invention devises, a kind of for the application of water-soluble location lysosomal Ratio-type pH fluorescent probe as the Ratio-type pH fluorescent probe at Hela cell.
The present invention devises, a kind of testing method for water-soluble location lysosomal Ratio-type pH fluorescent probe, its step: prepare above-mentioned water-soluble location lysosomal Ratio-type pH fluorescent probe, solution (ethanol/Britton--Robinson buffered soln, 40mM acetic acid, phosphoric acid at various ph values, boric acid, 1:4, volume ratio), try its responding ability to different pH value by Tissue with Spectrofluorometric;
Quantitatively AgNO3 is added respectively containing in the solution of pH fluorescent probe in acid condition to above-mentioned, Al (NO3) 3, Ba (NO3) 2, Cd (NO3) 2, Co (NO3) 2, Cu (NO3) 2, Fe (NO3) 3, HgCl2, Ni (NO3) 2, the aqueous solution of Pb (NO3) 2, Zn (NO3) 2, tests its immunity from interference;
Use the above-mentioned pH fluorescent probe of different concns to cultivate Hela cell, carry out fluorescence imaging respectively, fluorescence intensity change in observation of cell,
Use above-mentioned pH fluorescent probe and commercial lysosome probe Lysosensor GreenDND-189 to dye altogether to Hela cell of living, carry out dyeing location by fluorescence imaging,
Use the above-mentioned pH fluorescent probe of the different pH value of finite concentration to cultivate Hela cell, observe the change of fluorescence intensity and red fluorescence/blue-fluorescence ratio,
Use certain density above-mentioned pH fluorescent probe to cultivate Hela cell, under the continuous agitation of laser, observe the change of the ratio of blue-fluorescence/red fluorescence, test the light stability of this probe in Hela cell,
After using certain density above-mentioned pH fluorescent probe to cultivate Hela cell 6h, detection probes is on the impact of Hela cell survival rate.
Technique effect of the present invention is: the preferred Hela cell of described viable cell.Locate the response of lysosomal pH fluorescent probe rapidly, have excellent water-soluble, to many kinds of metal ions, there is good immunity from interference.Be changed significantly in 5.8 to 6.8 scopes at pH, under adapting to solutions of weak acidity, pH detects.This probe can be selected, by the lysosome dyeing in Hela cell, to be used in the detection of pH in viable cell.Above-mentioned probe has rhodamine-imidazo [1,5-α] pyridine structure, and when pH value is increased to 7.00 from 5.50, the blue-fluorescence of imidazo [1,5-α] pyridine groups is almost constant.Meanwhile, the red fluorescence of rhodamine group declines.And the ratio of red fluorescence/blue-fluorescence is increased to 7.00 from 5.50 and increases along with pH value.Can detect the fluorescence intensity change of two emission wavelengths, eliminate ectocine by self calibration, accuracy in detection is high simultaneously, to pH change response rapidly, has excellent water-soluble, strong to metal ion immunity from interference.Remarkable from fluorescence intensity change in 5.8 to 6.8 scopes at pH, under adapting to solutions of weak acidity, pH detects.Experiment proves that the lysosome in cell can selectivity dye by this probe, and the good light stability in cell, to cytotoxic.Probe of the present invention has important using value in pH value change in cell imaging and detection lysosome.
In the technical program, purification by column chromatography and silica gel, methylene chloride/methanol=10:1, v/v.
In the technical program, the chemical structural formula of Ratio-type pH fluorescent probe is important technical characteristic, in the technical field for water-soluble location lysosomal Ratio-type pH fluorescent probe, its preparation method, application and testing method, have novelty, creativeness and practicality, the term in the technical program is all to make an explanation with patent documentation in the art and to understand.
Four, accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 be intermediate compound III chemical structural formula:
The fluorescence spectrum (a) of Fig. 2 RMPM (10 μm) in the B-R buffered soln of different pH value (5.6-7.2) and the fluorescence intensity ratio (I of probe under corresponding pH value
580/ I
426) (b), (λ
ex=340nm, slit: 10nm/5nm), n=3:
Fig. 3 pH value is 5.6(a) and 7.2 (b) time, the fluorescence intensity ratio I of RMPM (10 μMs) in the B-R buffered soln containing different metal ion
580/ I
426, the concentration of all metallic cations is 100 μMs, (λ
ex=340nm, slit: 10nm/5nm):
Fig. 4 RMPM (10 μMs) fluorescence intensity ratio I
580/ I
426reaction times in the B-R buffered soln of pH5.6 and pH7.2:
Fluorescence microscopy images after the probe of Fig. 5 Hela cell and 5 μMs processes 2h at 37 DEG C.Ctr: do not have probe.The first row: the fluoroscopic image (400-555nm) of blue channel, the second row: the fluoroscopic image (560-700nm) of red channel, the third line: bright field image, fourth line: first, second and the overlay image of the third line, λ
ex=405nm.(*p<0.05;n=3)：
The location of Fig. 6 probe in Hela Cytolysosome.(a): RMPM probe (5 μMs), (b): LysoSensorsGreenDND-189 (1 μM), (c): the coverage diagram of (a) and (b), (d): bright field image, the common orientation factor figure of (e): RMPM and LysoSensorsGreenDND-189:
Fig. 7 after 2 μMs of probe process 2h, then known pH value containing 10 μMs of polyetherin As PBS damping fluid in cultivate 0.5h after, the fluorescence microscopy images (a) of Hela cell.The pH value of PBS damping fluid is respectively pH5.5 (a-d), pH6.0 (e-h), pH6.5 (i-l), and pH7.00 (m-p).First row: the fluoroscopic image (400-555nm) of blue channel; Secondary series: the fluoroscopic image (560 – 700nm) of red channel; 3rd row: bright field image; 4th row: the image of first, second, and third row superposition.Image j is used to analyze λ
ex=405nm fluorescence intensity ratio (b), result represents (* p<0.05 and * * p<0.01 with ± SE; N=3):
The photostabilization of Fig. 8 probe.After 2 μMs of probe process 2h, at λ
exthe photostabilization of Hela cell is tested under the continuous agitation of=405nm laser:
After 6h cultivated by the probe of Fig. 9 Hela cell and concentration known, probe is on the impact of Hela cell survival rate.(p>0.05;n=3)：
Figure 10 is the chemical structural formula for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM.
Five, embodiment
Below in conjunction with embodiment, further describe the present invention, following examples are intended to the present invention instead of limitation of the invention further are described.The steps include:
For first embodiment of its preparation method of water-soluble location lysosomal Ratio-type pH fluorescent probe,
Its step:
3-butyl-1-chlorine imidazo (1,5-α) pyridine-7-carboxylic acid ethyl ester and intermediate compound I 8mmol and NaOH18mmol be dissolved in the mixing solutions of ethanol 18ml and water 13ml, after back flow reaction 1.8h, gained solution is poured in 180ml water, be neutralized to neutrality with dilute hydrochloric acid 1.8M, have yellow solid to separate out to filter, wash 2 × 5mL with water, in an oven after 50 DEG C of dryings, obtain yellow solid and intermediate compound II, yield is 90.8%
By intermediate compound II 8mmol and CH
3i48mmol is dissolved in tetrahydrofuran (THF) 28ml solution, heating reflux reaction 7.8h under nitrogen protection, cool to room temperature; separate out yellow solid, by vacuum filtration, filter cake ethyl acetate 8ml washs 3 times; obtain yellow solid and intermediate compound III, yield is 73.0%
Intermediate compound V can pass through to be synthesized by the intermediate compound IV of method in the literature and reacting ethylenediamine,
Intermediate compound III 1.0mmol, HOBT1.0mmol, EDC1.0mmol, triethylamine 1.8mmol is dissolved in DMF18mL, stirring at room temperature 0.8h under nitrogen protection, obtain intermediate solution, then intermediate compound V 0.8mmol is joined in intermediate solution, under nitrogen protection, stirring at room temperature 18h, silica-gel plate is followed the tracks of and is determined reaction end, reaction solution reduction vaporization is concentrated to obtain thick terminated compound, extract in thick terminated compound methylene dichloride and water, collect organic phase, dry, concentrated, then purification by column chromatography is passed through, the faint yellow solid obtained and terminated compound RMPM, yield 45.2%, fusing point: 197-201 DEG C.
Wherein: intermediate compound III chemical structural formula is shown below:
；
Wherein: intermediate compound IV chemical structural formula is shown below:
Intermediate compound V chemical structural formula is shown below:
。
A kind of synthetic route of its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe is as follows:
FRET (fluorescence resonance energy transfer) FRET mechanism for water-soluble location lysosomal Ratio-type pH fluorescent probe:
For second embodiment of its preparation method of water-soluble location lysosomal Ratio-type pH fluorescent probe,
Its step:
3-butyl-1-chlorine imidazo (1,5-α) pyridine-7-carboxylic acid ethyl ester and intermediate compound I 8-12mmol and NaOH18-22mmol be dissolved in the mixing solutions of ethanol 18-22ml and water 13-17ml, after back flow reaction 1.8-2.2h, gained solution is poured in 180-220ml water, be neutralized to neutrality with dilute hydrochloric acid 1.8-2.2M, have yellow solid to separate out to filter, wash 2 × 5mL with water, in an oven after 50 DEG C of dryings, obtain yellow solid and intermediate compound II, yield is 90.8%
By intermediate compound II 8-12mmol and CH
3i48-52mmol is dissolved in tetrahydrofuran (THF) 28-32ml solution, heating reflux reaction 7.8-8.2h under nitrogen protection, cool to room temperature; separate out yellow solid, by vacuum filtration, filter cake ethyl acetate 8-12ml washs 3 times; obtain yellow solid and intermediate compound III, yield is 73.0%
Intermediate compound V can pass through to be synthesized by the intermediate compound IV of method in the literature and reacting ethylenediamine,
Intermediate compound III 1.0-1.4mmol, HOBT1.0-1.4mmol, EDC1.0-1.4mmol, triethylamine 1.8-2.2mmol is dissolved in DMF18-22mL, stirring at room temperature 0.8-1.2h under nitrogen protection, obtain intermediate solution, then intermediate compound V 0.8-1.2mmol is joined in intermediate solution, under nitrogen protection, stirring at room temperature 18-22h, silica-gel plate is followed the tracks of and is determined reaction end, reaction solution reduction vaporization is concentrated to obtain thick terminated compound, extract in thick terminated compound methylene dichloride and water, collect organic phase, dry, concentrated, then purification by column chromatography is passed through, the faint yellow solid obtained and terminated compound RMPM, yield 45.2%, fusing point: 197-201 DEG C.
For the 3rd embodiment of its preparation method of water-soluble location lysosomal Ratio-type pH fluorescent probe,
Its step:
3-butyl-1-chlorine imidazo (1,5-α) pyridine-7-carboxylic acid ethyl ester and intermediate compound I 12mmol and NaOH22mmol be dissolved in the mixing solutions of ethanol 22ml and water 17ml, after back flow reaction 2.2h, gained solution is poured in 220ml water, be neutralized to neutrality with dilute hydrochloric acid 2.2M, have yellow solid to separate out to filter, wash 2 × 5mL with water, in an oven after 50 DEG C of dryings, obtain yellow solid and intermediate compound II, yield is 90.8%
By intermediate compound II 12mmol and CH
3i52mmol is dissolved in tetrahydrofuran (THF) 32ml solution, heating reflux reaction 8.2h under nitrogen protection, cool to room temperature; separate out yellow solid, by vacuum filtration, filter cake ethyl acetate 12ml washs 3 times; obtain yellow solid and intermediate compound III, yield is 73.0%
Intermediate compound V can pass through to be synthesized by the intermediate compound IV of method in the literature and reacting ethylenediamine,
Intermediate compound III 1.4mmol, HOBT1.4mmol, EDC1.4mmol, triethylamine 2.2mmol is dissolved in DMF22mL, stirring at room temperature 1.2h under nitrogen protection, obtain intermediate solution, then intermediate compound V 1.2mmol is joined in intermediate solution, under nitrogen protection, stirring at room temperature 22h, silica-gel plate is followed the tracks of and is determined reaction end, reaction solution reduction vaporization is concentrated to obtain thick terminated compound, extract in thick terminated compound methylene dichloride and water, collect organic phase, dry, concentrated, then purification by column chromatography is passed through, the faint yellow solid obtained and terminated compound RMPM, yield 45.2%, fusing point: 197-201 DEG C.
For the 4th embodiment of its preparation method of water-soluble location lysosomal Ratio-type pH fluorescent probe,
Its step:
3-butyl-1-chlorine imidazo (1,5-α) pyridine-7-carboxylic acid ethyl ester and intermediate compound I 10mmol and NaOH20mmol be dissolved in the mixing solutions of ethanol 20ml and water 15ml, after back flow reaction 2.0h, gained solution is poured in 200ml water, be neutralized to neutrality with dilute hydrochloric acid 2.0M, have yellow solid to separate out to filter, wash 2 × 5mL with water, in an oven after 50 DEG C of dryings, obtain yellow solid and intermediate compound II, yield is 90.8%
By intermediate compound II 10mmol and CH
3i50mmol is dissolved in tetrahydrofuran (THF) 30ml solution, heating reflux reaction 8.0h under nitrogen protection, cool to room temperature; separate out yellow solid, by vacuum filtration, filter cake ethyl acetate 10ml washs 3 times; obtain yellow solid and intermediate compound III, yield is 73.0%
Intermediate compound V can pass through to be synthesized by the intermediate compound IV of method in the literature and reacting ethylenediamine,
Intermediate compound III 1.2mmol, HOBT1.2mmol, EDC1.2mmol, triethylamine 2.0mmol is dissolved in DMF20mL, stirring at room temperature 1.0h under nitrogen protection, obtain intermediate solution, then intermediate compound V 1.0mmol is joined in intermediate solution, under nitrogen protection, stirring at room temperature 20h, silica-gel plate is followed the tracks of and is determined reaction end, reaction solution reduction vaporization is concentrated to obtain thick terminated compound, extract in thick terminated compound methylene dichloride and water, collect organic phase, dry, concentrated, then purification by column chromatography is passed through, the faint yellow solid obtained and terminated compound RMPM, yield 45.2%, fusing point: 197-201 DEG C.
A kind of for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM, its chemical structural formula is shown below:
Spectrogram for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM is measured:
Nuclear magnetic resonance hydrogen spectruming determining:
1hNMR (DMSO-d
6, 300MHz), δ (ppm)=8.71-8.75 (m, 1H), 8.67 (d, J=7.5Hz, 1H), 8.14 (s, 1H), 7.80 (t, J=4.5Hz, 1H), 7.51 (t, J=4.5Hz, 2H), 7.39 (dd, J=1.5Hz, J=7.5Hz, 1H), 7.00 (t, J=7.5Hz, 1H), 6.30-6.43 (m, 6H), 4.03 (s, 3H), 3.39-3.48 (m, 4H), 3.18-3.32 (m, 10H), 1.56-1.64 (m, 2H), 1.39 (q, J=7.5Hz, 2H), 0.97-1.09 (m, 12H), 0.91 (t, J=7.5Hz, 3H).
Mr carbon spectrum measures:
13cNMR (DMSO-d
6, 75MHz), δ (ppm)=12.31 (4C), 13.48 (2C), 21.66 (2C), 25.12,28.57,29.96,43.51 (3C), 64.31,64.31,97.27 (2C), 104.74 (2C), 108.07 (2C), 110.93 (2C), 116.01,119.24,121.51,122.22,123.60,124.20,128.25,128.60,130.65 (2C), 131.45,132.68,138.47 (2C), 152.51 (2C), 153.87,163.76,167.69.
High resolution mass spectrum measures: Calcd. [M-I]
-: 733.3633; Foundvalue [M-I]
-: 733.3623.
A kind of testing method for water-soluble location lysosomal Ratio-type pH fluorescent probe, its step: prepare above-mentioned water-soluble location lysosomal Ratio-type pH fluorescent probe, solution (ethanol/Britton--Robinson buffered soln at various ph values, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), try its responding ability to different pH value by Tissue with Spectrofluorometric;
Quantitatively AgNO3 is added respectively containing in the solution of pH fluorescent probe in acid condition to above-mentioned, Al (NO3) 3, Ba (NO3) 2, Cd (NO3) 2, Co (NO3) 2, Cu (NO3) 2, Fe (NO3) 3, HgCl2, Ni (NO3) 2, the aqueous solution of Pb (NO3) 2, Zn (NO3) 2, tests its immunity from interference;
Use the above-mentioned pH fluorescent probe of different concns to cultivate Hela cell, carry out fluorescence imaging respectively, fluorescence intensity change in observation of cell,
Use above-mentioned pH fluorescent probe and commercial lysosome probe Lysosensor GreenDND-189 to dye altogether to Hela cell of living, carry out dyeing location by fluorescence imaging,
Use the above-mentioned pH fluorescent probe of the different pH value of finite concentration to cultivate Hela cell, observe the change of fluorescence intensity and red fluorescence/blue-fluorescence ratio,
Use certain density above-mentioned pH fluorescent probe to cultivate Hela cell, under the continuous agitation of laser, observe the change of the ratio of blue-fluorescence/red fluorescence, test the light stability of this probe in Hela cell,
After using certain density above-mentioned pH fluorescent probe to cultivate Hela cell 6h, detection probes is on the impact of Hela cell survival rate.
Mensuration embodiment 1 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
Preparation 20mL above-mentioned fluorescent probe RMPM (10 μMs) solution at various ph values (ethanol/Britton--Robinson buffered soln, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), try fluorescence spectrum by Tissue with Spectrofluorometric.Fig. 2 (a) and the fluorescence intensity ratio (I of probe in pH value (5.6-7.2) region
580/ I
426) Fig. 2 (b).
Result shows: for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM be that in the solution of 5.8 and 6.8, fluorescence intensity change is remarkable at pH, fluorescent emission compares I
580/ I
4264 minutes time, reach peak and at room temperature keep stable, this shows that RMPM can to pH value change response fast, and accuracy in detection is high.(Fig. 2,4)
Mensuration embodiment 2 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
To above-mentioned solution (ethanol/Britton-Robinson buffered soln containing pH fluorescent probe RMPM (10 μMs) under pH value is 5.6 and 7.2 conditions, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio) in add AgNO3 respectively, Al (NO3) 3, Ba (NO3) 2, Cd (NO3) 2, Co (NO3) 2, Cu (NO3) 2, Fe (NO3) 3, HgCl2, Ni (NO3) 2, Pb (NO3) 2, the aqueous solution of Zn (NO3) 2, the concentration of all metallic cations is 100 μMs, the fluorescence intensity ratio I of test in the solution containing different metal ion
580/ I
426(λ
ex=340nm, Fig. 3).
Result shows: almost do not respond above-mentioned metal ion under pH=5.6 and pH=7.2 condition for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM, prove that this probe is to H
+there is very high selectivity.Probe RMPM is to the strong interference immunity of common metal ion.
Mensuration embodiment 4 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
Hela cell and probe RMPM (1,2 or 5 μM) cultivate 2h under 37 DEG C of conditions.Observe under laser confocal microscope.Fixing excitation wavelength is 405nm, collects the fluoroscopic image (400-555nm) that emission band is respectively blue channel, the fluoroscopic image (560-700nm) of red channel.Fluorescence intensity blue and red in Hela cell all increases in the increase along with concentration and probe concentration, and this shows have for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM the ability (Fig. 5) dyeed by viable cell.
Mensuration embodiment 5 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
Hela cell and probe RMPM (5 μMs) cultivate 2h at 37 DEG C, then cultivate 1h further with the fresh culture containing Lysosensor GreenDND-189 (1 μM).(a) and the green fluorescence photo (Fig. 6 b) of Lysosensor GreenDND-189, by two photos superpositions, blue and green overlapping region shows cyan (Fig. 6 c) to Fig. 6 to obtain the blue fluorescence photograph of probe RMPM respectively.Result shows that two photo pigmented section can overlap well, and orientation factor is 0.931 altogether, namely can to lysosome selected marker (Fig. 6) for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM.
Mensuration embodiment 6 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
By Hela cell respectively at the RMPM(4.00 μM of different pH value (5.50,6.00,6.50,7.00), 5.00 μMs, 6.00 μMs and 7.00 μMs) middle cultivation 2h.Then the Hela cell processed is cultivated 0.5h in containing the PBS damping fluid of 10 μMs of polyetherin As.From the image of Laser Scanning Confocal Microscope shooting (Fig. 7 a), when pH value is increased to 7.00 from 5.50, the blue-fluorescence of imidazo [1,5-α] pyridine groups (first row) is almost constant, and the red fluorescence of rhodamine group (secondary series) declines simultaneously.And the ratio of red fluorescence/blue-fluorescence is increased to 7.00 from 5.50 and increases (Fig. 7 b) along with pH value.
Mensuration embodiment 7 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
After Hela cell and same procedure process above, at λ
exthe laser of=405nm is at different time (30S, 60S, 90S, 120S, 180S) under continuous agitation, from the image of Laser Scanning Confocal Microscope shooting, the ratio of blue-fluorescence/red fluorescence is constant (Fig. 8) substantially, shows the light stability had in Hela cell for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM.
Mensuration embodiment 8 for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM:
After Hela cell and probe RMPM (5 μMs) cultivate 6h at 37 DEG C, detect Hela cell survival rate with mtt assay, result shows do not have toxicity (Fig. 9) for water-soluble location lysosomal Ratio-type pH fluorescent probe and terminated compound RMPM to cell under experimental conditions.
The present invention has lower feature:
1, owing to devising Ratio-type pH fluorescent probe, the fluorescence intensity change of two emission wavelengths can be detected simultaneously, eliminate ectocine by self calibration, improve detection accuracy.。
2, owing to devising Ratio-type pH fluorescent probe, have excellent water-soluble, strong to metal ion immunity from interference.
3, owing to devising Ratio-type pH fluorescent probe, by the transmission ofenergy of FRET donor to the acceptor being in ground state, this process can reduce the error of fluoroscopic examination.
4, owing to devising the new compound structure of Ratio-type pH fluorescent probe, test shows that this compound structure feature achieves good technique effect.
5, owing to devising technical characteristic of the present invention, in the effect of the set separately and each other of technical characteristic, shown by test, property indices of the present invention is that existing property indices wants high, has good marketable value by assessment.
Or the akin technical characteristic identical with the chemical structural formula of Ratio-type pH fluorescent probe also having other is all one of embodiments of the invention, and each technical characteristic of the above embodiment can combine arbitrarily, for meeting the requirement of patent law, patent regulation and guidelines for examination, no longer the embodiment of all possible combination of each technical characteristic in above-described embodiment is all described.
Above-described embodiment is a kind of way of realization for water-soluble location lysosomal Ratio-type pH fluorescent probe, its preparation method, application and testing method provided by the present invention; according to other distortion of scheme provided by the present invention; the composition increased or reduce wherein or step; or the present invention is used for other the technical field close with the present invention, all belongs to protection scope of the present invention.
Claims (10)
1., for a water-soluble location lysosomal Ratio-type pH fluorescent probe, it is characterized in that: its chemical structural formula is shown below:
。
2. according to claim 1 for water-soluble location lysosomal Ratio-type pH fluorescent probe, it is characterized in that: have:
Nuclear magnetic resonance hydrogen spectruming determining result:
1hNMR (DMSO-d
6, 300MHz), δ (ppm)=8.71-8.75 (m, 1H), 8.67 (d, J=7.5Hz, 1H), 8.14 (s, 1H), 7.80 (t, J=4.5Hz, 1H), 7.51 (t, J=4.5Hz, 2H), 7.39 (dd, J=1.5Hz, J=7.5Hz, 1H), 7.00 (t, J=7.5Hz, 1H), 6.30-6.43 (m, 6H), 4.03 (s, 3H), 3.39-3.48 (m, 4H), 3.18-3.32 (m, 10H), 1.56-1.64 (m, 2H), (1.39 q, J=7.5Hz, 2H), 0.97-1.09 (m, 12H), 0.91 (t, J=7.5Hz, 3H)
Mr carbon spectrum measurement result:
13cNMR (DMSO-d
6, 75MHz), δ (ppm)=12.31 (4C), 13.48 (2C), 21.66 (2C), 25.12,28.57,29.96,43.51 (3C), 64.31,64.31,97.27 (2C), 104.74 (2C), 108.07 (2C), 110.93 (2C), 116.01,119.24,121.51,122.22,123.60,124.20,128.25,128.60,130.65 (2C), 131.45,132.68,138.47 (2C), 152.51 (2C), 153.87,163.76,167.69
High resolution mass spectrum measurement result: Calcd. [M-I]
-: 733.3633; Foundvalue [M-I]
-: 733.3623,
Compound.
3. for an its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe, it is characterized in that: its step:
Intermediate compound III, HOBT(1-hydroxybenzotriazole), EDC [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride], triethylamine be dissolved in DMF and obtain in intermediate solution, intermediate compound V is joined in intermediate solution, be obtained by reacting finalization compound RMPM
Wherein: intermediate compound III chemical structural formula is shown below:
；
Intermediate compound V chemical structural formula is shown below:
。
4. its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe according to claim 3, is characterized in that: its step:
Intermediate compound III 1.0-1.4mmol, HOBT1.0-1.4mmol, EDC1.0-1.4mmol, triethylamine 1.8-2.2mmol is dissolved in DMF18-22mL, stirring at room temperature 0.8-1.2h under nitrogen protection, obtain intermediate solution, then intermediate compound V 0.8-1.2mmol is joined in intermediate solution, under nitrogen protection, stirring at room temperature 18-22h, silica-gel plate is followed the tracks of and is determined reaction end, reaction solution reduction vaporization is concentrated to obtain thick terminated compound, extract in thick terminated compound methylene dichloride and water, collect organic phase, dry, concentrated, then purification by column chromatography is passed through, the faint yellow solid obtained and terminated compound RMPM, yield 45.2%, fusing point: 197-201 DEG C.
5. its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe according to claim 4, is characterized in that: its step:
3-butyl-1-chlorine imidazo (1,5-α) pyridine-7-carboxylic acid ethyl ester and intermediate compound I 8-12mmol and NaOH18-22mmol be dissolved in the mixing solutions of ethanol 18-22ml and water 13-17ml, after back flow reaction 1.8-2.2h, gained solution is poured in 180-220ml water, be neutralized to neutrality with dilute hydrochloric acid 1.8-2.2M, have yellow solid to separate out to filter, wash 2 × 5mL with water, in an oven after 50 DEG C of dryings, obtain yellow solid and intermediate compound II, yield is 90.8%
By intermediate compound II 8-12mmol and CH
3i48-52mmol is dissolved in tetrahydrofuran (THF) 28-32ml solution, heating reflux reaction 7.8-8.2h under nitrogen protection, cool to room temperature; separate out yellow solid, by vacuum filtration, filter cake ethyl acetate 8-12ml washs 3 times; obtain yellow solid and intermediate compound III, yield is 73.0%
Intermediate compound V can pass through to be synthesized by the intermediate compound IV of method in the literature and reacting ethylenediamine,
Wherein: intermediate compound IV chemical structural formula is shown below:
。
6. its preparation method for water-soluble location lysosomal Ratio-type pH fluorescent probe according to claim 3, is characterized in that: the synthetic route for its preparation method of water-soluble location lysosomal Ratio-type pH fluorescent probe is as follows:
。
7. the application for water-soluble location lysosomal Ratio-type pH fluorescent probe.
8. a kind of application for water-soluble location lysosomal Ratio-type pH fluorescent probe according to claim 6, is characterized in that: a kind of is the application of the fluorescent probe of 5.8 to 6.8 for water-soluble location lysosomal Ratio-type pH fluorescent probe as measuring pH.
9. a kind of application for water-soluble location lysosomal Ratio-type pH fluorescent probe according to claim 6, is characterized in that: a kind of for the application of water-soluble location lysosomal Ratio-type pH fluorescent probe as the Ratio-type pH fluorescent probe in Hela cell.
10. the testing method for water-soluble location lysosomal Ratio-type pH fluorescent probe, its step: prepare above-mentioned water-soluble location lysosomal Ratio-type pH fluorescent probe, solution (ethanol/Britton--Robinson buffered soln at various ph values, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), try its responding ability to different pH value by Tissue with Spectrofluorometric;
Quantitatively AgNO3 is added respectively containing in the solution of pH fluorescent probe in acid condition to above-mentioned, Al (NO3) 3, Ba (NO3) 2, Cd (NO3) 2, Co (NO3) 2, Cu (NO3) 2, Fe (NO3) 3, HgCl2, Ni (NO3) 2, the aqueous solution of Pb (NO3) 2, Zn (NO3) 2, tests its immunity from interference;
Use the above-mentioned pH fluorescent probe of different concns to cultivate Hela cell, carry out fluorescence imaging respectively, fluorescence intensity change in observation of cell,
Use above-mentioned pH fluorescent probe and commercial lysosome probe Lysosensor GreenDND-189 to dye altogether to Hela cell of living, carry out dyeing location by fluorescence imaging,
Use the above-mentioned pH fluorescent probe of the different pH value of finite concentration to cultivate Hela cell, observe the change of fluorescence intensity and red fluorescence/blue-fluorescence ratio,
Use certain density above-mentioned pH fluorescent probe to cultivate Hela cell, under the continuous agitation of laser, observe the change of the ratio of blue-fluorescence/red fluorescence, test the light stability of this probe in Hela cell,
After using certain density above-mentioned pH fluorescent probe to cultivate Hela cell 6h, detection probes is on the impact of Hela cell survival rate.
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