CN110183482A - A kind of near infrared fluorescent probe and its preparation method and application monitoring lysosomal pH - Google Patents
A kind of near infrared fluorescent probe and its preparation method and application monitoring lysosomal pH Download PDFInfo
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- CN110183482A CN110183482A CN201910567239.XA CN201910567239A CN110183482A CN 110183482 A CN110183482 A CN 110183482A CN 201910567239 A CN201910567239 A CN 201910567239A CN 110183482 A CN110183482 A CN 110183482A
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Classifications
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- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/12—Organo silicon halides
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- C07F7/122—Preparation or treatment not provided for in C07F7/14, C07F7/16 or C07F7/20 by reactions involving the formation of Si-C linkages
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- C—CHEMISTRY; METALLURGY
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- 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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- 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
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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
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- C—CHEMISTRY; METALLURGY
- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
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- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
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- C—CHEMISTRY; METALLURGY
- 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
- 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
- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
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- Health & Medical Sciences (AREA)
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Abstract
This patent discloses a kind of near infrared fluorescent probes and its preparation method and application for monitoring lysosomal pH, belong to technical field of analytical chemistry.Technical solution of the present invention main points are as follows: a kind of near infrared fluorescent probe for monitoring lysosomal pH, structural formula are as follows:The application of pH the invention further particularly discloses the preparation method of the near infrared fluorescent probe of the monitoring lysosomal pH and its in selective enumeration method water environment, biological cell system.Near infrared fluorescent probe of the invention has many advantages, such as the good transmitting of near infrared region, selectivity, good light stability, good reversibility and excellent lysosome targeting ability.
Description
Technical field
The present invention relates to a kind of near infrared fluorescent probes and its preparation method and application for monitoring lysosomal pH, belong to analysis
Technical field of chemistry.
Background technique
Lysosome is protein, nucleic acid, more as acidity (pH is about 3.8-5.5) organelle important in eukaryocyte
The decomposition place of the large biological molecules such as sugar, referred to as in cell " digestive organs ".Lysosome rises emphatically in many life process
The effect wanted, such as the growth of endocytosis, cell and apoptosis, autophagy, ion metabolism, oxidative stress.The unusual fluctuations meeting of lysosomal pH
Cause lysosome dysfunction, it is closely related with the diseases such as lysosome storage diseases, cancer.Therefore, exploitation being capable of real-time monitoring
The analytical technology of lysosomal pH is of great significance.
Fluorescent molecular probe is because its is easy to operate, fast response time, selectively height, high sensitivity, spatial and temporal resolution height etc.
Advantage, it has also become the indispensable tool in bio-imaging field.In recent years, researcher develops many for lyase in succession
The fluorescent molecular probe of the monitoring of body pH.But the excitation of most of probes in these probes and the shorter (λ of launch wavelengthex
λem), it is had some limitations in bio-imaging, there is that light injury to biological sample is big, tissue penetration depths are smaller, back
The problems such as interference of scape fluorescence is big.And near infrared fluorescent probe (near-infrared fluorescent probe) is right by its
The advantages that light injury of biological sample is small, tissue penetration depths are larger, background fluorescence interference is small, becomes fluorescent molecular probe field
Research hotspot.Based on this, researcher successively develops some based on traditional near-infrared cyanine dye (cyanine dyes)
PH fluorescence probe.But the photostability of the above-mentioned pH near-infrared probe based on near-infrared cyanine dye design is poor, it is difficult to
For a long time, the fluorescence signal of accurate stable is repeatedly obtained in imaging.And silicon rhodamine is a kind of tool newly reported in recent years
There is the nir dye of excellent light stability, photostability will be much better than cyanine dyes, be widely used in various analysis detections
The design of the near infrared fluorescent probe of object.In addition, silicon rhodamine is similar with common rhodamine, it is easy to spirocyclization, facilitates structure
Build the fluorescence probe for different test objects.
Summary of the invention
For the near infrared fluorescent probe problem encountered and status of current monitoring lysosome pH value, the present invention provides
A kind of near infrared fluorescent probe monitoring lysosomal pH, it is fixed which is targeted using morpholine group as lysosome
Position group, for the imaging of pH in cytase body, which has near infrared emission, the good, good light stability of selectivity, reversible
Property good, lysosome targeting ability it is excellent the features such as.
The present invention also provides the preparation method of the near infrared fluorescent probe of above-mentioned monitoring lysosomal pH and its in selectivity
The application of pH in detection water environment, biological cell body.
The present invention adopts the following technical scheme that solve above-mentioned technical problem, a kind of near-infrared fluorescent monitoring lysosomal pH
Probe, it is characterised in that the structural formula of the near infrared fluorescent probe is as follows:
The preparation method of the near infrared fluorescent probe of monitoring lysosomal pH of the present invention, it is characterised in that specific steps
Are as follows:
Step S1: in -78 DEG C, under protection of argon gas, by the bromo- n,N-Dimethylaniline of 6g 3- and 60 milliliters of anhydrous tetrahydros
Furans is added into 250 milliliters of dry round-bottomed flasks, makes it dissolve within magnetic agitation 5 minutes, then by 13 milliliters of molar concentrations
Hexane solution for the n-BuLi of 2.4mol/L is added dropwise in reaction solution, is reacted 2 hours after being added dropwise in 0 DEG C, then will
2.2 milliliters of dichlorodimethylsilane are dissolved in 10 milliliters of anhydrous tetrahydro furans, are then added dropwise in above-mentioned reaction solution, are added dropwise
After react to room temperature and be stirred overnight, add 50 milliliters of water quenching reactions, and reaction solution is extracted with ether, anhydrous sodium sulfate is done
Dry, decompression obtains crude product after being spin-dried for solvent, crude product is obtained compound 1 with silica gel column purification, structural formula is as follows:
Step S2: 500mg compound 1,1260mg 2- carboxyl benzaldehyde and 37.5mg copper bromide are added to 100 milliliters
In glass heavy wall pressure pipe, the cooled to room temperature after 140 DEG C of heating stirrings are reacted 5 hours is then molten by reaction mixture
In methylene chloride, is washed three times with the NaOH solution that mass concentration is 10%, recycle and be spin-dried for methylene chloride and mutually slightly produced
Crude product is obtained compound 2 with silica gel column purification by product, and structural formula is as follows:
Step S3: 443mg compound 2,20mL dry 1,2- dichloroethanes and 2mL are added in 100mL round-bottomed flask
Reaction solution in flask is heated to 85 DEG C and flowed back 4 hours by phosphorus oxychloride, and vacuum distillation removes solvent and obtains reaction residue, will
Reaction residue is dissolved in the dry acetonitrile of 20mL, and 5mL triethylamine is added, and then continues to be added dropwise thereto and contains 650mg 4-
The acetonitrile solution of the 10mL of (2- aminoethyl)-morpholine, reaction solution is stirred at room temperature overnight, removes solvent at reduced pressure conditions
Afterwards, with 50mL CH2Cl2Residue is dissolved, 50mL is saturated NaCl aqueous solution and washs 3 times, anhydrous Na2SO4It is dry, Rotary Evaporators
Solvent is evaporated off and obtains crude product, crude product is obtained into compound 3 with silica gel column purification, structural formula is as follows:
Step S4: at room temperature, 105mg compound 3 being dissolved in 5mL dry tetrahydrofuran, a small amount of that 76mg is repeatedly added
Lithium Aluminium Hydride after being stirred to react 6 hours at room temperature, is added the quenching reaction of 5mL methanol, reaction solution is evaporated under reduced pressure after removing solvent
Residue is obtained, residue is obtained into target fluorescent probe compound Lyso-NIR-pH with silica gel column purification.
The near infrared fluorescent probe of monitoring lysosomal pH of the present invention is in selective enumeration method water environment, biological cell body
The application of pH in system, wherein detection includes fluorescence detection in aqueous solution, cell imaging detection.
Compared with the prior art, the invention has the following beneficial effects: the synthesis of (1) near infrared fluorescent probe compares
It is easier to, and last handling process is relatively easy；(2) near infrared fluorescent probe realizes the highly selective quick detection of pH, has
Resist the ability of other materials interference in life entity；(3) the near infrared fluorescent probe fluorescence probe has nearly excellent lysosome
Targeting ability, the transmitting of near infrared region, excellent photostability can be applied to the image checking in Cytolysosome.This is close
Infrared fluorescence probes are steady to the light injury of biological sample, raising light by autofluorescence background interference, reduction in reduction life entity
The features such as qualitative, to obtain more accurate and stable optical signalling and imaging effect.Therefore, the near-infrared fluorescent in the present invention
Probe has broad application prospects in pH Imaging: Monitoring field, to lysosomal pH organism physiology and pathologic process effect
The research of mechanism is of great significance.
Detailed description of the invention
Fig. 1 is fluorescence spectrum of the fluorescent probe compounds Lyso-NIR-pH under condition of different pH made from embodiment 1
Figure；
Fig. 2 is that UV, visible light of the fluorescent probe compounds Lyso-NIR-pH made from embodiment 1 under condition of different pH is inhaled
Receive spectrogram；
It is glimmering at 675nm that Fig. 3, which is fluorescent probe compounds Lyso-NIR-pH made from embodiment 1 in launch wavelength,
The graph of relation that luminous intensity changes with pH；
Fig. 4 be fluorescent probe compounds Lyso-NIR-pH made from embodiment 1 under the conditions of pH=7.4 to various analyses
The response condition (from left to right) of object: 1, H+(pH=5)；2,Na+；3,K+；4,Ca2+；5,Mg2+；6,Fe3+；7,Cu2+；8,Zn2+；
9、Mn2+；10,Ni2+；11,Cd2+；12,Co2+；13,NH4+；14,Ac-；15,CO3 2-；16,SO4 2-；17,F-；18,Br-；19,I-；
20、S2O3 -；21,NO2 -；22,H2PO4-；23,HPO4 2-；24,Glutathione；25,Arginine；26,Valine；27,
Tryptophan；28,Cysteine；29,Glycine；30,Homocysteine.
Fig. 5 is the reversible response condition of pH of fluorescent probe compounds Lyso-NIR-pH made from embodiment 1.
Fig. 6 is lysosome common location of the fluorescent probe compounds Lyso-NIR-pH made from embodiment 1 in HeLa cell
Experiment；
Fig. 7 is that the photostability assessment of fluorescent probe compounds Lyso-NIR-pH made from embodiment 1 in the cell is real
It tests.
Fig. 8 is item of the fluorescent probe compounds Lyso-NIR-pH made from embodiment 1 in different pH value (pH=4,5,6)
Fluorescence imaging figure under part.
Fig. 9 is before drug chloroquine is added in HeLa cell in fluorescent probe compounds Lyso-NIR-pH made from embodiment 1
Fluorescence imaging situation afterwards.
Figure 10 is the cell that fluorescent probe compounds Lyso-NIR-pH is induced in agent dexamethasone made from embodiment 1
Fluorescence imaging situation in apoptotic process.
Specific embodiment
Above content of the invention is described in further details by the following examples, but this should not be interpreted as to this
The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on above content of the present invention belong to this hair
Bright range.
Embodiment 1
The synthesis of fluorescent probe compounds Lyso-NIR-pH
(1) synthesis of compound 1
In -78 DEG C, under protection of argon gas, the bromo- n,N-Dimethylaniline of 6g 3- and 60 milliliters of anhydrous tetrahydro furans are added
It into 250 milliliters of dry round-bottomed flasks, makes it dissolve within magnetic agitation 5 minutes, is then 2.4mol/ by 13 milliliters of molar concentrations
The hexane solution of the n-BuLi of L is added dropwise in reaction solution, is reacted 2 hours after being added dropwise in 0 DEG C, then by 2.2 milliliter two
Chlorodimethylsilane is dissolved in 10 milliliters of anhydrous tetrahydro furans, is then added dropwise in above-mentioned reaction solution, reacted after being added dropwise to
Room temperature is simultaneously stirred overnight, and adds 50 milliliters of water quenching reactions, and reaction solution ether is extracted (50 milliliters × 2), by the second of extraction
Ethereal solution washs (50 milliliters × 3), anhydrous Na with saturation NaCl aqueous solution2SO4It is dry, and solvent is evaporated off with Rotary Evaporators and obtains
Crude product, by crude product silica gel column purification, silica gel particle size is 200-300 mesh, and eluant, eluent volume proportion is petroleum ether/second
Acetoacetic ester=80:1 obtains compound 1, and yellow oil, 3.35g, yield 75%, synthetic route is as follows:
(2) synthesis of compound 2
By 500mg compound 1 (1.68mmol), 1260mg 2- carboxyl benzaldehyde (8.4mmol) and 37.5mg copper bromide
(0.168mmol) is added in 100 milliliters of glass heavy wall pressure pipes, is placed into oil bath pan after tube sealing in 140 DEG C of heating stirrings 5
Reaction mixture, is then dissolved in 50 milliliters of methylene chloride by cooled to room temperature after hour, is 10% with mass concentration
NaOH solution wash (50 milliliters × 3), remove the acidic by-products such as unreacted 2- carboxyl benzaldehyde, the dichloromethane that will be obtained
Alkane mutually uses anhydrous Na2SO4It is dry, and solvent is evaporated off with Rotary Evaporators and obtains crude product, by crude product silica gel column purification, silica gel
Granular size is 200-300 mesh, and eluant, eluent volume proportion is petrol ether/ethyl acetate=2:1, obtains compound 2, and green is solid
Body, 0.33g, yield 45%, synthetic route is as follows:
(3) synthesis of compound 3
In 100mL round-bottomed flask, dry 1, the 2- dichloroethanes of 443mg compound 2,20mL, 2mL trichlorine oxygen is first added
Phosphorus.Reaction solution in flask is heated to 85 DEG C to flow back 4 hours, vacuum distillation removes solvent, obtains reaction residue.It will react residual
Excess is dissolved in the dry acetonitrile of 20mL, and 5mL triethylamine is added, and then continues to be added dropwise thereto and contains 650mg 4- (2- ammonia second
Base)-morpholine 10mL acetonitrile solution.Reaction solution is stirred at room temperature overnight, after removing solvent at reduced pressure conditions, is used
50mL CH2Cl2Residue is dissolved, and washs (50 milliliters × 3), anhydrous Na with saturation NaCl aqueous solution2SO4It is dry, then use
Rotary Evaporators are evaporated off solvent and obtain crude product.By crude product silica gel column purification, silica gel particle size is 200-300 mesh, elution
Agent proportion is methylene chloride/methanol=25:1, obtains compound 3, celadon solid chemical compound, 277.0mg, yield 51.3%.
Its synthetic route is as follows:
(4) synthesis of compound 4
At room temperature, 105mg compound 3 is dissolved in 5mL dry tetrahydrofuran, a small amount of total amount that is repeatedly added is 76mg tetra-
Hydrogen aluminium lithium.After being stirred to react 6 hours at room temperature, the quenching reaction of 5mL methanol is added.Reaction solution is evaporated under reduced pressure after removing solvent and is obtained
Residue silica gel column purification is obtained into target fluorescent probe compound Lyso-NIR-pH, 38.0mg, yield to residue
36.2%.Its synthetic route is as follows:
Embodiment 2
The measurement of fluorescent probe compounds Lyso-NIR-pH fluorescence spectra under condition of different pH
The measurement of fluorescence spectrum measurement in 40mM Britton-Robinson buffer solution (containing 1%DMSO).It will
Lyso-NIR-pH fluorescence probe is dissolved in dimethyl sulfoxide (DMSO), and 500 μM of stock solutions are made.Test solution Lyso-NIR-pH
(5.0 μM) are diluted by above-mentioned 500 μM of stock solutions to be made.Different pH is obtained by the way that the HCl or NaOH of micro isoconcentration is added.Examination
It tests liquid and saves 30min at room temperature, measure fluorescence spectrum under 620nm excitation wavelength.Fluorescence emission spectral limit be 640~
800nm, excitation seam are 3nm, and transmitting seam is 3nm.Fluorescence spectrum is visited as shown in Figure 1, when the pH value of BR buffer is greater than 7.4
Needle Lyso-NIR-pH is substantially without fluorescence, because it is stable non-fluorescence spirane structure.When pH drops to 3 from 7.4
When, occur the near-infrared fluorescent signal significantly increased at 675nm, this is because H+The helical ring of induction is opened, and fluorescence is strong
Degree increases by more than 1400 times.Fluoremetry instrument used is Perkin Elmer LS55 sepectrophotofluorometer.
Embodiment 3
The measurement of fluorescent probe compounds Lyso-NIR-pH ultraviolet-visible absorption spectroscopy figure under condition of different pH
Fig. 2 is the ultraviolet-visible absorption spectroscopy figure of probe Lyso-NIR-pH (5 μM) under the conditions of pH 7.4-pH 3.From figure
2 can be seen that the reduction with pH, it can be observed that an absorption peak gradually increased at 655nm.This shows probe
Lyso-NIR-pH produces response to pH, and spirane structure is in proton H+It is opened under effect, absorbs and increase.UV, visible light is inhaled
The instrument for receiving spectroscopic assay is TU-1900 type ultraviolet-uisible spectrophotometer (Beijing Purkinje General
Instrument Co.,Ltd.)。
Embodiment 4
The pH titration curve of fluorescent probe compounds Lyso-NIR-pH
Fig. 3 is the pH titration curve of probe Lyso-NIR-pH.From figure 3, it can be seen that with reduction (the pH 7.4- of pH
PH3), fluorescence intensity of the probe at 675nm gradually increases.We are by the fluorescence intensity of probe and pH according to Henderson-
Hasselbalch equation maps to obtain Fig. 3, and further acquires probe Lyso-NIR-pH to the pK of pHaIt is 4.63.It is molten
The pH of enzyme body is generally 3.8-5.5, and the pK of probe Lyso-NIR-pHaJust within the scope of this, be conducive to lysosomal pH
Fluorescence monitoring imaging.
Embodiment 5
The selectivity of fluorescent probe compounds Lyso-NIR-pH is investigated
Selectivity is to assess an important indicator of fluorescence probe performance.As shown in figure 4, Lyso-NIR-pH (5 μM) is in pH
Fluorescence intensity when=7.4 is shown, 20 times of equivalent Common Cations (Na are being added+、K+、Ca2+、Mg2+、Fe3+、Cu2+、Zn2+、
Al3+、Mn2+、Ni2+、Cd2+、Co2+、NH4 +) after, fluorescence is without obviously increasing.In addition, when being separately added into 20 times of equivalent Common Anions
(Ac afterwards-、CO3 2-、SO4 2-、F-、I-、S2O3 -、NO2-、Cl-、Br-、H2PO4-、HPO4 2-), the fluorescence intensity of Lyso-NIR-pH increases
Also it can be neglected.200 times of various amino acid of equivalent (valine, tryptophan, cysteine, glycine, homocysteine) with
Glutathione also can be ignored the fluorescence interference of Lyso-NIR-pH.These results indicate that Lyso-NIR-pH is to acidity
PH has the fluorescence response of specificity, and other analytes, without significantly interfering with, can satisfy pH in practical biological sample and monitor to probe
Demand.
Embodiment 6
The invertibity of fluorescent probe compounds Lyso-NIR-pH is investigated
Then, we have investigated the invertibity (Fig. 5) of Lyso-NIR-pH.It can be seen that probe Lyso-NIR-pH (5 μ
M the pH of buffer system locating for) (pH 7 arrives pH 3.5) after cyclic switching three times, fluorescence intensity level remain to maintain initial
Value 90% or more, song results showed that probe Lyso-NIR-pH to pH have preferable invertibity, can be used in the more of pH
Secondary measurement.
Embodiment 7
The lysosome common location of fluorescent probe compounds Lyso-NIR-pH is tested
In order to investigate Lyso-NIR-pH probe in cell to the targeting ability of lysosome, we utilize probe Lyso-
NIR-pH (5 μM) and commercialization lysosome dyestuff LysoTracker Green DND-26 (500nM) and mitochondrial dye Mito
(1 μM) of Tracker Green FM comparison, has carried out common location experiment.Firstly, we are carrying out lysosome to HeLa cell
Common location experiment.As shown in figures 6 a-6d, in green channel it can be observed that have green fluorescence (510-550nm) issue (Fig. 6 a),
This is the light of LysoSensor Green DND-26 transmitting, and has been observed that red fluorescence (650- in red channel
(Fig. 6 b) 720nm) is issued, this is the feux rouges that probe Lyso-NIR-pH is issued, and the two is handled with software it can be concluded that two
The common location coefficient of kind dyestuff is 0.90 (Fig. 6 c).Meanwhile linear point of the fluorescence of fluorescence probe and commercialization lysosome dyestuff
Cloth shows apparent synchronism (Fig. 6 d).This illustrates that probe Lyso-NIR-pH is mainly gathered in lysosome, can navigate to
In lysosome.Then, We conducted the common locations of probe and commercialization mitochondrial dye to test (6e-6h).As shown, visiting
Needle and commercialization mitochondria positioning agent fluorescence are without obvious overlapping (Fig. 6 g), and linear distribution is asynchronous (Fig. 6 h), by Fig. 6 g through soft
Part handles to obtain common location coefficient between the two and there was only 0.43, this confirms that Lyso-NIR-pH is targeted with lysosome from further
Property, rather than Mitochondrially targeted property.
Embodiment 8
The photostability of fluorescent probe compounds Lyso-NIR-pH is investigated
By with traditional nir dye Cy5-N3Comparison, we have investigated fluorescent probe compounds Lyso-NIR-pH
Photostability (Fig. 7).As shown, the lasting excitation by 50 times is imaged, fluorescent probe compounds Lyso-NIR-pH's is glimmering
Luminous intensity remains within the 88% of initial value, and traditional nir dye Cy5-N3Fluorescence intensity then show significantly
Decline (for the 10% of initial value).This result shows that fluorescent probe compounds Lyso-NIR-pH have preferable photostability,
It can satisfy the demand being imaged for a long time in vivo, obtain stable fluorescence signal.Excitation wavelength is 635nm, power 20%.
Embodiment 9
Fluorescent probe compounds Lyso-NIR-pH grinds the imaging for the Cytolysosome pH value that different pH buffer solutions act on
Study carefully
In HeLa cell, the fluorescence imaging situation that probe Lyso-NIR-pH changes Cytolysosome pH value is investigated.Tool
Steps are as follows for gymnastics work: thin with Lyso-NIR-pH (5 μM) 37 DEG C of incubation A549 cells and HeLa in PBS buffer solution (pH7.4)
Then born of the same parents 30min is containing 10 μM of nigericins (Nigericin, a kind of H+/K+Ionophore makes intracellular and extracellular
PH homogenization) different pH value (4.0,5.0 and 6.0) buffer in, at 37 DEG C, be incubated for 30min.Then burnt with copolymerization
Microscope is imaged, and imaging results are as shown in Figure 8.It can be seen that fluorescence signal gradually subtracts with the raising of system pH
It is small.Excitation wavelength is 635nm, and red channel wavelength capture range is 650-720nm.
Embodiment 10
The imaging contexts for the lysosomal pH variation that fluorescent probe compounds Lyso-NIR-pH induces drug chloroquine
Fluorescent probe compounds Lyso-NIR-pH (5 μM) is applied to the lysosomal pH variation of alkaline drug chloroquine induction
Monitoring (Fig. 9).As shown, the fluorescence intensity of cell is decreased obviously after (100 μM) additions of chloroquine in 150 seconds, this knot
Fruit illustrates alkaline drug chloroquine induction of the raising of lysosomal pH, and lead to probe is in spirane structure, and fluorescence is remarkably decreased, also table
The ability of pH in probe real time monitoring lysosome is illustrated.
Embodiment 11
Imaging contexts of the fluorescent probe compounds Lyso-NIR-pH to the lysosomal pH variation of apoptosis-inducing
Finally, probe Lyso-NIR-pH (5 μM) is used for the monitoring (figure that lysosomal pH changes in apoptosis process
10).In apoptosis process, proton leakage can occur for lysosomal pH, and pH is caused to rise.As shown, when 2 μM of ground plugs are added
For meter Song (drug induced cell apoptosis) afterwards in 2 hours, apparent variation is had occurred in cell morphology, shows that cell has occurred really
Process of Apoptosis.At the same time, the fluorescence of cell is also gradually reduced.The above result shows that probe Lyso-NIR-pH is successfully
Realize the monitoring of lysosomal pH uphill process in apoptosis process.
Embodiment above describes basic principles and main features of the invention and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within
In the scope of protection of the invention.
Claims (3)
1. a kind of near infrared fluorescent probe for monitoring lysosomal pH, it is characterised in that the structural formula of the near infrared fluorescent probe is such as
Under:
2. a kind of preparation method of the near infrared fluorescent probe of monitoring lysosomal pH described in claim 1, it is characterised in that tool
Body step are as follows:
Step S1: in -78 DEG C, under protection of argon gas, by the bromo- n,N-Dimethylaniline of 6g3- and 60 milliliters of anhydrous ethers be added to
It in 250 milliliters of dry round-bottomed flasks, makes it dissolve within magnetic agitation 5 minutes, is then 2.4mol/L by 13 milliliters of molar concentrations
The hexane solution of n-BuLi be added dropwise in reaction solution, reacted 2 hours after being added dropwise in 0 DEG C, then by 2.2 milliliters of dichloros
Dimethylsilane is dissolved in 10 milliliters of anhydrous ethers, is then added dropwise in above-mentioned reaction solution, is reacted to room temperature simultaneously after being added dropwise
It is stirred overnight, adds 50 milliliters of water quenching reactions, and reaction solution ether is extracted, anhydrous sodium sulfate drying, decompression is spin-dried for solvent
After obtain crude product, crude product is obtained into compound 1 with silica gel column purification, structural formula is as follows:
Step S2: 500mg compound 1,1260mg2- carboxyl benzaldehyde and 37.5mg copper bromide are added to 100 milliliters of glass thickness
In wall pressure pipe, then reaction mixture is dissolved in dichloro by the cooled to room temperature after 140 DEG C of heating stirrings are reacted 5 hours
In methane, is washed three times with the NaOH solution that mass concentration is 10%, recycle and be spin-dried for methylene chloride and mutually obtain crude product, it will be thick
Product obtains compound 2 with silica gel column purification, and structural formula is as follows:
Step S3: in 100mL round-bottomed flask, 443mg compound 2,20mL dry 1,2- dichloroethanes and 2mL tri- is first added
Reaction solution in flask is heated to 85 DEG C and flowed back 4 hours by chlorethoxyfos, and vacuum distillation removes solvent and obtains reaction residue, will be anti-
It answers residue to be dissolved in the dry acetonitrile of 20mL, and 5mL triethylamine is added, then continue to be added dropwise thereto and contain 650mg4- (2- ammonia
Ethyl)-morpholine 10mL acetonitrile solution, reaction solution is stirred at room temperature overnight, at reduced pressure conditions remove solvent after, use
50mL CH2Cl2Residue is dissolved, 50mL is saturated NaCl aqueous solution and washs 3 times, anhydrous Na2SO4Dry, Rotary Evaporators are evaporated off
Solvent obtains crude product, crude product is obtained celadon solid chemical compound 3 with silica gel column purification, structural formula is as follows:
Step S4: at room temperature, 105mg compound 3 being dissolved in 5mL dry tetrahydrofuran, and it is 76mg tetra- that total amount, which is repeatedly added,
Hydrogen aluminium lithium, is added the quenching reaction of 5mL methanol after being stirred to react at room temperature 3 hours, reaction solution Rotary Evaporators are removed solvent
After obtain residue, residue is obtained into target fluorescent probe compound Lyso-NIR-pH with silica gel column purification.
3. the near infrared fluorescent probe of monitoring lysosomal pH described in claim 1 is in selective enumeration method water environment, biological cell
The application of pH in system, wherein detection includes fluorescence detection and cell imaging detection in aqueous solution.
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| CN113527350A (en) * | 2021-07-27 | 2021-10-22 | 大连理工大学 | Near-infrared lysosome fluorescence indicator and application |
| CN113527350B (en) * | 2021-07-27 | 2022-06-07 | 大连理工大学 | Near-infrared lysosome fluorescence indicator and application |
| US11584762B1 (en) | 2021-07-27 | 2023-02-21 | Dalian University Of Technology | Near-infrared fluorescent indicators for lysosome and application thereof |
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