CN106243085B - Double target spot fluorescence probes of copper ion and sulphion based on chinoline backbone and its preparation method and application - Google Patents

Double target spot fluorescence probes of copper ion and sulphion based on chinoline backbone and its preparation method and application Download PDF

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CN106243085B
CN106243085B CN201610621552.3A CN201610621552A CN106243085B CN 106243085 B CN106243085 B CN 106243085B CN 201610621552 A CN201610621552 A CN 201610621552A CN 106243085 B CN106243085 B CN 106243085B
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qlba
target spot
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CN106243085A (en
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蒋宇扬
谭英
谭春燕
刘海洋
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Shenzhen Graduate School Tsinghua University
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Abstract

The present invention provides copper ion and the double target spot fluorescence probes of sulphion based on chinoline backbone and its preparation method and application, which has structure shown in Formulas I.Double target spot fluorescence probes of the invention can be with efficient identification, detection copper ion and sulphion, and realizes identify both ions in the cell simultaneously.It is simple, convenient, it is reproducible, stability is high.

Description

The double target spot fluorescence probes of copper ion and sulphion based on chinoline backbone and its preparation side Method and application
Technical field
The present invention relates to biological fluorescent labeling fields, more particularly, to a kind of copper ion and sulphur based on chinoline backbone Double target spot fluorescence probes of ion and preparation method thereof and the application of double target spot fluorescence probes in cell imaging.
Background technique
The Copper in Body element overwhelming majority is with Cu2+Performance, while copper is the most abundant microelement in human body again One of.Copper ion plays vital role during physiological metabolism, for example, can serve as active oxygen removing toxic substances species, Neurotransmitters biosynthesis and degradation.However, the disorder of intracellular copper ion will seriously endanger human health, especially Serious neurodegenerative disease can be induced, such as Alzheimer, Menkes and Wilson disease.Equally, sulphion is answered extensively The fields such as the manufacture for sulfuric acid industry, chemical fertilizer production and dyestuff, cosmetics the metabolism of sulfide-oxidizing enzyme and contain in human body The metabolism of sulphur amino acid can all generate sulphion.The raising of sulphion concentration can also seriously affect the health of the mankind, example in human body It such as will cause human muscle's damage, pain, mucosal tissue damage, can cause to suffocate when serious.Therefore in detection human body cell Copper ion and sulphion have great significance.
Fluorescence detection has the advantages such as highly sensitive, highly selective, the simple, quick response of operation.Cell fluorescence imaging is made It is concerned for efficient detection means a kind of in biology and pharmaceutical science field.Although in recent years, a large amount of related copper ion, The fluorescence probe of sulphion largely reported, but there has been no can detect simultaneously both ions and be used for into the cell at Picture, the fluorescence probe for identifying both ions.
Summary of the invention
The object of the present invention is to provide a kind of copper ions based on chinoline backbone and the double target spot fluorescence probes of sulphion, and The preparation method and its application in cell imaging of double target spot fluorescence probes are provided.
Quinoline is a big conjugated system, can easily generate the electron transition of π to π *, quinoline radical derivative is not Only there is stronger fluorescence, and its heterocyclic nitrogen atom can participate in being coordinated, i.e., there is identification and fluorescent functional simultaneously.Of the invention Inventor designs according to this characteristic, has synthesized a simple chinoline backbone class probe, can be with efficient identification, detection copper ion And sulphion, and realize identify both ions in the cell simultaneously.It is simple, convenient, it is reproducible, stability is high.
The first aspect of the present invention is to provide a kind of copper ion based on chinoline backbone and the double target spot fluorescence probes of sulphion (hereinafter referred to as QLBA), double target spot fluorescence probes have structure shown in Formulas I:
The second aspect of the present invention is to provide the preparation method of above-mentioned double target spot fluorescence probes, this method comprises:
In the presence of an organic, compound shown in Formula II is contacted with quinaldinic acid acyl chlorides and is reacted, obtain Formulas I institute Show compound,
The third aspect of the present invention is to provide application of the double target spot fluorescence probes in cell imaging.
The features of the present invention is shown:
(1) fluorescence probe QLBA synthesis is simple.
(2) QLBA is to Cu2+With highly sensitive and highly selective recognition detection.
(3) QLBA-Cu obtained2+Compound can further identify sulphion.
(4) with identification of the probe to copper ion of the technological means detailed analysis such as high resolution mass spectrum, nuclear-magnetism, DET calculating Mechanism.
(5) copper ion and sulphion can be identified in living cells.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its Its purpose, feature and advantage will be apparent.
The hydrogen that Fig. 1 (a) and Fig. 1 (b) is QLBA is composed.
The carbon that Fig. 2 (a) and Fig. 2 (b) is QLBA is composed.
Fig. 3 (a) and Fig. 3 (b) is respectively QLBA in CH3Ultraviolet suction in OH-HEPES (20mM, pH 7.2) solution system Receive spectrogram and fluorescent emission spectrogram.
Fig. 4 (a) is the CH that 50 μM of different metal ions are added to QLBA (10 μM)3OH-HEPES (20mM, pH 7.2) Fluorescent emission spectrogram after solution.Fig. 4 (b) is QLBA (10 μM) in CH3In OH-HEPES (20mM, pH 7.2) solution with Cu2+ The fluorescent emission spectrogram of (30 μM) and other metal ions (30 μM), 1, QLBA；2,Cu2+；3,Cu2++Na+；4,Cu2++K+；5, Cu2++Mg2+；6,Cu2++Ca2+；7,Cu2++Co2+；8,Cu2++Zn2+；9,Cu2++Mn2+；10,Cu2++Fe3+；11,Cu2++Al3+； 12,Cu2++Cr3+；13,Cu2++Pb2+；14,Cu2++Hg2+；15,Cu2++Cd2+(λ ex=370nm).
Fig. 5 (a) shows the CH in QLBA (10 μM)3Cu is constantly added dropwise in OH-HEPES (20mM, pH 7.2) solution2+'s Fluorescent emission spectrogram, wherein illustration is in QLBA (10 μM) in CH3Cu is constantly added dropwise in OH-HEPES (20mM, pH 7.2) solution2 +Fluorescent value and Cu at 508nm2+The titration curve of concentration is added dropwise.It is glimmering at 0~10 μM that Fig. 5 (b) shows copper ion concentration The relationship of luminous intensity (435nm) and copper ion concentration.
Fig. 6 (a) is shown in QLBA-Cu2+The CH of (10 μM)3It is added in OH-HEPES (20mM, pH7.2) solution different normal See the fluorescence pattern of anion, 1, QLBA；2,QLBA+Cu2+；3,QLBA+Cu2++S2-；then addition of 4,F-；5, Cl-；6,Br-；7,NO3 -；8,NO2 -；9,SO4 2-；10,SO3 2-；11,OAc-；12,H2PO4 -；13,ClO4 -；14, lysine；15, half Cystine).Fig. 6 (b) shows QLBA-Cu2+(10 μM) are in CH330 μM are added in OH-HEPES (20mM, pH 7.2) solution S2-, then it is added (4, F-；5,Cl-；6,Br-；7,I-；8,NO2 -；9,SO4 2-；10,SO3 2-；11,OAc-；12,H2PO4 -；13, ClO4 -；14, lysine；15, cysteine) CH3The fluorescence emission spectrum of OH-HEPES buffer solution (1/9, v/v, pH 7.2) Figure.(λ ex=370nm)
Fig. 7 (a) shows the CH at (10 μM)3Constantly be added dropwise in OH-HEPES (20mM, pH 7.2) buffer solution sulphur from The fluorescent emission spectrogram of son.Fig. 7 (b) show sulphion concentration within the scope of 0~10 μM with its fluorescence intensity (435nm) be in Linear relationship.
Fig. 8 is QLBA and 2 equivalent Cu2+Mass spectrum.
Fig. 9 (a) is the nucleus magnetic hydrogen spectrum of QLBA, and Fig. 9 (b) is QLBA+0.5 equivalent Cu2+Nucleus magnetic hydrogen spectrum, Fig. 9 (c) be QLBA + 1.0 equivalent Cu2+And Fig. 9 (d) is QLBA+2.0 equivalent Cu2+In DMSO-d6Nucleus magnetic hydrogen spectrum in solvent.
Figure 10 (a) and Figure 10 (b) shows the QLBA/QLBA-Cu that DFT calculates analysis2+Optimization structure.
Figure 11 shows the cytotoxicity of various concentration QLBA.
Figure 12 a is the Laser Scanning Confocal Microscope imaging that HeLa cell is tested as a control group；Figure 12 b is HeLa and 20 μM QLBA is incubated for the Laser Scanning Confocal Microscope imaging after 4h altogether；Figure 12 c is that HeLa and 20 μM of QLBA is incubated for 4h altogether, and 20 μM of Cu are added2+ Laser Scanning Confocal Microscope imaging；Figure 12 d is that HeLa and 20 μM of QLBA is incubated for 4h altogether, and 20 μM of Cu are added2+It is incubated for 30min, then The Laser Scanning Confocal Microscope imaging of 10 μM of sulphions is added；Figure 12 e is that HeLa and 20 μM of QLBA is incubated for 4h altogether, and 20 μM of Cu are added2+ It is incubated for 30min, the Laser Scanning Confocal Microscope imaging of 20 μM of sulphions is then added.
Specific embodiment
The preferred embodiment that the present invention will be described in more detail below with reference to accompanying drawings.Although showing the present invention in attached drawing Preferred embodiment, however, it is to be appreciated that may be realized in various forms the present invention without the embodiment party that should be illustrated here Formula is limited.
The present invention provides a kind of copper ion based on chinoline backbone and the double target spot fluorescence probes of sulphion, double target spot fluorescence Probe has structure shown in Formulas I:
The present invention also provides the preparation methods of above-mentioned double target spot fluorescence probes, this method comprises:
In the presence of an organic, compound shown in Formula II is contacted with quinaldinic acid acyl chlorides and is reacted, obtain Formulas I institute Show compound,
The organic solvent can be the aprotic organic solvent of this field routine, preferably methylene chloride, toluene and just At least one of hexane.During organic synthesis, above-mentioned organic solvent used is required to by being dried.
Preferably, the molar ratio of compound shown in the Formula II and quinaldinic acid acyl chlorides is 1:0.8-1.2.
Preferably, which comprises the mixed solution of quinaldinic acid acyl chlorides and organic solvent is added dropwise to shown in Formula II In the mixed solution of compound and organic solvent.
Preferably, the dropwise addition carries out under cryogenic, preferably ice bath stirring.
Preferably, the temperature of the reaction is -4 to 5 DEG C, and the time is 4-8 hours.
Preferably, the condition of the reaction is ice bath stirring.
Compound shown in Formula II of the present invention and quinaldinic acid acyl chlorides can be commercially available.
Method of the invention can also include conventional post-processing step, such as cooling, revolving solvent, column chromatography etc..
The present invention also provides application of the double target spot fluorescence probes in cell imaging.
The reaction equation of a kind of preferred embodiment according to the present invention, the method is as follows,
The following steps are included:
Compound shown in Formula II is dissolved in methylene chloride, is added in reaction flask, stirring at normal temperature is subsequently placed under condition of ice bath Stirring.Quinaldinic acid acyl chlorides is dissolved in methylene chloride, is added drop-wise in the above reaction flask dropwise with constant pressure funnel.It drips Finish, is stirred under condition of ice bath.Then, revolving removes methylene chloride, obtains brown solid and goes out product, and passes through column chromatography point From purifying, the QLBA of light yellow solid powder is obtained.
By following embodiment, invention is further explained.
Embodiment 1
Compound shown in Formula II (1.05g, 5mmol) is dissolved in methylene chloride (30mL), is added to the two-mouth bottle of 200mL In, it is put into magnetic rotor, stirring at normal temperature 10 minutes, is subsequently placed under condition of ice bath and stirs.By prepare in advance etc. amount of substance Quinaldinic acid acyl chlorides is dissolved in 20mL methylene chloride, is added drop-wise in the above reaction flask dropwise with constant pressure funnel.It is added dropwise, ice It is stirred 5 hours under the conditions of bath.Then, revolving removes methylene chloride, obtains brown solid and goes out product, and passes through column chromatography point From purifying, 1.12g light yellow solid powder QLBA, yield 62% are obtained.
The hydrogen that Fig. 1 (a) and Fig. 1 (b) is QLBA is composed.The carbon that Fig. 2 (a) and Fig. 2 (b) is QLBA is composed.
Test case 1
The optical physics of 1QLBA characterizes
The absorption spectrogram of 1.1 test QLBA.CH3OH-HEPES (20mM, pH 7.2) solution of the QLBA of 10 μM of configuration, takes 2mL is added in quartz colorimetric utensil, tests it using ultraviolet-visible spectrophotometer and absorbs spectrogram.
Test the fluorescent emission spectrogram of QLBA.The QLBA solution of 10 μM of configuration is molten in CH3OH-HEPES (20mM, pH 7.2) Liquid takes 2mL to be added in cuvette, and the fluorescent emission spectrogram of QLBA is tested on sepectrophotofluorometer.Excitation wavelength is 360nm, transmitting section are 390-700nm.
Test case 2
Selection Journal of Sex Research of the 2QLBA to different metal ions
There are 2 milliliters of CH in 2.1 detection architectures, in cuvette3OH-HEPES (20mM, pH 7.2) solution.It is each when detection Contain different metal ions, Na in cuvette+, K+, Mg2+, Zn2+, Ca2+, Fe2+, Mn2+, Hg2+, Cd2+, Co2+, Al3+, Pd2+, Cu2+, Fe3+Concentration of salt solution is 30 μM, simultaneously containing 10 μM of QLBA.Fluorescence intensity is carried out on sepectrophotofluorometer Detection (excitation wavelength 360nm).
2.2 acquire and handle data.
Test case 3
3Cu2+And S2-Titration experiments
3.1 10 μM of QLBA solution of configuration, solvent CH3OH-HEPES (20mM, pH 7.2) solution takes 1mL that colorimetric is added In ware.
The CuCl of 3.2 configuration 20mM2With the Na of 20mM2S aqueous solution.
3.3 Cu is added dropwise into cuvette respectively2+, Cu2+Concentration range be 0~30 μM, on sepectrophotofluorometer according to Secondary test fluorescent emission spectrogram.
10 μM of Cu are added dropwise into cuvette respectively for 3.4 same methods2+, then it is added dropwise based on solution system according to this S2-, test fluorescent emission spectrogram.
3.5 acquire and handle data.
Test case 4
4QLBA and Cu2+Mechanism of action analysis
The CuCl of 4.1 configuration QLBA and 5 equivalents2Solution, be analyzed by mass spectrometry.Mass spectral analysis obtains QLBA and Cu2+'s In conjunction with ratio.
The D6-DMSO solution of 4.2 configuration QLBA, surveys nucleus magnetic hydrogen spectrum, CuCl is then added dropwise2D6-DMSO solution, often It is added dropwise once, tests a hydrogen spectrum.Resulting nuclear-magnetism will be tested longitudinally to compare, find out changing rule.
4.3, which carry out density functional theory (DFT) using 09 software of Gauss on computers, calculates analysis.
Test case 5
5 cell culture processes
HeLa cell is placed in 37 DEG C, carbon dioxide content is in 5% incubator.Culture medium is containing 10%FBS's RPMI-1640.It is passed on when cell density is about 90% using pancreatin cell dissociation buffer, passage in average 2 days is primary.
Test case 6
The detection of 6 cytotoxicities
Logarithmic phase HeLa cell is collected, adjustment cell solution concentration to 50000/mL, every hole is added 100 in 96 orifice plates μL.In 5%CO2, after 37 DEG C of incubator overnight incubation, the QLBA100 μ L of various concentration gradient is added, makes each gradient concentration (0,2,5,10,20,50,100 μM).Five multiple holes of each gradient.After culture for 24 hours, 3- (4, the 5- dimethyl of 20 μ L are added in every hole Thiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT) PBS solution (5mg/mL), be incubated for 4h altogether.Supernatant is sucked, every hole is added The DMSO of 150 μ L sets low speed on shaking table and shakes 10 minutes, dissolves crystal sufficiently.In enzyme-linked immunosorbent assay instrument OD 570nm Place measures the light absorption value in each hole.
Test case 7
7 cell imagings
7.1QLBA and HeLa cytosis imaging experiment.HeLa cell is passaged to the copolymerization coke glass bottom that diameter is 20mm Culture dish, in 5%CO2, after 37 DEG C of incubator culture for 24 hours.QLBA is added in culture medium, the concentration of QLBA is 20 μM, is incubated After educating 6h, culture solution is removed, is cleaned cell six times with 1 × PBS.(the Olympus FV1000- under Laser Scanning Confocal Microscope IX81confocallaser scanning microscope) observation.Exciting light selects mercury laser (72.0%405nm).
7.2 intracellular detection QLBA and Cu2+And S2-Cell imaging experiment.It is 20mm's that HeLa cell, which is passaged to diameter, It is copolymerized burnt Glass bottom culture dish, in 5%CO2, after 37 DEG C of incubator culture for 24 hours.QLBA is added in culture medium, the concentration of QLBA It is 20 μM, after being incubated for 4 hours, 20 μM of Cu is added2+Culture solution is removed after being incubated for 30min, is cleaned cell 4 times with 1 × PBS.Altogether (Olympus FV1000-IX81confocal laser scanning microscope) is observed under focusing microscope.Excitation Light selects mercury laser (72.0%405nm).Two groups of above-mentioned parallel laboratory test is carried out, after copper ion incubation 30min is added, then 10 μM and 20 μM of sulphions are added into this two groups of parallel laboratory tests respectively, removes culture solution after being incubated for 30min, is cleaned with 1 × PBS Cell 4 times.It is observed under Laser Scanning Confocal Microscope.
8 arrange data and analysis
8.1QLBA is in CH3UV absorption and fluorescent emission spectrogram in OH-HEPES (20mM, pH 7.2) solution system. As shown in Fig. 3 (a) and Fig. 3 (b), QLBA possesses the absorption region of 300~400nm.It is its maximum emission wavelength at 508nm.
The selection Journal of Sex Research of 8.2QLBA and different metal ions
QLBA is designed based on chinoline backbone, and the quinoline of heterocyclic nitrogen atom can be used as the chela of metal ion Coincidence point, so carrying out the selection Journal of Sex Research between QLBA and different metal ions, the present invention has chosen 14 kinds of common metals altogether Ion (Na+, K+, Mg2+, Zn2+, Ca2+, Cr3+, Mn2+, Hg2+, Cd2+, Co2+, Al3+, Pd2+, Cu2+, Fe3+)。
As shown in Fig. 4 (a) and Fig. 4 (b), after joined different metal ions, Cu2+The fluorescence display of QLBA has been gone out and has been shown What is write is quenched, and does not change significantly to other ions.Then other ions are continuously added in this system not draw Other significant changes are played, this result illustrates QLBA to Cu2+There is a special response, and under the conditions of existing for other ions, Bu Huigan QLBA is disturbed to Cu2+Special response.
8.3Cu2+To the titration experiments of QLBA
As shown in Fig. 5 (a) and Fig. 5 (b), in the CH of QLBA (10 μM)3OH-HEPES (20mM, pH 7.2) solution system In, with Cu2+Dropwise addition concentration be continuously increased, the fluorescent value at the maximum emission wavelength (435nm) of QLBA constantly declines, when Cu2+Dropwise addition concentration when reaching 10 μM, the fluorescence intensity of QLBA is quenched substantially.Work as Cu2+Dropwise addition concentration at 0~10 μM, The maximum emission wavelength (435nm) and Cu of QLBA2+Concentration presents preferable linear.Its detection limit is calculated by LOD=3*Sb/S, QLBA is to Cu2+Detection be limited to 2.2 × 10-7M。
8.4QLBA-Cu2+System is to S2-Detection
As shown in Fig. 6 (a), QLBA-Cu2+After joined different anion in (10 μM) system, S2-It can make QLBA- Cu2+The fluorescence being originally quenched significantly increases, and other ions do not change significantly.Then it is continuously added in this system Other ions do not cause other significant changes, and as shown in Fig. 6 (b), this result illustrates QLBA-Cu2+Sulphion is specifically rung It answers, and under the conditions of existing for other ions, QLBA will not be interfered to Cu2+Special response.
As shown in Fig. 7 (a) and Fig. 7 (b), in QLBA-Cu2+The CH of (10 μM)3OH-HEPES (20mM, pH 7.2) solution body In system, as the dropwise addition concentration of sulphion is continuously increased, fluorescent value of the system at 435nm is gradually increased, when the drop of sulphion When concentration being added to reach 10 μM, QLBA-Cu2+The fluorescence intensity of system basically reaches maximum value.Also, when within the scope of 0~10 μM, The maximum emission wavelength (435nm) and sulphion concentration of system present preferable linear.Its detection is calculated by LOD=3*Sb/S Limit, QLBA is to Cu2+Detection be limited to 0.46 μM.
8.5QLBA and Cu2+And Fe3+Study on mechanism
The mass spectral results of Fig. 8 show that m/z 365.1389 (calculated value 365.1397) corresponds to [QLBA+H]+,m/z 426.0533 (calculated values 426.0536) correspond to [QLBA+Cu-H]+, this Mass spectrometry experiments result can be explicitly shown probe QLBA It is the coordination relationship of 1:1 between copper ion.
Fig. 9 shows that the hetero atom N on benzimidazole, the N in N and amido bond in quinoline unit is participated in and copper ion Coordination combine.
It is calculated and is analyzed by DFT, QLBA and Cu2+Binding site such as Figure 10 (a) and Figure 10 (b) shown in.Cu2+And QLBA The distance of N (1) in structure, N (2), N (3) atom is respectively .QLBA in Figure 10, and Compound QLBA-Cu2+Structure is optimum structure.
8.6 cell imaging
8.6.1 cytotoxicity
The cytotoxicity of QLBA, as a result as shown in figure 11, the incubation when QLBA concentration reaches 20 μM are studied by mtt assay After for 24 hours, cell survival rate is up to 90% or more.
8.6.2QLBA intracellular identification copper ion and sulphion is further identified
In Figure 12 a- Figure 12 e carried out after 4h after QLBA and HeLa cell are incubated for altogether as the result is shown copolymerization coke it is micro- at Picture, cell are displayed in blue fluorescence, and fluorescence is quenched after equivalent copper ion is added, and sulphion is then added and co-cultures, fluorescence meeting again Restore.QLBA can be used as identifying Cu into the cell as the result is shown for this2+, while sulphion can be further identified again.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill Many modifications and changes are obvious for the those of ordinary skill in art field.

Claims (8)

1. pair target spot fluorescence probe is preparing the application in cell imaging reagent, which is characterized in that double target spot fluorescence probes are Copper ion and the double target spot fluorescence probes of sulphion, with structure shown in Formulas I:
2. application according to claim 1, which is characterized in that the preparation method of double target spot fluorescence probes includes:
In the presence of an organic, compound shown in Formula II is contacted with quinaldinic acid acyl chlorides and is reacted, obtain Formulas I shownization Object is closed,
3. application according to claim 2, wherein the organic solvent be methylene chloride, toluene and n-hexane in extremely Few one kind.
4. application according to claim 2, wherein the molar ratio of compound shown in the Formula II and quinaldinic acid acyl chlorides is 1:0.8-1.2.
5. application according to claim 2, wherein the described method includes: by the mixed of quinaldinic acid acyl chlorides and organic solvent Solution is closed to be added dropwise in the mixed solution of compound and organic solvent shown in Formula II.
6. application according to claim 5, wherein the condition of the dropwise addition is ice bath stirring.
7. application according to claim 2, wherein the temperature of the reaction is -4 to 5 DEG C, and the time is 4-8 hours.
8. application according to claim 7, wherein the condition of the reaction is ice bath stirring.
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