CN110128402A - Branch shape fluorescent dye compound containing pyrazole ring - Google Patents
Branch shape fluorescent dye compound containing pyrazole ring Download PDFInfo
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- CN110128402A CN110128402A CN201910465139.6A CN201910465139A CN110128402A CN 110128402 A CN110128402 A CN 110128402A CN 201910465139 A CN201910465139 A CN 201910465139A CN 110128402 A CN110128402 A CN 110128402A
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- pyrazole ring
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1011—Condensed systems
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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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
Abstract
Branch shape fluorescent dye compound containing pyrazole ring, the fluorescent dye have the structure of general formula I.The fluorochrome has lower fluorescence background in the non-mitochondria of living cells, has stronger fluorescence signal in living cells Mitochondria region, and to O in living cells2It is marked with very strong specificity.This kind of compound has the water solubility of certain level, while having good permeability of cell membrane.This kind of compound of the invention also has lower bio-toxicity, phototoxicity, photobleaching simultaneously.Its spectral region and the spectral region of biological sample have sufficiently large difference.
Description
Technical field
The present invention relates to the preparation method of fluorescent dye of the one kind containing pyrazole ring and its gaseous signal molecule detection in
Application, especially to O2The identification of molecule and quantitative, dynamic and visual detection.
Background technique
Oxygen (O2), the most wide gas molecule of distributed in nature participates in the respiratory of human body and animal, is that human body carries out
The key substance of metabolism, is the primary need of human life activity.In addition to this, oxygen presence is widely answered in life
With, such as: smelting process, chemical industry, health care etc., which all have, to play an important role.In recent years, oxygen exists
It plays an important role in chemistry and biochemical reaction, either reactant or product, all gradually causes scientific research work
The idea of authors.Currently, the generation of many diseases and oxygen have close connection.Anoxic is extremely normal in clinical various diseases
A kind of pathologic process seen.Anoxic refers to metabolism, function so as to cause tissue because of the oxygen supply deficiency of tissue or with oxygen obstacle
The pathologic process of variation can be abnormal with morphosis.The life-critical organ hypoxia such as brain, heart is also to cause body dead
The major reason died.Cell hypoxia causes the oxygen content of tissue and cell inadequate, this is not only to cause the basic of disease and cancer
Reason, and will also result in the constitution of degenerative disorders, anoxic is immune and degenerative disorders a significant factor.Cancer is thin
Born of the same parents are with the maximum difference of normal cell: cancer cell is anaerobic cell, only oxygen is insufficient, oxygen concentration is too in blood
Just it can spread and divide when low or number of free radical is too high；And normal cell then needs sufficient oxygen.All chronic aches,
Slight illness is all because lacking caused by the oxygen of normal cell physiology dosage.Coronary heart disease and cardiopathic reason are also the oxygen that heart obtains
Caused by tolerance is insufficient.In recent years, concern of the oxygen in physiology and field of pathology by numerous workers.So building
Stand a kind of simplicity, quickly, effective, sensitive, specific recognition and O quantitative, in dynamic monitoring cell or tissue2With
It and can be an important job to its techniques or methods for carrying out quantitative imaging.
Due to its importance in the various applications of life science and environment science, it is reliable that people have been directed to exploitation
Oxygen detection technology, and increasingly focus on them.Traditional oxygen detection method has titration, and amperometry is changed
Learn luminous and thermoluminescence.However, there is many limitations in these conventional methods, such as the oxygen consumption in sensing process, phase
To the trend of longer response time and electrode.Since optical oxygen sensing allows to carry out non-invasive measurement to inanimate object,
The parallel monitoring of multiple samples and imaging, therefore it causes sizable interest in the past few decades.In addition to this, very
Multi detection technology can not pass through cell tissue outer membrane, cannot achieve dynamic monitoring in real time.In recent years, fluorescent dye combined continuous
The micro-imaging technique of development provides a kind of feasible important Microobservation tool to eliminate drawbacks described above, is made with fluorescent dye
It is O for the optical molecular imaging technology of core2Related basic research provide a kind of potential visualization tool.Currently, luxuriant and rich with fragrance
The fluorescent dyes such as pyridine class (EB, PI), acridine (AO) and the Hua Jingjia same clan (Cy, TOTO, SYTO) all have become fluorescent dye
The kernel object molecule of research.However, there is also some the shortcomings that can not changing for these dyestuffs.Such as: in phenanthridines dye class
Ethidium bromide (EB) has good optical property, but its biological carcinogenicity with higher as fluorescent dye.It is prior
It is that these traditional fluorescent dyes are in fluorescent stability, the excitation of near-infrared targeting, high-resolution dark-field imaging, biological sample
Autofluorescence interference etc. have very big defect (Nature, 1999,2:989-996.；Science,1997,275:
530.；Angewandte Chemie International Edition,2001,40:2098.).Therefore, design is synthesized and is made
Standby toxicity low/no, good photostability, high membrane permeability, highly selective identification and quantitative, real-time dynamic monitoring living biological samples
O in (cell or tissue)2Fluorescent dye still have huge challenge.
Summary of the invention
The purpose of the present invention, which first consists in, to be provided one kind and can selectively position in the biological sample and to O2It can produce
The molecule of fluorescence response signal strong enough.For this purpose, the branch shape fluorescent dye chemical combination present invention firstly provides one kind containing pyrazole ring
Object, the compound have the structure of general formula I:
In general formula I:
The R1, R2It is each independently selected from :-H ,-CN ,-NH2、-OH、-(CH2)xCH3、-(CH2)xNH2、-(CH2)xCN、-(CH2)xCOOH、-(CH2)xOH、-(CH2)xOCH3、-(CH2)xCOCH3、-NH(CH2)xCH3、-(CH2OCH2)xCOOH、-
(CH2OCH2)xOH、-COO(CH2)xCH3、-CH[(CH2)xCOOH]2With-CH [(CH2)xOH]2, wherein x is independently selected from 1-8's
Integer；
The R3It is selected from :-(C6H5)、-(C6H4)CH3、-(C6H4)(CH2)yCH3、-(C6H4)(CH2)yNH2、-(C6H4)N
(CH3)2、-(C6H4)NHCH3、-(C6H4)NHCOOH、-(C6H4)N(COOH)2、-(C6H4)NHOH、-(C6H4)NH(CH2)yCH3、-
(C6H4)NH2、-(C6H4)NHOH、-(C6H4)N(COOH)2、-(C6H4)NH(CH2)yNH2, wherein y is independently selected from the whole of 1-8
Number；
The M is selected from M1~M5:
The L is selected from L1~L5:
According to the usual cognition of this field, described-(C6H5) it is phenyl ,-(C6H4)-be substituted-phenyl, substituent group is in benzene
Position on ring can be ortho position, contraposition or meta position.
In the structural formula of above-mentioned group, " --- " is to indicate saturation singly-bound that the group is connect with compound other parts.
The branch shape fluorescent dye compound containing pyrazole ring of aforementioned present invention can be in cell and tissue to O2Have strong
Fluorescence response signal, and this fluorochrome has good water-soluble, photostability and outstanding cell membrane penetration
Property.Also there is lower bio-toxicity, phototoxicity, photobleaching simultaneously.Its spectral region and the spectral region of biological sample have
Sufficiently large difference, can be to avoid the generation of archebiosis fluorescence.Based on this, another aspect of the present invention is intended to provide above-mentioned containing pyrrole
The fluorescent dye compound of azoles ring is preparing the application in gaseous signal molecule detection reagent.The wherein gaseous signal molecule
Especially O2。
Application in gaseous signal molecule detection.Particularly preferably being applied to object is O in living cells or tumor tissues2
Movable real-time dynamic monitoring and label.
In another aspect, the present invention is intended to provide the preparation method of the above-mentioned fluorescent dye compound containing pyrazole ring, including such as
Lower step:
1)R1- M-CHO and R2-L-COCH31:1-1:10 reacts in molar ratio, prepare compound R1- M-CH=CHCO-L-
R2；
Reaction temperature be 0-100 DEG C, the reaction time be 1-12 hour, reaction dissolvent selected from ethyl alcohol, methylene chloride, methanol,
Or mixtures thereof acetone, dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid；
2) compound R1- M-CH=CHCO-L-R2With R3-NHNH21:1-1:10 reacts in molar ratio, prepares the change of general formula I
Close object；Reaction temperature is 0-150 DEG C, and the reaction time is 1-12 hours, and reaction dissolvent is selected from ethyl alcohol, methylene chloride, methanol, third
Or mixtures thereof ketone, dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid.
Detailed description of the invention
14 width of attached drawing of the present invention:
Fig. 1 is the general structure I of the fluorescent dye compound of the invention containing pyrazole ring.
Fig. 2 is compound A1Water-soluble detection test result figure.
Fig. 3 is compound A1In aqueous solution to O2Detection test result figure.
Fig. 4 is compound A1Imaging test result figure in living cells.Wherein, figure (a) is S-band imaging examination
Test result figure；Scheming (b) is L-band imaging test result figure.
Fig. 5 is compound A1To O in living cells2Detection test result figure.Wherein, figure (a) and figure (b) are short respectively
Wavelength and L-band are without O2The cell imaging result figure of processing；Scheming (c) and figure (d) is short wavelength and long wave respectively
Section is through O2Cell imaging result figure that treated.
Fig. 6 is compound A2Water-soluble detection test result figure.
Fig. 7 is compound A2In aqueous solution to O2Detection test result figure.
Fig. 8 is compound A2Imaging test result figure in living cells.Wherein, figure (a) is S-band imaging
Test result figure；Scheming (b) is L-band imaging test result figure.
Fig. 9 is compound A3Photostability detect test result figure.
Figure 10 is compound A3Imaging test result figure in living cells.Wherein, figure (a) is S-band imaging examination
Test result figure；Scheming (b) is L-band imaging test result figure.
Figure 11 is compound A3To O in living cells2Detection test result figure.Wherein, figure (a) and figure (b) are short respectively
Wavelength and L-band are without O2The cell imaging result figure of processing；Scheming (c) and figure (d) is short wavelength and long wave respectively
Section is through O2Cell imaging result figure that treated.
Figure 12 is compound A4To the detection test result figure of living cells toxicity.
Figure 13 is compound A4Imaging test result figure in living cells.Wherein: figure (a) is S-band imaging examination
Test result figure；Scheming (b) is L-band imaging test result figure.
Figure 14 is compound A4To O in living cells2Detection test result figure.Wherein, figure (a) and figure (b) are short respectively
Wavelength and L-band are without O2The cell imaging result figure of processing；Scheming (c) and figure (d) is short wavelength and long wave respectively
Section is through O2Cell imaging result figure that treated.
Specific embodiment
Present invention is primarily aimed at provide a kind of branch shape fluorescent dye containing pyrazole ring, the structure with general formula I.
General formula I such as attached drawing 1:
In specific embodiment, the R1Selected from-H ,-CN ,-NH2、-OH、-(CH2)xCH3With-(CH2)xNH2；It is preferred that
R1It is-H ,-CN ,-OH or-(CH2)xCH3；Particularly preferably-H or-(CH2)xCH3, the integer of the preferred 1-4 of x.
In another specific embodiment, the R2Selected from-H ,-CN ,-NH2、-OH、-(CH2)xCH3With-(CH2)xNH2；
It is preferred that R1It is-H ,-CN ,-OH or-(CH2)xCH3；Particularly preferably-H or-(CH2)xCH3, the integer of the preferred 1-4 of x.
In yet another embodiment, the R3Selected from-(C6H5)、-(C6H4)CH3、-(C6H4)(CH2)yCH3、-
(C6H4)(CH2)yNH2With-(C6H4)N(CH3)2；It is preferred that R3For-(C6H5)、-(C6H4)CH3、-(C6H4)(CH2)yCH3Or-(C6H4)
(CH2)yNH2；More preferably-(C6H5)、-(C6H4)CH3；Wherein y is preferably the integer of 1-4.
In the specific embodiment of another aspect, the M is selected from M1~M5；Preferably M1、M2、M3Or M5；Particularly preferably
M2Or M3。
In the specific embodiment of another further aspect, the L is selected from L1~L5, preferably L1、L2、L3For L5, more preferable L1Or
L2:
Each preferred feature described above can be combined with each other, and gained technical solution should be included in this hair completely
Bright addressed range.
The combination of preferred feature, obtains most preferred embodiment of the present invention about fluorescent dye, and described contains pyrrole
The fluorescent dye of azoles ring is represented by A1、A2、A3And A4:
On the other hand, invention further provides the preparation method of the above-mentioned fluorescent dye containing pyrazole ring, including it is as follows
Step:
1) compound R1- M-CHO and R2-L-COCH31:1-1:10 reacts in molar ratio, prepare compound R1- M-CH=
CHCO-L-R2；
Reaction temperature be 0-100 DEG C, the reaction time be 1-12 hour, reaction dissolvent selected from ethyl alcohol, methylene chloride, methanol,
Or mixtures thereof acetone, dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid；
In specific embodiment, preferred 0-80 DEG C of the reaction temperature of the step 1), more preferable 10-60 DEG C, most preferably 20-
50℃；The reaction time preferred 1-10h, more preferable 1-8h, most preferably 2-6h；The reaction dissolvent preferred alcohol, two
Or mixtures thereof chloromethanes, methanol, acetone, dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid, more preferable ethyl alcohol, dichloromethane
Or mixtures thereof alkane, methanol, acetone, dimethyl sulfoxide, acetic acid, most preferred ethanol, methylene chloride, methanol, acetone or its mixing
Object；Compound R1- M-CHO and R2-L-COCH3Carry out reaction preferred molar ratio 1:1-1:7, more preferable 1:1-1:6, most preferably 1:
1-1:4。
2) compound R1- M-CH=CHCO-L-R2With R3-NHNH21:1-1:10 reacts in molar ratio, prepares the change of general formula I
Close object；
Reaction temperature be 0-150 DEG C, the reaction time be 1-12 hour, reaction dissolvent selected from ethyl alcohol, methylene chloride, methanol,
Or mixtures thereof acetone, dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid.
In specific embodiment, preferred 10-120 DEG C of the reaction temperature of the step 2), more preferable 30-100 DEG C, most preferably
50-90℃；The reaction time preferred 1-10h, more preferable 2-8h, most preferably 3-7h；The reaction dissolvent preferred alcohol,
Or mixtures thereof methylene chloride, methanol, acetone, dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid, more preferable ethyl alcohol, dichloro
Or mixtures thereof methane, methanol, acetone, dimethyl sulfoxide, acetic acid, most preferred ethanol, methylene chloride, methanol, acetone or it is mixed
Close object；Compound R1- M-CH=CHCO-L-R2With R3-NHNH2Carry out reaction preferred molar ratio 1:1-1:7, more preferable 1:1-1:
6, most preferably 1:1-1:4.
To the present invention using the fluorescent dye compound containing pyrazole ring of above method synthesis, using nmr spectrum or
Mass spectrum confirms its structure.
Fluorescent dye of the present invention containing pyrazole ring has following advantages:
The dye composition introduces and O2The reaction site of effect improves dyestuff to O in living cells and tissue2Inspection
The specificity surveyed and identified.
The dye composition has excellent optical property, floats when applied to biological sample imaging with low bio-light
White, light injury and bio-toxicity.
The dye composition side effect is small, and raw material is easy to get, and structure is simple, easily prepared, easy industrialization.
In consideration of it, fluorescent dye of the present invention can be used for O in living cells2The label of signaling molecule.In addition to herein
Described in form be directly used in O in living cells2Label, the composition containing fluorescent dye compound of the invention can also be with
For O in living cells2Label.It should include a effective amount of fluorescent dye compound provided by the present invention in the composition
One of.Furthermore it is also possible to include other components, such as solvent, pH adjusting agent etc. required for biological sample dyeing.These components
It is all that current row is known in the art.Above-mentioned composition can exist as an aqueous solution, or can be to be formulated as before use with water
Other suitable forms of solution exist.
The present invention also provides using, the fluorescent dye compound of aforementioned present invention is marked and dynamic realtime monitors in living cells
O2Method, this method includes the steps that contacting the compound with biological sample.Term " contact " packet used herein
It includes but is not limited to contact in solution or solid phase.
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
Embodiment 1
Fluorescent dye A1Synthetic route and method:
(1) compound A1- 1 synthesis
The 1-2 of the compound 1-1 and 20mmol of 10mmol are added to the 3mol/L sodium hydrate aqueous solution containing 6mL
In ethyl alcohol (30mL) solution, 3h is stirred at room temperature.It is mixed by reaction product filtering, drying, and from ethyl acetate/acetic acid (1:1v/v)
It closes crystallization in solution and obtains yellow crystals.
(2) compound A1Synthesis
By the A of 3mmol1The phenylhydrazine of -1 and 9mmol is added in the ethyl alcohol containing 37%HCl (40mL) solution.It will mixing
Object is stirred at reflux 6h at 78 DEG C, obtains yellow solid.Crude product is separated, and is recrystallized to give glassy yellow from ethyl acetate and consolidates
Body.Yield: 80%；
(3) compound A1Characterization
1H NMR(CDCl3): 8.57-8.48 (m, 3H), 8.31 (q, 2H), 8.11 (t, 1H), 7.99 (t, 1H), 7.78 (d,
1H), 7.63 (t, 1H), 7.37 (m, 2H), 6.98 (m, 4H), 6.67 (d .2H), 5.19 (t, 1H), 3.92-3.67 (d, 2H).
Embodiment 2
Fluorescent dye compound A1Water-soluble detection test
The fluorescent dye compound A that will be synthesized in above-described embodiment1It is made into mother liquor, different volumes mother liquor is pipetted and is added to water
In, it is configured to the chemical combination A that concentration is respectively 1,3,5,7,9,11,13,15 and 17 μM1Aqueous solution；Sequentially determining these not
With A under concentration1Absorbance under the maximum absorption wavelength of aqueous solution.Test result is Fig. 2, as the result is shown: as compound A1Concentration
When greater than 15 μM, absorbance value linearly shifts, i.e. fluorescent dye compound A1Maxima solubility in water is 15 μM.
The present embodiment instrument is 8453 ultraviolet specrophotometer of Agilent.
Embodiment 3
Fluorescent dye A1In aqueous solution to O2Detection test
Pipette the fluorescent dye compound A synthesized in above-described embodiment1The mother liquor being made into 3mL quartz colorimetric utensil, according to
The secondary O for being passed through different volumes2(0uL, 40uL, 80uL, 120uL, 160uL) measures different volumes O2Under the conditions of emission spectrum.
As a result as shown in Figure 3: with being passed through O2The increase of volume, long wavelength's fluorescence intensity gradually decrease, and short wavelength's fluorescence intensity is gradually
Increase.Fig. 3 is different volumes O2Act on lower A1Fluorescence spectrum variation.
Embodiment 4
Fluorescent dye A1Imaging test in living cells
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A1, at 37 DEG C, 5%CO2Under conditions of be incubated for 2 cell 0.5h of HepG.Then laser confocal imaging test is carried out.Choosing
Representative area is taken, is observed with oil mirror (60 ×), in triplicate, the fluorescence that acquisition wave band is 410-490nm and 500-600nm.
Test result such as Fig. 4, wherein Fig. 4 a fluorescent collecting wave band is 410-490nm, and shows stronger fluorescence signal；Fig. 4 b fluorescence
Acquisition wave band is 500-600nm, and fluorescence signal intensity is weaker.The result shows that: fluorescent dye compound A1It is thin to can be used for HepG 2
The imaging test of born of the same parents.
Embodiment 5
Fluorescent dye compound A1To in living cells to O2Detection test
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A1, at 37 DEG C, 5%CO2Under conditions of be incubated for HepG2 cell 0.5h.40 μ L O are passed through in experimental group2After being incubated for 0.5h.
Laser confocal imaging.Representative area is chosen, is observed with oil mirror (60 ×), (fluorescent collecting wave band is 410- in triplicate
490nm and 500-600nm).Excitation wavelength is 373nm.
Experimental result is as shown in figure 5, Fig. 5 a and Fig. 5 b are respectively control group, fluorescent dye compound A1In control group (figure
5a) intermediate waves strong point (410-490nm) has an obvious and hyperfluorescence signal, and long wave strong point (Fig. 5 b, 500-600nm) have and compared with
Weak fluorescence signal.When (Fig. 5 c and 5d) is passed through O in experimental group2Afterwards, luminescent dye molecule A1With O2Reaction, with reaction
It carries out, the fluorescence signal at short wavelength (410-490nm, Fig. 5 c) is remarkably reinforced, and obviously weakens in the fluorescence of long wave strong point
(500-600nm, Fig. 5 d).This is the experimental results showed that fluorescent dye compound A1It can be to O in living cells2Carry out recognition imaging.
Embodiment 6
Fluorescent dye A2Synthetic route and method:
(1) compound A2- 1 synthesis
The 1-2 of the compound 1-1 and 20mmol of 10mmol are added to the 3mol/L sodium hydrate aqueous solution containing 6mL
In ethyl alcohol (30mL) solution, 3h is stirred at room temperature.It is mixed by reaction product filtering, drying, and from ethyl acetate/acetic acid (1:1v/v)
It closes crystallization in solution and obtains yellow crystals.
(2) compound A2Synthesis
By the A of 3mmol2The phenylhydrazine of -1 and 9mmol is added in the ethyl alcohol containing 37%HCl (40mL) solution.It will mixing
Object is stirred at reflux 6h at 78 DEG C, obtains yellow solid.Crude product is separated, and is recrystallized to give glassy yellow from ethyl acetate and consolidates
Body.Yield: 80%.
(3) compound A2Characterization
1H NMR(CDCl3): 8.57-8.48 (m, 3H), 8.31 (q, 2H), 8.11 (t, 1H), 7.99 (t, 1H), 7.78 (d,
1H), 7.63 (t, 1H), 7.19 (m, 2H), 6.98 (m, 4H), 6.95 (s, 3H), 6.83 (d .2H), 5.19 (t, 1H), 3.92-
3.67 (d, 2H).
Embodiment 7
Fluorescent dye compound A2Water-soluble detection test
The fluorescent dye compound A that will be synthesized in above-described embodiment2It is made into mother liquor, different volumes mother liquor is pipetted and is added to water
In, it is configured to the chemical combination A that concentration is respectively 1,3,5,7,9,11,13,15,17,19 and 21 μM2Aqueous solution, measure in difference
A under concentration2Absorbance under the maximum absorption wavelength of aqueous solution.Test result is Fig. 6, as the result is shown: as compound A2Concentration is big
When 19 μM, absorbance value linearly shifts, i.e. fluorescent dye compound A2Maxima solubility in water is 19 μM.
The present embodiment instrument is 8453 ultraviolet specrophotometer of Agilent.
Embodiment 8
Fluorescent dye A2In aqueous solution to O2Detection test
Pipette the fluorescent dye compound A of above-described embodiment synthesis2The mother liquor being made into is in the quartz colorimetric utensil containing 3mL water
In, successively it is passed through the O of different volumes2(0uL, 40uL, 80uL, 120uL, 160uL) measures different volumes O2Under the conditions of transmitting
Spectrum.As a result as shown in Figure 7: with being passed through O2The increase of volume, long wavelength's fluorescence intensity gradually decrease, short wavelength's fluorescence intensity
It gradually increases.Fig. 7 shows different volumes O2Act on lower A2Fluorescence spectrum variation.
Embodiment 9
Fluorescent dye A2Imaging test in living cells
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A2, at 37 DEG C, 5%CO2Under conditions of be incubated for 2 cell 0.5h of HepG.Then laser confocal imaging test is carried out.Choosing
Representative area is taken, is observed with oil mirror (60 ×), in triplicate, the fluorescence that acquisition wave band is 410-490nm and 500-600nm.
Test result such as Fig. 8, wherein Fig. 8 a fluorescent collecting wave band is 410-490nm, and shows stronger fluorescence signal；Fig. 8 b fluorescence
Acquisition wave band is 500-600nm, and fluorescence signal intensity is weaker.The result shows that: fluorescent dye compound A2It is thin to can be used for HepG 2
The imaging test of born of the same parents.
Embodiment 10
Fluorescent dye A3Synthetic route and method:
(1) compound A3- 1 synthesis
The 1-2 of the compound 1-1 and 20mmol of 10mmol are added to the 3mol/L sodium hydrate aqueous solution containing 6mL
In ethyl alcohol (30mL) solution, 3h is stirred at room temperature.It is mixed by reaction product filtering, drying, and from ethyl acetate/acetic acid (1:1v/v)
It closes crystallization in solution and obtains yellow crystals.
(2) compound A3Synthesis
By the A of 3mmol3The phenylhydrazine of -1 and 9mmol is added in the ethyl alcohol containing 37%HCl (40mL) solution.It will mixing
Object is stirred at reflux 6h at 78 DEG C, obtains yellow solid.Crude product is separated, and is recrystallized to give glassy yellow from ethyl acetate and consolidates
Body.Yield: 80%.
(3) compound A3Characterization
1H NMR(CDCl3): 9.50 (s, 1H), 9.42 (d, 2H), 8.12 (d, 1H), 7.82 (d, 1H), 7.55 (t, 1H),
7.19 (t, 2H), 6.97-6.91 (d, 3H), 6.83 (d, 2H), 5.19 (t, 1H), 3.92-3.67 (d, 2H), 3.05 (t, 2H),
1.68(q,2H),0.95(t,3H)。
Embodiment 11
Fluorescent dye compound A3Photostability detect test
Pipette the fluorescent dye compound A of above-described embodiment synthesis of certain volume3The mother liquor being made into is in containing 3mL
In the four-way quartz colorimetric utensil of DMSO, make its final concentration of 10 μM.Measure its different time (0.5h, 1.0h, 1.5h, 2h,
3h, 4h, 5h, 6h and 7.0h) under A3Fluorescence spectrum variation.Test result is Fig. 9, as the result is shown: with irradiation time
Gradually extend, compound A3Fluorescence intensity remain unchanged, time sustainable 7.0h.
Instrument is Agilent sepectrophotofluorometer.
Embodiment 12
Fluorescent dye A3Imaging test in living cells
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A3, at 37 DEG C, 5%CO2Under conditions of be incubated for 2 cell 0.5h of HepG.Then laser confocal imaging test is carried out.Choosing
Representative area is taken, is observed with oil mirror (60 ×), in triplicate, the fluorescence that acquisition wave band is 410-490nm and 500-600nm.
Test result such as Figure 10, wherein Figure 10 a fluorescent collecting wave band is 410-490nm, and shows stronger fluorescence signal；Figure 10 b
Fluorescent collecting wave band is 500-600nm, and fluorescence signal intensity is weaker.The result shows that: fluorescent dye compound A3It can be used for HepG
The imaging test of 2 cells.
Embodiment 13
Fluorescent dye compound A3To in living cells to O2Detection test
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A3, at 37 DEG C, 5%CO2Under conditions of be incubated for HepG2 cell 0.5h.40 μ L O are passed through in experimental group2After being incubated for 0.5h.
Laser confocal imaging.Representative area is chosen, is observed with oil mirror (60 ×), (fluorescent collecting wave band is 410- in triplicate
490nm and 500-600nm).Excitation wavelength is 373nm.
Experimental result is as shown in figure 11, and experimental result is as shown in figure 11, and Figure 11 a and Figure 11 b are respectively control group, fluorescence dye
Expect compound A3Have obviously and hyperfluorescence signal in control group (Figure 11 a) intermediate waves strong point (410-490nm), and in long wave strong point
(Figure 11 b, 500-600nm) has and weaker fluorescence signal.When (Figure 11 c and 11d) is passed through O in experimental group2Afterwards, fluorescent dye
Molecule A3With O2Reaction, with the progress of reaction, the fluorescence signal at short wavelength (410-490nm, Figure 11 c) is remarkably reinforced, and
Obviously weaken (500-600nm, Figure 11 d) in the fluorescence of long wave strong point.This is the experimental results showed that fluorescent dye compound A3In work
It into the cell can be to O2Carry out recognition imaging.
Embodiment 14
Fluorescent dye A4Synthetic route and method:
(1) compound A4- 1 synthesis
The 1-2 of the compound 1-1 and 20mmol of 10mmol are added to the 3mol/L sodium hydrate aqueous solution containing 6mL
In ethyl alcohol (30mL) solution, 3h is stirred at room temperature.It is mixed by reaction product filtering, drying, and from ethyl acetate/acetic acid (1:1v/v)
It closes crystallization in solution and obtains yellow crystals.
(2) compound A4Synthesis
By the A of 3mmol4The phenylhydrazine of -1 and 9mmol is added in the ethyl alcohol containing 37%HCl (40mL) solution.It will mixing
Object is stirred at reflux 6h at 78 DEG C, obtains yellow solid.Crude product is separated, and is recrystallized to give glassy yellow from ethyl acetate and consolidates
Body.Yield: 80%；
(3) compound A4Characterization
1H NMR(CDCl3): 8.92 (s, 2H), 8.01-7.97 (m, 3H), 7.57 (t, 1H), 7.49 (d, 2H), 7.45 (d,
2H), 7.25 (t, 1H), 6.95 (d .1H), 5.19 (t, 1H), 3.91-3.66 (d, 2H), 2.75 (q, 2H), 2.35 (s, 3H),
1.31(t,3H)。
Embodiment 15
Fluorescent dye compound A4Detection test to living cells toxicity
Select HepG 2 and Chinese hamster ovary celI as research object respectively.Dyestuff is characterized to cytotoxicity with cell survival rate
Size.With 5*104The cell density of a/mL is inoculated in 96 orifice plates, and the volume in every hole is 100 μ L, in 37 DEG C of 5%CO2Item
It is cultivated for 24 hours under part.Dye molecule A is then added with final concentration (5 μM, 10 μM, 15 μM, 20 μM) gradient4In culture medium, each
5 multiple holes are arranged in gradient concentration, and blank control is arranged, and the survival rate of cell is detected after culture.It is first added in every hole when detection
20 μ L 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT) solution, 37 DEG C of 5%CO2Under the conditions of cultivate
4h.Then it removes original culture solution and DMSO (150 hole μ L/) is added, then measure OD value with microplate reader, in triplicate, as a result
As shown in figure 12.The result shows that: the fluorescent dye A of above-mentioned synthesis4For living cells almost without toxicity.
Embodiment 16
Fluorescent dye A4Imaging test in living cells
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A4, at 37 DEG C, 5%CO2Under conditions of be incubated for HepG2 cell 0.5h.Then laser confocal imaging test is carried out.Choosing
Representative area is taken, is observed with oil mirror (60 ×), in triplicate, the fluorescence that acquisition wave band is 410-490nm and 500-600nm.
Test result such as Figure 13, wherein Figure 13 a fluorescent collecting wave band is 410-490nm, and shows stronger fluorescence signal；Figure 13 b
Fluorescent collecting wave band is 500-600nm, and fluorescence signal intensity is weaker.The result shows that: fluorescent dye compound A4It can be used for HepG
The imaging test of 2 cells.
Embodiment 17
Fluorescent dye compound A4To in living cells to O2Detection test
2 cell of HepG is chosen as research object.The fluorescent dye synthesized with final concentration of 15.0 μM above-described embodiment
Compound A4, at 37 DEG C, 5%CO2Under conditions of be incubated for HepG2 cell 0.5h.40 μ L O are passed through in experimental group2After being incubated for 0.5h.
Laser confocal imaging.Representative area is chosen, is observed with oil mirror (60 ×), (fluorescent collecting wave band is 410- in triplicate
490nm and 500-600nm).Excitation wavelength is 373nm.
Experimental result is as shown in figure 14, and Figure 14 a and Figure 14 b are respectively control group, fluorescent dye compound A4In control group
(Figure 14 a) intermediate waves strong point (410-490nm) has obviously and hyperfluorescence signal, and at long wave strong point (Figure 14 b, 500-600nm)
Have and weaker fluorescence signal.When (Figure 14 c and 14d) is passed through O in experimental group2Afterwards, luminescent dye molecule A4With O2Reaction, with
The progress of reaction, the fluorescence signal at short wavelength (410-490nm, Figure 14 c) is remarkably reinforced, and in the fluorescence of long wave strong point
It is obvious to weaken (500-600nm, Figure 14 d).This is the experimental results showed that fluorescent dye compound A4It can be to O in living cells2Known
It is not imaged.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.It is a kind of purposes of noval chemical compound of the present invention, and it cannot be said that the compound of the present invention is only used for as fluorescent dye
Fluorescent dye is being used as fluorescence based on the compounds of this invention for those of ordinary skill in the art to which the present invention belongs
Under the considerations of identical mechanism of action of dyestuff, several simple inferences can also be made, other for obtaining the compound of the present invention are answered
With purposes, protection scope of the present invention all shall be regarded as belonging to.
Claims (10)
1. the branch shape fluorescent dye compound containing pyrazole ring, the structure with general formula I:
In general formula I:
The R1, R2It is each independently selected from :-H ,-CN ,-NH2、-OH、-(CH2)xCH3、-(CH2)xNH2、-(CH2)xCN、-
(CH2)xCOOH、-(CH2)xOH、-(CH2)xOCH3、-(CH2)xCOCH3、-NH(CH2)xCH3、-(CH2OCH2)xCOOH、-
(CH2OCH2)xOH、-COO(CH2)xCH3、-CH[(CH2)xCOOH]2With-CH [(CH2)xOH]2, wherein x is independently selected from 1-8's
Integer；
The R3It is selected from :-(C6H5)、-(C6H4)CH3、-(C6H4)(CH2)yCH3、-(C6H4)(CH2)yNH2、-(C6H4)N
(CH3)2、-(C6H4)NHCH3、-(C6H4)NHCOOH、-(C6H4)N(COOH)2、-(C6H4)NHOH、-(C6H4)NH(CH2)yCH3、-
(C6H4)NH2、-(C6H4)NHOH、-(C6H4)N(COOH)2、-(C6H4)NH(CH2)yNH2, wherein y is independently selected from the whole of 1-8
Number；
The M is selected from M1~M5:
The L is selected from L1~L5:
2. compound according to claim 1, which is characterized in that the M is selected from M1、M2、M3And M5:
3. compound according to claim 1, which is characterized in that the L is selected from L1、L2、L3And L5:
4. compound according to claim 1, which is characterized in that the R1And R2Be each independently selected from :-H ,-CN ,-
NH2、-OH、-(CH2)xCH3、-(CH2)xNH2, wherein x is selected from the integer of 1-4.
5. compound according to claim 4, which is characterized in that the R1And R2Be each independently selected from :-H ,-CN ,-
OH and-(CH2)xCH3。
6. compound according to claim 1, which is characterized in that the R3It is selected from :-(C6H5)、-(C6H4)CH3、-
(C6H4)(CH2)yCH3、-(C6H4)(CH2)yNH2、-(C6H4)N(CH3)2, wherein y is selected from the integer of 1-4.
7. compound according to claim 6, which is characterized in that the R3It is selected from :-(C6H5)、-(C6H4)CH3、-
(C6H4)(CH2)yCH3With-(C6H4)(CH2)yNH2。
8. compound according to claim 1 is selected from compound A1、A2、A3And A4:
9. the preparation method of the fluorescent dye compound described in claim 1 containing pyrazole ring, includes the following steps:
1)R1- M-CHO and R2-L-COCH31:1-1:10 reacts in molar ratio, prepare compound R1- M-CH=CHCO-L-R2；
Reaction temperature be 0-100 DEG C, the reaction time be 1-12 hour, reaction dissolvent selected from ethyl alcohol, methylene chloride, methanol, acetone,
Or mixtures thereof dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid；
2) compound R1- M-CH=CHCO-L-R2With R3-NHNH21:1-1:10 reacts in molar ratio, prepares compounds of formula I；
Reaction temperature be 0-150 DEG C, the reaction time be 1-12 hour, reaction dissolvent selected from ethyl alcohol, methylene chloride, methanol, acetone,
Or mixtures thereof dimethyl sulfoxide, glycerine, ethyl acetate, acetic acid.
10. the branch shape fluorescent dye compound in claim 1-8 described in any claim containing pyrazole ring is preparing gas
Application in signaling molecule detection reagent.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548974A (en) * | 2009-05-26 | 2009-10-07 | 上海师范大学 | Compound having fluorescent characteristic and anti-cancer activity, preparation thereof and application thereof |
| CN102796045A (en) * | 2012-09-11 | 2012-11-28 | 山东大学 | Pyrazoline derivatives, and preparation method and application thereof |
| CN106478731A (en) * | 2016-09-07 | 2017-03-08 | 大连理工大学 | The double emission ratios type oxygen probe of one class and application |
| CN110054591A (en) * | 2018-12-26 | 2019-07-26 | 浙江工业大学 | A kind of embedded photoluminescent material and the preparation method and application thereof |
-
2019
- 2019-05-30 CN CN201910465139.6A patent/CN110128402B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548974A (en) * | 2009-05-26 | 2009-10-07 | 上海师范大学 | Compound having fluorescent characteristic and anti-cancer activity, preparation thereof and application thereof |
| CN102796045A (en) * | 2012-09-11 | 2012-11-28 | 山东大学 | Pyrazoline derivatives, and preparation method and application thereof |
| CN106478731A (en) * | 2016-09-07 | 2017-03-08 | 大连理工大学 | The double emission ratios type oxygen probe of one class and application |
| CN110054591A (en) * | 2018-12-26 | 2019-07-26 | 浙江工业大学 | A kind of embedded photoluminescent material and the preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| M. V. JYOTHI等: ""Synthesis and Antimicrobial activity Evaluation of some Novel Pyrazolines"", 《J. CHEM. PHARM. RES.》 * |
| R. STEPHEN DAVIDSON等: ""AN EXAMPLE OF HOW PHOTOPHYSICAL PROPERTIES CAN INFLUENCE THE MAGNITUDE OF SOLVENT REACTIONS ISOTOPE EFFECTS UPON PHOTO-OXIDATION"", 《PHOTOCHEMISTRY AND PHOTOBIOLOGY》 * |
| VALERY F. TRAVEN等: ""Efficient Photooxidation of Aryl(hetaryl)pyrazolines by Benzoquinone"", 《PHOTOCHEMISTRY AND PHOTOBIOLOGY》 * |
| YUE YU等: ""Highly efficient luminescent benzoylimino derivative and fluorescent probe from a photochemical reaction of imidazole as an oxygen sensor"", 《CHEMCOMM》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114199653A (en) * | 2021-10-28 | 2022-03-18 | 济南德亨医学科技有限公司 | Microbial immunofluorescence staining solution for vagina |
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