CN109516969A - One kind coumarin derivative containing carboxyl and its synthetic method and application - Google Patents
One kind coumarin derivative containing carboxyl and its synthetic method and application Download PDFInfo
- Publication number
- CN109516969A CN109516969A CN201811324486.9A CN201811324486A CN109516969A CN 109516969 A CN109516969 A CN 109516969A CN 201811324486 A CN201811324486 A CN 201811324486A CN 109516969 A CN109516969 A CN 109516969A
- Authority
- CN
- China
- Prior art keywords
- cysteine
- solution
- added
- fluorescence
- containing carboxyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
-
- 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
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
-
- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Optics & Photonics (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of coumarin derivative containing carboxyl and its synthetic method and applications, the Chinese of the derivative is (Z) -2- cyano -3- (4- ((E) -3- (7- (4- (carbethoxyl group) piperazine -1- base) -2- oxo -2H- chromene -3- base) -3- oxo propyl- 1- alkene -1- base) phenyl) acrylic acid, English name is (Z) -2-cyano-3- (4- ((E) -3- (7- (4- (ethoxycarbonyl) piperazin-1-yl) -2-oxo-2H-chromen-3-yl) -3-oxoprop-1-en-1-yl) phenyl) acrylic acid, it is named as XI-2.The present invention provides a kind of derivative XI-2 to the method for cysteine specific detection, in the solution of PBS-DMSO (4:1, v/v, pH 7.4), passes through the content of sepectrophotofluorometer quantitative detection cysteine.The detection process is recyclable, easy, sensitive, quick, and testing result is accurate.The present invention also provides derivative XI-2 to prepare the application in cell cysteine detection reagent, i.e. cysteine of the XI-2 under NEM regulating and controlling effect in high-effective penetrating cell membrane realization detection cytoplasm.
Description
Technical field
The present invention relates to coumarin derivatives, particularly belong to one kind coumarin derivative containing carboxyl and its synthetic method and N-
High-effective penetrating cell membrane under ethyl maleimide (NEM) regulating and controlling effect is distinguished on detection cell membrane and half Guang ammonia in cytoplasm
The application of acid.
Background technique
The change of cysteine homeostasis is related with a variety of diseases and cell function, therefore, cysteine dynamic realtime
Living cells imaging intracellular and quantitative analysis are for understanding that pathology, physiology course are extremely important.Therefore, whether cysteine probe has
High-effective penetrating is vital.
Studies have shown that cell membrane is made of phospholipid bilayer and the protein of insertion, protein passes through cell membrane and inhales
It is attached to cell membrane surface.Integrated membrane protein in memebrane protein on the cysteine residues of cytoplasmic matrix side with fat molecule
Covalent bond is inserted between double-layer of lipoid, a small number of albumen and glycolipid covalent bond.N-ethylmaleimide (NEM) is used as egg
The covalent modification gene of white matter cysteine residues is reacted with memebrane protein sulfydryl after cultivating cell, changes cell
The prototype structure of film, improves permeability of cell membranes, and probe being capable of cysteine in efficient detection cytoplasm.
However, there is presently no the reports about the specificity fluorescent probe of cysteine in efficient detection cytoplasm.This
It is still a challenge.
Summary of the invention
The object of the present invention is to provide a kind of coumarin derivative containing carboxyl and preparation method thereof, the tonka-beans containing carboxyl
Plain derivatives selectively is good, and high sensitivity is, it can be achieved that quantitative cycle detection cysteine and efficient detection is thin under NEM regulation
Cysteine in cytoplasm.
One kind coumarin derivative containing carboxyl provided by the invention, Chinese are (Z) -2- cyano -3- (4- ((E) -3-
(7- (4- (carbethoxyl group) piperazine -1- base) -2- oxo -2H- chromene -3- base) -3- oxo propyl- 1- alkene -1- base) phenyl) propylene
Acid, English name are:
(Z)-2-cyano-3-(4-((E)-3-(7-(4-(ethoxycarbonyl)piperazin-1-yl)-2-oxo-
2H-chromen-3-yl) -3-oxo prop-1-en-1-yl) phenyl) acrylic acid, it is named as XI-2.Structural formula
Are as follows:
The synthetic method of XI-2 of coumarin derivative containing carboxyl provided by the invention a kind of, includes the following steps:
1) ethyl chloroformate and sodium bicarbonate are dissolved in tetrahydrofuran, are slowly dropped to hydroxy phenyl piperazine between containing
In tetrahydrofuran and the mixed solution of deionized water, stirred overnight at room temperature；Extraction, dry, vacuum distillation obtains white needles crystalline substance
Body compound A:4- (3- hydroxy phenyl) piperazine -1- Ethyl formate；Wherein ethyl chloroformate, sodium bicarbonate and hydroxy phenyl piperazine
The molar ratio 1.4-1.7:1.1-1.4:1 of piperazine；
2) under the conditions of ice-water bath, excessive phosphorus oxychloride is slowly dropped in n,N-Dimethylformamide, is stirred, it will
0 DEG C of compound A being dissolved in n,N-Dimethylformamide is added in above-mentioned solution, stirring；System is poured into ice water, is adjusted
PH filters to a large amount of white solids are precipitated, obtains compound B:4- (4- formoxyl -3- hydroxy phenyl) piperazine -1- Ethyl formate；
3) compound B obtained by step 2) and ethyl acetoacetate are added in ethyl alcohol 1:1.5-2.0 in molar ratio, and addition is urged
The piperidines of change amount, is back to fully reacting；It is cooled to room temperature, yellow needles solid is precipitated, filters to obtain compound C:4- (3- acetyl
Base -2- oxo -2H- chromene -7- base) piperazine -1- Ethyl formate；
4) in molar ratio 1:1.5-2.0 by compound C obtained by step 3) and dissolving terephthalaldehyde in ethanol, addition is urged
The piperidines of change amount, is back to fully reacting；It is cooling, yellow solid is precipitated, yellow powdery solid is filtered to obtain, through pillar layer separation
Obtain compound D:(E) -4- (3- (3- (4- Fonnylphenyl) acryloyl group) -2- oxo -2H- chromene -7- base) piperazine -1- formic acid
Ethyl ester；
5) compound D and 2- cyanoacetic acid obtained by step 4) is dissolved in ethyl alcohol by 1:2.5-3.5 in molar ratio, and catalysis is added
The piperidines of amount, is back to fully reacting；System evaporated under reduced pressure solvent obtains crude product, through pillar layer separation, purifies to obtain targeted
Close object XI-2.
Tetrahydrofuran and deionized water volume ratio are preferably 1:1 in the step 1)；The ethyl chloroformate, bicarbonate
The molar ratio of sodium and hydroxy phenyl piperazine is preferably 1.5:1.25:1.
The molar ratio of compound C and terephthalaldehyde is preferably 1:1.5, the elution of the column chromatography in the step 4)
Agent is preferably 15:1 for the volume ratio of methylene chloride and ethyl acetate.
The molar ratio of compound D and 2- cyanoacetic acid is preferably 1:3 in the step 5), the eluant, eluent of the column chromatography
Volume ratio for methylene chloride and methanol is preferably 10:1.
In the present invention, by change in fluorescence before and after cysteine and compound nucleophilic substitution, cysteine is realized
Specific detection；The efficient detection to realize cytoplasm cysteine is adjusted by n-ethylmaleimide (NEM).
A method of detection cysteine, step are as follows:
(1), it prepares pH=7.4, the PBS buffer solution that concentration is 10mM, prepares the aqueous cystein solution of 20mM, configure
The DMSO solution of the XI-2 of 2mM；
(2), the DMSO solution of PBS/DMSO (v/v, 4:1, pH 7.4) solution, 10 μ L XI-2 for taking 2mL be added to one it is glimmering
It in light cuvette, is detected on Fluorescence spectrophotometer, the fluorescence intensity with the addition to test sample cysteine, at 500nm
Gradually increase；
(3), in 6 cuvettes, each PBS/DMSO (v/v, 4:1, pH 7.4) solution that 2mL is added is separately added into half
The volume of cystine solution measures fluorescence intensity at 500nm in Fluorescence Spectrometer after being 0,2,4,6,8,10 μ L, 5min
50.46,285.0,555.4,845.7,1087,1321, using cysteine ??acid salinity as abscissa, using fluorescence intensity as ordinate
Figure is drawn, the working curve of semicystinol concentration is obtained；Equation of linear regression are as follows: the unit of F=12.92c+44.92667, c is
10-6mol/L；
(4), when measuring sample solution, the fluorescence intensity measured is substituted into equation of linear regression, cysteine can be acquired
Concentration.
The XI-2 extracorporal circulatory system of coumarin derivative containing carboxyl detects cysteine:
The cysteine solution for preparing 0.2M, prepares the n-ethylmaleimide solution of 0.2M；Take the PBS/DMSO of 2mL
(v/v, 4:1, pH 7.4) solution, 10 μ L XI-2 DMSO solution be added in a fluorescence cuvette, be added 10 μ L cysteines
Before and after solution, the fluorescence intensity measured at 500nm on fluorescence spectrum is respectively 37.77,2669, and 10 μ L N- second are then added
Base maleimide amine aqueous solution, fluorescence intensity is 333.5 at 500nm.2 times subsequent circulation fluorescence intensities are respectively 2824,350,
2720、212.9。
Present invention coumarin derivative containing carboxyl XI-2 can also carry out the cysteine in water environment and cytoplasm special
Opposite sex detection；The detection includes that fluorescence detection and cell imaging detect.
Compared with prior art, the invention has the advantages that and effect:
1, the synthesis of the coumarin derivative of the invention containing carboxyl is simple, low in cost, easy to operate；
2, detection method is able to achieve the specific detection of cysteine, and shows high sensitivity and fabulous
Selectivity；
3, detection method is able to achieve the extracorporal circulatory system detection of cysteine, grinds in the detection of cysteine pathology
Studying carefully center has great potential；
4, detection method realizes that NEM regulates and controls cysteine in lower efficient detection cytoplasm for the first time, in half Guang ammonia
It has broad application prospects in sour pathology and Physiologic Studies；
5, detection means of the present invention is simple, it is only necessary to can be realized by Fluorescence Spectrometer and laser confocal microscope.
Detailed description of the invention
The nucleus magnetic hydrogen spectrum figure of XI-2 prepared by Fig. 1 embodiment 1.
The nuclear-magnetism carbon spectrogram of XI-2 prepared by Fig. 2 embodiment 1.
The mass spectrogram of XI-2 prepared by Fig. 3 embodiment 1.
The fluorescent emission figure of Fig. 4 XI-2 and cysteine effect.
Fig. 5 XI-2 and cysteine and NEM act on fluorescent emission figure at any time.
Fig. 6 XI-2 and cysteine act on and NEM recycles fluorogram.
The fluorescence histogram of Fig. 7 XI-2 and various analytes.
The working curve of Fig. 8 XI-2 measurement cysteine.
The fluorescent emission figure of Fig. 9 XI-2 measurement sample.
Figure 10 XI-2 carries out specific image to cell cysteine.
Figure 11 is to imaging cells Fluorescence Intensity Assays figure in Figure 10 Green channel.
Fluorescence intensity segment analysis figure is imaged to cell membrane cysteine in Figure 10 in Figure 12.
Figure 13 XI-2 is to mouse image.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
Embodiment 1
The preparation and characterization of XI-2
1) ethyl chloroformate (0.488g, 4.50mmol) and sodium bicarbonate (0.315g, 3.75mmol) are dissolved in 10mL
In tetrahydrofuran, the volume ratio of hydroxy phenyl piperazine (0.535g, 3.00mmol) between containing is slowly dropped to constant pressure funnel
For in the tetrahydrofuran of 1:1 and solution (40mL) round-bottomed flask of deionized water, stirred overnight at room temperature；After the reaction was completed, dichloro
Methane extraction, anhydrous sodium sulfate is dry, and vacuum distillation obtains white needle-like crystals A:4- (3- hydroxy phenyl) piperazine -1- formic acid second
Ester.
2) under conditions of ice-water bath, phosphorus oxychloride (5mL) is slowly dropped in 10mL n,N-Dimethylformamide,
After stirring 30min, 0 DEG C of the A (0.500g, 2.00mmol) being dissolved in 10mL n,N-Dimethylformamide is slowly added to
Into above-mentioned solution, 2h is stirred after mixing completely；System is imported in the ice water of 100mL, with saturated sodium bicarbonate solution tune pH
To 8, a large amount of white solids are precipitated, filters, obtains compound B:4- (4- formoxyl -3- hydroxy phenyl) piperazine -1- Ethyl formate.
3) B (0.557g, 2.00mmol) obtained by step 2) and ethyl acetoacetate (400 μ L, 3.00mmol) are added
In 20mL ethyl alcohol, 100 μ L piperidines are added and are catalyst, 78 DEG C of reflux 6h to fully reacting；It is cooled to room temperature, yellow needles is precipitated
Solid filters, obtains compound C:4- (3- acetyl group -2- oxo -2H- chromene -7- base) piperazine -1- Ethyl formate.
4) C (0.690g, 2.00mmol) and terephthalaldehyde (0.400g, 3.00mmol) obtained by step 3) are dissolved in
In 20mL ethyl alcohol, 100 μ L piperidines are added and are catalyst, 90 DEG C of reflux 72h to fully reacting；It is cooling, yellow solid is precipitated, takes out
Filter, obtains yellow powdery solid, purifies to obtain compound through silica gel column chromatography (dichloromethane/ethyl acetate, 15/1, V/V, elution)
D:(E) -4- (3- (3- (4- Fonnylphenyl) acryloyl group) -2- oxo -2H- chromene -7- base) piperazine -1- Ethyl formate.
5) D (0.230g, 0.500mmol) and 2- cyanoacetic acid (0.065g, 1.50mmol) obtained by step 4) are dissolved in
In 20mL ethyl alcohol, 100 μ L piperidines are added and are catalyst, 78 DEG C of reflux 4h to fully reacting；System evaporated under reduced pressure solvent obtains slightly
Product purifies to obtain red powder solid XI-2 through silica gel column chromatography (methylene chloride/methanol, 10/1, V/V, elution) purifying.
1H NMR (600MHz, DMSO) δ (ppm): 8.65 (s, 1H), 7.97 (t, J=12.5Hz, 4H), 7.85 (d, J=
8.2Hz, 2H), 7.76 (d, J=9.0Hz, 1H), 7.71 (d, J=15.7Hz, 1H), 7.05 (dd, J=9.0,2.0Hz, 1H),
6.90 (s, 1H), 4.08 (q, J=7.1Hz, 2H), 3.58-3.50 (m, 8H), 1.21 (t, J=7.1Hz, 3H) (Fig. 1)13C
NMR(151MHz,DMSO-d6)δ(ppm):186.3,160.0,158.2,155.3,155.1,148.8,141.4,132.5,
(130.5,129.3,126.8,117.9,112.1,109.7,98.9,61.4,46.5,15.0. Fig. 2) .HR-MS [probe+H]+:m/
Z Calcd 528.5405, Found 528.17674. (Fig. 3)
Embodiment 2
It prepares pH=7.4, the PBS buffer solution that concentration is 10mM, prepares the DMSO solution of 2mM XI-2, prepare 20mM half
Cystine aqueous solution；The DMSO solution of PBS-DMSO (10mM, pH=7.4,4:1, v/v) solution, 10 μ L XI-2 for taking 2mL adds
Into fluorescence cuvette, the volume for being gradually added into cysteine solution is 0,5,10,15,20,25,30,35,40,45 μ L, simultaneously
In Fluorescence Spectrometer measure 500nm place fluorescence intensity be 50.23,898.8,1590,1917,2360,2567,2754,2952,
2962,3081, fluorescence intensity gradually increases.Fluorescent emission figure is shown in Fig. 4.
Embodiment 3
It prepares pH=7.4, the PBS buffer solution that concentration is 10mM, prepares the DMSO solution of 2mM XI-2, prepare 0.2M's
Aqueous cystein solution prepares n-ethylmaleimide (NEM) solution of 0.2M；The DMSO solution of 10 μ L XI-2 is added
Into the fluorescence cuvette of 2mL PBS-DMSO (10mM, pH=7.4,4:1, v/v), 10 μ L cysteine solutions are taken, in addition
It states in buffer solution, when system fluorescence intensity is no longer changed, takes 0.5 μ L NEM solution, be added in above-mentioned solution,
When system fluorescence intensity is no longer changed, 2 μ L NEM solution are continuously added, are no longer changed to system fluorescence intensity
When, it repeats addition 2 μ L NEM solution 4 times and is not changing to system fluorescence intensity；10 μ L cysteine solutions are added again
So that the fluorescence of system increases again.Fluorescent emission figure is shown in Fig. 5.A fluorescence cuvette separately is taken, the DMSO 10 μ L XI-2 is molten
Liquid is added in the fluorescence cuvette of 2mL PBS-DMSO (10mM, pH=7.4,4:1, v/v), takes 10 μ L cysteine solutions,
It is added in above-mentioned buffer solution, when system fluorescence intensity is no longer changed, takes 10 μ L n-ethylmaleimide
(NEM) solution is added in this cuvette, is detected on Fluorescence spectrophotometer, with the addition of n-ethylmaleimide,
Fluorescence intensity reduces at 500nm, and system fluorescence is gradually restored to the fluorescence intensity of probe itself.Fluorescent emission figure is shown in Fig. 6.
Embodiment 4
It prepares pH=7.4, the PBS buffer solution that concentration is 10mM, prepares the DMSO solution of 2mM XI-2, prepare 0.2M's
Aqueous cystein solution prepares 0.2M Ala, Asn, Asp, Arg, Gln, Glu, Gly, Ile, Leu, Met, Pro, Ser respectively,
Thr,Trp,Tyr,Val,GSH,Hcy,K+, Na+, Mg2+, Mn2+,Ca2+, Cu2+, Cl-, SO4 2-, NO3 -, Br-, OH-, CNS-,
CO3 2-, S2O3 2-, SO3 2-, S2-Aqueous solution；In 35 fluorescence cuvettes, each PBS-DMSO (4:1, pH 7.4) that 2mL is added
The DMSO solution and the 50 various analytes of μ L of solution and 10 μ L XI-2: Ala, Asn, Asp, Arg, Gln, Glu, Gly, Ile,
Leu,Met,Pro,Ser,Thr,Trp,Tyr,Val,GSH,Hcy,K+, Na+, Mg2+, Mn2+,Ca2+, Cu2+, Cl-, SO4 2-, NO3 -,
Br-, OH-, CNS-, CO3 2-, S2O3 2-, SO3 2-, S2-And 5 μ L Cys, detected on Fluorescence spectrophotometer, draw different analyses
Fluorescence intensity histogram at the corresponding 500nm of object (see Fig. 7).Cysteine makes detection architecture fluorescence intensity at 500nm bright
Aobvious to increase, other analytes do not cause the variation of detection architecture fluorescence intensity substantially.
Embodiment 5
It prepares pH=7.4, the PBS buffer solution that concentration is 10mM, and prepares the solution of 2mM XI-2 with DMSO, prepare
20mM aqueous cystein solution；In 6 cuvettes, each PBS/DMSO (v/v, 4:1, pH 7.0) solution, 10 μ L that 2mL is added
The DMSO solution of XI-2, after the volume for being separately added into cysteine solution is 0,1,2.5,3.3,5,6,7.5,9,10 μ L, 5min
It is 50.46,285.0,555.4,845.7,1087,1321 that fluorescence intensity at 500nm is measured in Fluorescence Spectrometer, with half Guang ammonia
Acid concentration is abscissa, draws Fig. 8 by ordinate of fluorescence intensity, obtains the working curve of semicystinol concentration；Linear regression
Equation are as follows: the unit of F=12.92c+44.92667, c are 10-6mol/L；
Embodiment 6
It prepares pH=7.4, the PBS buffer solution that concentration is 10mM, and prepares the solution of 2mM XI-2 with DMSO, prepare
20mM aqueous cystein solution；The DMSO solution of 10 μ L XI-2 be added to 2mL PBS-DMSO (10mM, pH=7.4,4:1,
V/v in fluorescence cuvette), the 6.5 μ L of solution of cysteine is taken, is added in this cuvette with microsyringe, while glimmering
It is 898.8 that 500nm fluorescence intensity is measured on photothermal spectroscopic analyzer, by the equation of linear regression of embodiment 5, acquire c=66.09 ×
10-6mol/L.Deviation is 1.6%.See Fig. 9.
Embodiment 7
It prepares pH=7.4, the PBS buffer solution that concentration is 10mM, prepares the DMSO solution of 2mM XI-2, buffered with PBS
Solution prepares the glutathione of 20mM cysteine solution, 15 μM of homocysteine, 1mM respectively, is matched with PBS buffer solution
1mM NEM (n-ethylmaleimide, mercaptan scavenger) solution processed；The DMSO solution of 10 μ L XI-2 is added to 1990 μ L
PBS in；Probe solution is added in HepG-2 cell culture fluid, so that its concentration is 10 μM, with HepG-2 cell at 37 DEG C
Under, it is incubated for 30min, system is shown under phosphorimager green fluorescence generation on cell membrane, i.e. carboxyl and film on XI-2
The amino of albumen forms peptide chain, is stuck in cell membrane, responds with the cysteine on cell membrane, generates green fluorescence, such as Figure 10
(A)；The NEM solution of 2mL is added in HepG-2 cell culture fluid, 30min is incubated at 37 DEG C, siphons away solution, rinsed with PBS
Once, the probe solution for adding 10 μM of 2mL, is incubated for 30min at 37 DEG C, and system shows that green is glimmering under phosphorimager
In light distribution cytoplasm, i.e. after NEM effect, the permeability of after birth is improved, and XI-2 being capable of half Guang ammonia in efficient detection cytoplasm
Acid, such as Figure 10 (B)；The NEM solution of 2mL is added in HepG-2 cell culture fluid, 30min is incubated at 37 DEG C, siphons away solution,
It is rinsed once with PBS, 20 μ L 20mM cysteine solutions is added in the PBS of 1980 μ L；Cysteine solution is added
In HepG-2 cell culture fluid, so that its concentration is 200 μM, with HepG-2 cell at 37 DEG C, it is incubated for 30min, is rinsed with PBS
Once, the probe solution for adding 10 μM of 2mL, is incubated for 30min at 37 DEG C, and system shows that green is glimmering under phosphorimager
Light distribution is in cytoplasm, i.e. after NEM effect, XI-2 enters cell, not only responds with the cysteine in cytoplasm, moreover it is possible to
Exogenous cysteine is reacted, such as Figure 10 (C)；The NEM solution of 2mL is added in HepG-2 cell culture fluid, at 37 DEG C
Lower incubation 30min, siphons away solution, is rinsed once with PBS, and it is thin that 15 μM of the homocysteine solution of 2mL is incubated for HepG-2
Born of the same parents are incubated for 30min at 37 DEG C, are rinsed once with PBS, add 10 μM of 2mL of probe solution, be incubated at 37 DEG C
30min, system show that green fluorescence is distributed in cytoplasm under phosphorimager, comparison diagram 10 (B) and Figure 10 (C) discovery
After NEM effect, XI-2 enters cell, only responds with the cysteine in cytoplasm, such as Figure 10 (D)；The NEM solution of 2mL is added
Enter in HepG-2 cell culture fluid, be incubated for 30min at 37 DEG C, siphon away solution, is rinsed with PBS once, by the paddy of the 1mM of 2mL
The sweet peptide solution of Guang is incubated for HepG-2 cell, at 37 DEG C, is incubated for 30min, is rinsed once with PBS, add 10 μM of 2mL of spy
Needle solution is incubated for 30min at 37 DEG C, and system shows that green fluorescence is distributed in cytoplasm under phosphorimager, comparison diagram
After 10 (B) and Figure 10 (C) discovery NEM effect, XI-2 enters cell, only responds with the cysteine in cytoplasm, such as Figure 10
(E)。
Figure 10 be the test of confocal fluorescent imager for the detection of cell membrane cysteine (green fluorescence is mainly distributed
On cell membrane) and NEM effect after, probe in cytoplasm cysteine high-efficiency fluorescence response (green fluorescence is mainly distributed
In cytoplasm) and after NEM is acted on again and in cytoplasm and the response of the cysteine of external source (display green fluorescence) imaging
Figure.Therefore, it from result above, proposes under NEM regulating and controlling effect, XI-2 can efficiently, continuously monitor half Guang ammonia in cytoplasm
Acid.
Figure 11 is imaging cells Fluorescence Intensity Assays figure in Figure 10 Green channel.By each imaging in Figure 10 Green channel
As a result it is analyzed, concrete operations are as follows: the cell in every green fluorescence image is individually calculated into average fluorescent strength
(only circle cellular portions), i.e., first calculate the fluorescence intensity of each cell, then sum it up and calculate average value again.By comparison
5 average values find that the cysteine in cytoplasm will be more than the content of cell membrane cysteine；And whether homotype half
The result of cystine or the cell average fluorescent strength of glutathione processing is all and only with the result one of NEM and XI-2 processing
Sample, therefore can also be concluded that: NEM be only involved in the whole process reacted with the sulfydryl of memebrane protein into
And change permeability of cell membranes, without the mercaptan in scavenger-cell matter.
Figure 12 is the individual cells fluorescence distribution analysis chart of Figure 10 middle probe incubated cell result.Probe is directly incubated for carefully
Born of the same parents, green fluorescence are mainly distributed on cell membrane.It may be to be led since the amino of carboxyl and memebrane protein on probe forms peptide chain
Probe is caused to be stuck in cell membrane surface.
Embodiment 8
Mouse is subcutaneously injected with yellow Jackets (100 μ L 0.5mL/0.03%) and is anaesthetized.By XI-2 solution (20 μ L,
0.1mM), it is subcutaneous to be injected at mouse.Mouse only adds XI-2 to clap one group of photo under phosphorimager, then injects at same position
Cysteine (20 μ L, 0.2mM) does time series.In temporal sequence 0.5,5,10,20,25,30min records fluorogram respectively
Picture.The experimental results showed that when cysteine is added, observe that fluorescence intensity enhances in 20 minutes time ranges, then by
Body maintains, and illustrates that XI-2 can be used successfully to cysteine in Mice Body and be imaged.(a) in Figure 13 is without the naked of any processing
XI-2 (b) is only subcutaneously injected in mouse, and (c-h) is respectively that cysteine 0.5,5,10,20,25,30min is subcutaneously injected.
Claims (8)
1. a kind of XI-2 of coumarin derivative containing carboxyl, which is characterized in that structural formula are as follows:
2. a kind of synthetic method of the XI-2 of coumarin derivative containing carboxyl as described in claim 1, which is characterized in that step is such as
Under:
1) ethyl chloroformate and sodium bicarbonate are dissolved in tetrahydrofuran, are slowly dropped to the tetrahydro of hydroxy phenyl piperazine between containing
In furans and the mixed solution of deionized water, stirred overnight at room temperature；Extraction, dry, vacuum distillation obtains white needle-like crystals
Close object A:4- (3- hydroxy phenyl) piperazine -1- Ethyl formate；Wherein ethyl chloroformate, sodium bicarbonate and hydroxy phenyl piperazine
Molar ratio 1.4-1.7:1.1-1.4:1；
2) under the conditions of ice-water bath, excessive phosphorus oxychloride is slowly dropped in n,N-Dimethylformamide, is stirred, by 0 DEG C
The compound A being dissolved in n,N-Dimethylformamide is added in above-mentioned solution, stirring；System is poured into ice water, pH is adjusted
To a large amount of white solids are precipitated, filter, obtain compound B:4- (4- formoxyl -3- hydroxy phenyl) piperazine -1- Ethyl formate；
3) compound B obtained by step 2) and ethyl acetoacetate are added in ethyl alcohol 1:1.5-2.0 in molar ratio, and catalytic amount is added
Piperidines, be back to fully reacting；It is cooled to room temperature, yellow needles solid is precipitated, filters to obtain compound C:4- (3- acetyl group-
2- oxo -2H- chromene -7- base) piperazine -1- Ethyl formate；
4) in molar ratio 1:1.5-2.0 by compound C obtained by step 3) and dissolving terephthalaldehyde in ethanol, catalytic amount is added
Piperidines, be back to fully reacting；It is cooling, yellow solid is precipitated, filters to obtain yellow powdery solid, must change through pillar layer separation
Close object D:(E) -4- (3- (3- (4- Fonnylphenyl) acryloyl group) -2- oxo -2H- chromene -7- base) piperazine -1- formic acid second
Ester；
5) compound D and 2- cyanoacetic acid obtained by step 4) is dissolved in ethyl alcohol by 1:2.5-3.5 in molar ratio, and catalytic amount is added
Piperidines is back to fully reacting；System evaporated under reduced pressure solvent obtains crude product, through pillar layer separation, purifies to obtain target compound
XI-2。
3. the synthetic method of the XI-2 of coumarin derivative containing carboxyl as claimed in claim 2, which is characterized in that the step 1)
Middle tetrahydrofuran and deionized water volume ratio are 1:1；The ethyl chloroformate, sodium bicarbonate and hydroxy phenyl piperazine rub
You are than being 1.5:1.25:1.
4. the preparation method of the D of coumarin derivative containing carboxyl as claimed in claim 2, which is characterized in that C in the step 4)
It is 1:1.5 with the molar ratio of terephthalaldehyde, the eluant, eluent of column chromatography is that the volume ratio of methylene chloride and ethyl acetate is 15:1
5. the preparation method of the XI-2 of coumarin derivative containing carboxyl as claimed in claim 2, which is characterized in that the step 5)
The molar ratio of middle D and terephthalaldehyde is 1:1.5, and the eluant, eluent of column chromatography is that the volume ratio of methylene chloride and methanol is 10:1.
6. a kind of method of specific detection cysteine, includes the following steps:
(1), it prepares pH=7.4, the PBS buffer solution that concentration is 10mM, prepares the aqueous cystein solution of 20mM, configure 2mM
XI-2 DMSO solution；
(2), take the mixed solution that 2mL volume ratio is the PBS and DMSO of the pH 7.4 of 4:1 into a fluorescence cuvette, 10 μ L
The DMSO solution of XI-2 is added in above-mentioned system, is detected on Fluorescence spectrophotometer, with adding to test sample cysteine
Enter, the fluorescence intensity at 500nm gradually increases；
(3), in 6 cuvettes, the mixed solution of each PBS and DMSO that the pH 7.4 that 2mL volume ratio is 4:1 is added, respectively
The volume that cysteine solution is added measures fluorescence at 500nm in Fluorescence Spectrometer after being 0,2,4,6,8,10 μ L, 5min strong
Degree is 50.46,285.0,555.4,845.7,1087,1321, is vertical sit with fluorescence intensity using cysteine ??acid concentration as abscissa
Drawing is marked and drawed, the working curve of semicystinol concentration is obtained；Equation of linear regression are as follows: the unit of F=12.92c+44.92667, c
It is 10-6mol/L；
(4), when measuring sample solution, the fluorescence intensity measured is substituted into equation of linear regression, the dense of cysteine can be acquired
Degree.
7. the XI-2 of coumarin derivative containing carboxyl as described in claim 1 is in preparation extracorporal circulatory system detection cysteine reagent
Application.
8. the XI-2 of coumarin derivative containing carboxyl is preparing answering in cell cysteine detection reagent as described in claim 1
With.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811324486.9A CN109516969B (en) | 2018-11-08 | 2018-11-08 | Carboxyl-containing coumarin derivative and synthesis method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811324486.9A CN109516969B (en) | 2018-11-08 | 2018-11-08 | Carboxyl-containing coumarin derivative and synthesis method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109516969A true CN109516969A (en) | 2019-03-26 |
| CN109516969B CN109516969B (en) | 2022-03-18 |
Family
ID=65774498
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811324486.9A Active CN109516969B (en) | 2018-11-08 | 2018-11-08 | Carboxyl-containing coumarin derivative and synthesis method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109516969B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105376A (en) * | 2019-06-10 | 2019-08-09 | 山西大学 | A kind of fluorescein derivative and its synthetic method and application |
| CN111233804A (en) * | 2020-03-03 | 2020-06-05 | 山西大学 | Benzopyranoylium ion-coumarin derivative and synthetic method and application thereof |
| CN111303072A (en) * | 2020-02-27 | 2020-06-19 | 山西大学 | Reagent for distinguishing and detecting cysteine and synthetic method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108358906A (en) * | 2018-03-14 | 2018-08-03 | 中南大学 | One species specificity distinguishes the fluorescence probe of different mercaptan |
-
2018
- 2018-11-08 CN CN201811324486.9A patent/CN109516969B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108358906A (en) * | 2018-03-14 | 2018-08-03 | 中南大学 | One species specificity distinguishes the fluorescence probe of different mercaptan |
Non-Patent Citations (1)
| Title |
|---|
| YANG YANG,等: "Dual-Site and Dual-Excitation Fluorescent Probe That Can Be Tuned for Discriminative Detection of Cysteine, Homocystein, and Thiophenols", 《ANAL. CHEM.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105376A (en) * | 2019-06-10 | 2019-08-09 | 山西大学 | A kind of fluorescein derivative and its synthetic method and application |
| CN110105376B (en) * | 2019-06-10 | 2021-07-02 | 山西大学 | Fluorescein derivative and synthetic method and application thereof |
| CN111303072A (en) * | 2020-02-27 | 2020-06-19 | 山西大学 | Reagent for distinguishing and detecting cysteine and synthetic method and application thereof |
| CN111233804A (en) * | 2020-03-03 | 2020-06-05 | 山西大学 | Benzopyranoylium ion-coumarin derivative and synthetic method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109516969B (en) | 2022-03-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107235946A (en) | A kind of glutathione fluorescence probe and its preparation method and application | |
| CN105153214B (en) | A kind of silicon substrate rhodamine nitric oxide fluorescent probe and its preparation method and application | |
| CN106632326B (en) | Double pyrene modification imide derivative fluorescence probes and its synthetic method and application | |
| CN106950210B (en) | A kind of reagent detecting glutathione and its synthetic method and application | |
| CN109516969A (en) | One kind coumarin derivative containing carboxyl and its synthetic method and application | |
| CN110003060A (en) | A kind of malononitrile derivative species near-infrared hydrogen sulfide fluorescence probe and the preparation method and application thereof | |
| CN107652220A (en) | A kind of preparation method and application technology for the fluorescence probe for detecting cysteine | |
| CN109438319A (en) | A kind of compound and its preparation method and application detecting leucine amino peptidase | |
| CN106432164B (en) | A kind of coumarin derivative DOCOPA and its preparation method and application | |
| CN106518855B (en) | It is a kind of using half river cyanines and flavonols as the Sulfur Dioxide-derivatives scale fluorescence probe of fluorogen and its application | |
| CN110143954A (en) | A kind of coumarin derivative and its synthetic method and application | |
| CN107459483A (en) | A kind of cell membrane targets H2S fluorescence probes and its preparation method and application | |
| CN111072648A (en) | Fluorescent probe for detecting biological thiol in lysosome as well as preparation method and application thereof | |
| CN109897625A (en) | Selective enumeration method cysteine fluorescence probe and its synthetic method and application | |
| CN111217799A (en) | Indole salt-coumarin derivative and synthesis method and application thereof | |
| CN101012207A (en) | Fluorescent probe for detecting ultra-oxygen anion free radical, synthesis method and use | |
| CN105601658A (en) | Application and preparation method of novel fluorescent probe capable of distinguishing biological mercaptans | |
| CN106478505B (en) | A kind of two-photon GSH probe and its preparation and application | |
| CN108530457A (en) | Amino acid detection reagent naphthalimide derivative and its synthetic method and application | |
| CN103951673A (en) | Reagent and application thereof in mercaptan detection | |
| Chen et al. | Responsive luminescent silver-based metal-organic frameworks for highly sensitive and selective detection of hydrogen sulfide in biological system via a self-assembled headspace separation device | |
| CN105777607B (en) | A kind of double indoles salt compounded of iodine of triphenylamine and its synthetic method and application | |
| CN106047339B (en) | Amphipathic pyrene derivatives fluorescence probe and its synthetic method based on cholic acid modification and application | |
| CN110372681B (en) | Application of self-assembled nano fluorescent probe for selectively detecting human serum albumin | |
| CN106008463A (en) | Maleimide derivative and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |