CN109608414A - Detect the fluorescence probe and its preparation method and application of peroxynitrite - Google Patents
Detect the fluorescence probe and its preparation method and application of peroxynitrite Download PDFInfo
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- CN109608414A CN109608414A CN201811600544.6A CN201811600544A CN109608414A CN 109608414 A CN109608414 A CN 109608414A CN 201811600544 A CN201811600544 A CN 201811600544A CN 109608414 A CN109608414 A CN 109608414A
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- 239000000523 sample Substances 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 title claims abstract 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000001514 detection method Methods 0.000 claims abstract description 39
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 claims abstract description 7
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 28
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 18
- 235000019253 formic acid Nutrition 0.000 claims description 18
- 238000003384 imaging method Methods 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 12
- 239000007795 chemical reaction product Substances 0.000 claims description 11
- 229940125904 compound 1 Drugs 0.000 claims description 9
- 229940125782 compound 2 Drugs 0.000 claims description 9
- 201000007270 liver cancer Diseases 0.000 claims description 9
- 208000014018 liver neoplasm Diseases 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- NCGICGYLBXGBGN-UHFFFAOYSA-N 3-morpholin-4-yl-1-oxa-3-azonia-2-azanidacyclopent-3-en-5-imine;hydrochloride Chemical group Cl.[N-]1OC(=N)C=[N+]1N1CCOCC1 NCGICGYLBXGBGN-UHFFFAOYSA-N 0.000 claims description 4
- 229940126214 compound 3 Drugs 0.000 claims description 4
- 238000010898 silica gel chromatography Methods 0.000 claims description 4
- -1 benzo phenoxazine Piperazine Chemical compound 0.000 claims description 3
- 238000010226 confocal imaging Methods 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 230000004044 response Effects 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 5
- YOSZEPWSVKKQOV-UHFFFAOYSA-N 12h-benzo[a]phenoxazine Chemical compound C1=CC=CC2=C3NC4=CC=CC=C4OC3=CC=C21 YOSZEPWSVKKQOV-UHFFFAOYSA-N 0.000 abstract description 4
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 17
- 238000012360 testing method Methods 0.000 description 12
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 239000002904 solvent Substances 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000010792 warming Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000012921 fluorescence analysis Methods 0.000 description 2
- 238000012632 fluorescent imaging Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 2
- 229940116269 uric acid Drugs 0.000 description 2
- TZMSYXZUNZXBOL-UHFFFAOYSA-N 10H-phenoxazine Chemical compound C1=CC=C2NC3=CC=CC=C3OC2=C1 TZMSYXZUNZXBOL-UHFFFAOYSA-N 0.000 description 1
- VRVRGVPWCUEOGV-UHFFFAOYSA-N 2-aminothiophenol Chemical class NC1=CC=CC=C1S VRVRGVPWCUEOGV-UHFFFAOYSA-N 0.000 description 1
- NZTPZUIIYNYZKT-UHFFFAOYSA-N 6-aminonaphthalene-2-carboxylic acid Chemical compound C1=C(C(O)=O)C=CC2=CC(N)=CC=C21 NZTPZUIIYNYZKT-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 150000004893 oxazines Chemical class 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
- C07D265/38—[b, e]-condensed with two six-membered rings
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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"
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Abstract
The invention belongs to synthesize and detection technique field, more particularly to the fluorescence probe and its preparation method and application of detection peroxynitrite.The present invention provides the universal recognition group of detection peroxynitrite, and it is connect with common fluorogen and has constructed three kinds of new fluorescence probes, it is respectively designated as BT-FAP, NA-FAP, NR-FAP, they are respectively with benzothiazole-amino naphthalenes (BT), naphthalimide (NA), benzo phenoxazine (NR) for fluorogen, using formamide (FAP) as recognition group, it can be achieved that and ONOO‑Transient response (< 4s), and high sensitivity, selectivity are good, are highly suitable for ONOO in cell‑Fluorescence imaging research.
Description
Technical field
The invention belongs to synthesize and detection technique field, more particularly to based on formamide recognition group, can be used for detecting
The fluorescence probe and the preparation method and application thereof of oxygen nitrite anions.
Background technique
In vivo, peroxynitrite (ONOO-) be important active nitrogen species, by Superoxide radicalanion and
Nitric oxide reaction generates, can contemporary table oxidative stress and nitrification stress level.Moreover, existing studies have shown that ONOO-
It is closely related with the occurrence and development of a variety of diseases, including neurodegenerative disease, diabetes, cancer etc..ONOO-In physiology item
Half-life short and Css under part is extremely low, so that the ONOO in detection living cells and living tissue-Become very difficult.Base
In fluorescence probe fluorescence analysis can in cell and animal model real-time visual molecular events, be the above problem solution
Certainly provide effective analysis method.However, detection ONOO reported at present-The generally existing following problems of fluorescence probe:
(1) recognition group does not have general applicability, is difficult to be generalized to other fluorophors, increases the design difficulty of new probe；(2)
Probe chemical structure is complicated, and synthetic route is long, and synthesis is difficult, cannot achieve large-scale production.These problems cause existing
ONOO-The practical application of fluorescence probe be greatly limited, be unfavorable for large-scale promotion application, therefore, it is necessary to develop
New is used to detect ONOO-Fluorescence probe and preparation method thereof.
Summary of the invention
For above-mentioned problems of the prior art, the present invention is intended to provide the fluorescence probe of detection peroxynitrite
And its preparation method and application.The present invention provide detection peroxynitrite universal recognition group, and by its with commonly use it is glimmering
Three kinds of new fluorescence probes have been constructed in light blob connection, are respectively designated as BT-FAP, NA-FAP, NR-FAP, they are respectively with benzene
And thiazole-amino naphthalenes (BT), naphthalimide (NA), benzo phenoxazine (NR) they are fluorogen, using formamide (FAP) as identification base
Group is, it can be achieved that and ONOO-Transient response (< 4s), and high sensitivity, selectivity are good, are highly suitable for ONOO in cell-It is glimmering
Light imaging research.
An object of the present invention is to provide the fluorescence of three kinds of detection peroxynitrites based on formamide recognition group
Probe.
The second object of the present invention is to provide the fluorescence of the above-mentioned detection peroxynitrite based on formamide recognition group
The preparation method of probe.
The third object of the present invention is to provide the fluorescence of the above-mentioned detection peroxynitrite based on formamide recognition group
Imaging method of the probe in liver cancer cells.
The fourth object of the present invention is to provide the fluorescence probe of the detection peroxynitrite based on formamide recognition group
And its application of the imaging method in liver cancer cells.
For achieving the above object, specifically, the invention discloses following technical proposals:
Firstly, the present invention discloses the fluorescence probe of the first detection peroxynitrite based on formamide recognition group,
It is named as BT-FAP, structural formula are as follows:
Secondly, the present invention discloses the fluorescence probe of second of detection peroxynitrite based on formamide recognition group,
It is named as NA-FAP, structural formula are as follows:
Again, the present invention discloses the fluorescence probe of the third detection peroxynitrite based on formamide recognition group,
It is named as NR-FAP, structural formula are as follows:
Thirdly, the fluorescence that the present invention discloses the above-mentioned three kinds detection peroxynitrites based on formamide recognition group is visited
The preparation method of needle, reaction equation are respectively as follows:
Wherein, the compound 1 indicates that benzothiazole-amino naphthalenes, compound 2 indicate that naphthalimide, compound 3 indicate benzene
And phenoxazine.
Reaction step are as follows: the compound 1,2 or 3 in above-mentioned reaction equation is dissolved in formic acid respectively, after mixing evenly,
Mixed liquor, react setting time to get.
Further, in above-mentioned reaction step, the ratio of the compound 1,2 or 3 and formic acid are as follows: 0.2-1.5mM:0.5-
5mL。
Further, in above-mentioned reaction step, the reaction temperature is 60-100 DEG C, reaction time 3-6h；Preferably,
The temperature of the reaction is 75-85 DEG C, and the time of reaction is 4.5-5h.
Further, in above-mentioned reaction step, further include the steps that for reaction product solution being spin-dried for, purify.It is preferred that
Ground purifies reaction product using silica gel column chromatography.
It is highly preferred that the eluant, eluent of the silica gel column chromatography be methylene chloride and methanol mixed liquor, and compound 1,2 or
3 volume ratios for purifying methylene chloride and methanol used with three reaction products of formic acid respectively are followed successively by 80-130:1,75- respectively
125:1,40-60:1；It is respectively preferably 100:1,100:1,50:1.
In addition, the fluorescence probe of invention additionally discloses the above-mentioned detection peroxynitrite based on formamide recognition group exists
To ONOO in living cells-Application in imaging.Specifically, the application is imaging method of the fluorescence probe in liver cancer cells,
Include the following steps: cultured liver cancer cells ONOO-Releasing agent is incubated for the dimethyl sulfoxide solution of probe again after being incubated for,
After being incubated for setting time, the dimethyl sulfoxide solution of probe is removed, and cleaned with PBS, then carry out laser confocal imaging, i.e.,
?.
Preferably, in above-mentioned imaging method, the ONOO-Releasing agent is SIN-1.
Preferably, in above-mentioned imaging method, the time of the incubation is 20-40min.
Finally, the present invention disclose it is above-mentioned based on formamide recognition group detection peroxynitrite fluorescence probe and its
Application of the imaging method in field of biological detection in liver cancer cells.
Compared with prior art, the beneficial effect that the present invention obtains is:
(1) three kinds of probe molecule maximum emission wavelengths proposed by the present invention are located at 500nm, 560nm, 620nm,
With moderate Stokes shift (about 50nm), it can be effectively reduced self-absorption, improve the accuracy of imaging.
(2) three kinds of probe molecules proposed by the present invention can be realized to ONOO-Quick response (reacted in 4s
Finish), and to ONOO-With highly sensitive and selectivity, probe dyeing effect in living cells is good, and dyeing time is shorter
(20min), dyeing efficiency is higher, is highly suitable for ONOO in cell-Fluorescence imaging research.
(3) three kinds of probe molecules proposed by the present invention can be realized ONOO in the liver cancer cells being incubated for SIN-1-Concentration
Raised instruction, so that the ONOO in detection living cells and living tissue-It is more easier, realizes fluorescence analysis well
Method real-time visual molecular events in cell and animal model.
(4) synthesis step of three kinds of probe molecules of the invention is simple, yield is high, easy purification；And preparation of the invention
Directly using formic acid as reactant in method, while using formic acid as solvent, other complementary solvents or reagent, cost are not used
Low, environmental pollution is small.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation are not constituted an undue limitation on the present application for explaining the application.
Fig. 1 is ONOO of three kinds of fluorescence probes to various concentration of embodiment 1-Fluorescence response spectrogram；Abscissa is
Wavelength (nm), ordinate are fluorescent emission intensity.
Fig. 2 be embodiment 1 three kinds of fluorescence probes respectively the fluorescence intensity at 500nm, 560nm and 620nm from it is different
The ONOO of concentration-Linear relationship；Abscissa is ONOO-Concentration (μM), ordinate are probe in respective wavelength (500nm, 560nm
And 620nm) at fluorescent emission intensity.
Fig. 3 is that the fluorescence intensity at 500nm, 560nm and 620nm is at any time respectively for three kinds of fluorescence probes of embodiment 1
Real-time change figure；Abscissa is the time (s), and ordinate is three kinds of probes at respective wavelength (500nm, 560nm and 620nm)
Fluorescent emission intensity.
Fig. 4 be in the presence of variety classes substance, three kinds of fluorescence probes of embodiment 1 respectively in 500nm, 560nm and
Fluorescence intensity situation at 620nm.Abscissa is variety classes substance, and ordinate is probe in respective wavelength (500nm, 560nm
And 620nm) at fluorescent emission intensity.
Fig. 5 is fluorescence imaging figure of the three kinds of fluorescence probes of embodiment 1 in liver cancer cells (HepG2).
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, currently, existing detection ONOO-The generally existing following problems of fluorescence probe:
(1) recognition group does not have general applicability, is difficult to be generalized to other fluorophors, increases the design difficulty of new probe；(2)
Probe chemical structure is complicated, and synthetic route is long, and synthesis is difficult, cannot achieve large-scale production.For this purpose, present invention proposition detected
The fluorescence probe and its preparation method and application of oxygen nitrite anions, with reference to the accompanying drawings and detailed description to the present invention into one
Walk explanation.
It should be understood that the present invention is using existing existing raw material compound 1,2,3, by a step in the following example
Reaction simple efficiently synthesizes, and finally prepares three kinds of probes BT-FAP, NA-FAP and NR-FAP by silica gel chromatograph post separation.Example
Such as, compound 1 is heated to 150 DEG C, reaction obtains overnight by 6- amino -2- naphthoic acid and near amino thiophenols in polyphosphoric acids
?.Compound 2 reference literature Cui, L.；Peng,Z.；Ji,C.；Huang,J.；Huang,D.；Ma,J.；Zhang,S.；Qian,
X.；Xu, Y.Chem.Commun.2014,50,1485-1487 synthesis.Compound 3 reference literature Greenspan, P.；Fowler,
S.D.J.Lipid Res.1985,26,781-789 synthesis.
Embodiment 1
The preparation method of the fluorescence probe of three kinds of detection peroxynitrites based on formamide recognition group, reactional equation
Formula is respectively as follows:
Wherein, compound 1 indicates that benzothiazole-amino naphthalenes, compound 2 indicate that naphthalimide, compound 3 indicate benzo pheno
Oxazines.
Specifically, the preparation step of the above-mentioned three kinds of fluorescence probes of the present embodiment are as follows:
(1) 1mmol compound 1 or 2 or 3 (respectively 274mg or 268mg or 263mg) are mixed with 3mL formic acid respectively；
(2) three kinds of mixed liquors for then obtaining step (1) are warming up to 80 DEG C, react 5h；
(3) after completion of the reaction, three kinds of reaction solutions are cooled to room temperature, then concentration is spin-dried for solvent, the solid that will be obtained
Column chromatography for separation is carried out, three kinds of product solids: BT-FAP, NA-FAP, NR-FAP are respectively obtained, is as identified based on formamide
The fluorescence probe of the detection peroxynitrite of group；Wherein, compound 1,2 or 3 is mentioned with three reaction products of formic acid respectively
The volume ratio of pure methylene chloride used and methanol is followed successively by 100:1,100:1,50:1 respectively.
After testing, the quality of the present embodiment obtains BT-FAP, NA-FAP, NR-FAP be respectively 243mg, 257mg,
235mg；Being equivalent to yield is respectively 80%, 87%, 81%.
Embodiment 2
The preparation method of the fluorescence probe of three kinds of detection peroxynitrites based on formamide recognition group, reactional equation
Formula/principle is the same as embodiment 1, specific preparation step are as follows:
(1) 1.2mmol compound 1 or 2 or 3 are mixed with 5mL formic acid respectively；
(2) three kinds of mixed liquors for then obtaining step (1) are warming up to 60 DEG C, react 6h；
(3) after completion of the reaction, three kinds of reaction solutions are cooled to room temperature, then concentration is spin-dried for solvent, the solid that will be obtained
Column chromatography for separation is carried out, three kinds of product solids: BT-FAP, NA-FAP, NR-FAP are respectively obtained, is as identified based on formamide
The fluorescence probe of the detection peroxynitrite of group；Wherein, compound 1,2 or 3 is mentioned with three reaction products of formic acid respectively
The volume ratio of pure methylene chloride used and methanol is followed successively by 80:1,110:1,40:1 respectively.
After testing, the yield of the present embodiment obtains BT-FAP, NA-FAP, NR-FAP is respectively 84%, 86%,
82%.
Embodiment 3
The preparation method of the fluorescence probe of three kinds of detection peroxynitrites based on formamide recognition group, reactional equation
Formula/principle is the same as embodiment 1, specific preparation step are as follows:
(1) 1.5mmol compound 1 or 2 or 3 are mixed with 4mL formic acid respectively；
(2) three kinds of mixed liquors for then obtaining step (1) are warming up to 85 DEG C, react 4.5h；
(3) after completion of the reaction, three kinds of reaction solutions are cooled to room temperature, then concentration is spin-dried for solvent, the solid that will be obtained
Column chromatography for separation is carried out, three kinds of product solids: BT-FAP, NA-FAP, NR-FAP are respectively obtained, is as identified based on formamide
The fluorescence probe of the detection peroxynitrite of group；Wherein, compound 1,2 or 3 is mentioned with three reaction products of formic acid respectively
The volume ratio of pure methylene chloride used and methanol is followed successively by 120:1,125:1,55:1 respectively.
After testing, the yield of the present embodiment obtains BT-FAP, NA-FAP, NR-FAP is respectively 87%, 84%,
85%.
Embodiment 4
The preparation method of the fluorescence probe of three kinds of detection peroxynitrites based on formamide recognition group, reactional equation
Formula/principle is the same as embodiment 1, specific preparation step are as follows:
(1) 0.2mmol compound 1 or 2 or 3 are mixed with 0.5mL formic acid respectively；
(2) three kinds of mixed liquors for then obtaining step (1) are warming up to 100 DEG C, react 3h；
(3) after completion of the reaction, three kinds of reaction solutions are cooled to room temperature, then concentration is spin-dried for solvent, the solid that will be obtained
Column chromatography for separation is carried out, three kinds of product solids: BT-FAP, NA-FAP, NR-FAP are respectively obtained, is as identified based on formamide
The fluorescence probe of the detection peroxynitrite of group；Wherein, compound 1,2 or 3 is mentioned with three reaction products of formic acid respectively
The volume ratio of pure methylene chloride used and methanol is followed successively by 130:1,90:1,60:1 respectively.
After testing, the yield of the present embodiment obtains BT-FAP, NA-FAP, NR-FAP is respectively 82%, 88%,
83%.
Embodiment 5
The preparation method of the fluorescence probe of three kinds of detection peroxynitrites based on formamide recognition group, reactional equation
Formula/principle is the same as embodiment 1, specific preparation step are as follows:
(1) 0.5mmol compound 1 or 2 or 3 are mixed with 1mL formic acid respectively；
(2) three kinds of mixed liquors for then obtaining step (1) are warming up to 75 DEG C, react 4.5h；
(3) after completion of the reaction, three kinds of reaction solutions are cooled to room temperature, then concentration is spin-dried for solvent, the solid that will be obtained
Column chromatography for separation is carried out, three kinds of product solids: BT-FAP, NA-FAP, NR-FAP are respectively obtained, is as identified based on formamide
The fluorescence probe of the detection peroxynitrite of group；Wherein, compound 1,2 or 3 is mentioned with three reaction products of formic acid respectively
The volume ratio of pure methylene chloride used and methanol is followed successively by 90:1,75:1,45:1 respectively.
After testing, the yield of the present embodiment obtains BT-FAP, NA-FAP, NR-FAP is respectively 86%, 83%,
87%.
Product property test, three kinds of fluorescence probes BT-FAP, NA-FAP, the NR-FAP prepared to embodiment 1 are tested,
As a result as follows:
(1) mass spectrum and nuclear-magnetism characterization:
BT-FAP: a pair of of rotational isomer.1H NMR(400MHz,DMSO-d6):δ10.56(s,0.7H),10.49(d,J
=10.9Hz, 0.3H), 9.05 (d, J=10.8Hz, 0.3H), 8.63 (s, 1H), 8.41 (d, J=9.2Hz, 1.7H), 8.20-
8.12 (m, 2.7H), 8.09 (d, J=8.1Hz, 1H), 8.02 (d, J=8.6Hz, 0.7H), 7.97 (d, J=8.5Hz, 0.3H),
7.78 (s, 0.3H), 7.68 (d, J=8.7Hz, 0.7H), 7.60-7.52 (m, 1.3H), 7.48 (t, J=7.4Hz, 1H)13C
NMR(100MHz,DMSO-d6):δ167.35,162.73,160.11,153.66,137.88,137.49,135.02,134.85,
134.48,130.53,129.86,129.55,129.10,128.93,128.47,128.06,127.25,127.08,126.68,
125.49,124.73,124.52,122.78,122.35,120.68,119.38,115.19,112.36.HRMS(ESI):
calculated for C18H13N2OS+(M+H+)305.0743,found 305.0746.
NA-FAP:1H NMR(400MHz,DMSO-d6): δ 10.96 (s, 1H), 8.66 (d, J=6.7Hz, 2H), 8.45 (d,
J=25.5Hz, 3H), 7.87 (s, 1H), 4.01 (br s, 2H), 1.70-1.50 (m, 2H), 1.41-1.29 (m, 2H), 0.93
(t, J=7.1Hz, 3H)13C NMR(100MHz,DMSO-d6):δ169.55,163.38,162.81,140.26,131.54,
130.73,129.10,128.20,126.25,123.82,122.17,119.12,117.30,39.25,29.65,19.81,
13.71.HRMS(ESI):calculated for C17H17N2O3 +(M+H+)297.1234,found 297.1239.
NR-FAP: a pair of of rotational isomer.1H NMR(400MHz,DMSO-d6):δ10.80(s,0.6H),10.66(d,J
=10.8Hz, 0.4H), 10.54 (s, 0.4H), 9.06 (d, J=10.7Hz, 0.4H), 8.62 (d, J=7.8Hz, 1H), 8.41
(s, 0.6H), 8.15 (d, J=7.4Hz, 1H), 7.92-7.80 (m, 3.6H), 7.50 (ddd, J=12.9,8.7,2.1Hz,
1H),6.47-6.40(m,1H).HRMS(ESI):calculated for C17H11N2O3 +(M+H+)291.0764,found
291.0758.
(1) three kinds of probes prepared by embodiment 1 are to ONOO-Fluorescence response experiment:
Probe (stock solution 1.0mM, 200 μM) is diluted with water after PBS (pH 7.4) buffering is added, and various concentration is added
ONOO-, make 2.0 μM of probe ultimate density, PBS concentration is 50mM, as a result as shown in Figure 1, probe BT-FAP is swashed with 385nm
ONOO when hair, in the basic unstressed configuration transmitting of 500nm or so, with various concentration-(the ONOO being corresponding in turn to from bottom to top-Concentration
Be 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM) be incubated for after, fluorescence significantly increases；Probe
ONOO when NA-FAP is excited with 435nm, in the basic unstressed configuration transmitting of 560nm or so, with various concentration-(from bottom to top successively
Corresponding ONOO-Concentration be 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM) be incubated for after, fluorescence is aobvious
Write enhancing；ONOO when probe NR-FAP is excited with 530nm, in the basic unstressed configuration transmitting of 620nm or so, with various concentration-(from
Under the ONOO that is up corresponding in turn to-Concentration be 0 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM) be incubated for
Afterwards, fluorescence significantly increases.And three kinds of probes fluorescence intensity and ONOO at 500nm, 560nm and 620nm respectively-Concentration presents good
Good linear correlation (Fig. 2).Calculating detection limit according to 3 σ of formula/k is respectively 64nM, 28nM and 102nM, illustrates three kinds of probes
With endogenous trace ONOO in detection biosystem-Ability.
(2) three kinds of probes prepared by embodiment 1 are to ONOO-Dynamic response experiment:
On the basis of above-mentioned (1), invention further contemplates three kinds of probes to ONOO-Dynamic response performance,
As a result as shown in figure 3, three kinds of probes (2.0 μM) itself fluorescence is weaker at 500nm, 560nm and 620nm respectively, work as addition
ONOO-After (test b T-FAP is added 10 μM, and test NA-FAP is added 10 μM, and test NR-FAP is added 4 μM), fluorescence increases rapidly
By force, vertex is reached in 4s, shows that probe being capable of transient response ONOO-, reactivity height, half in physiological environment can be captured
Decline phase short ONOO-。
(3) three kinds of probes prepared by embodiment 1 are selectively tested:
On the basis of above-mentioned (1), three kinds of probes of further progress of the present invention are to ONOO-Selection Journal of Sex Research, as a result such as
Shown in Fig. 4, in Fig. 4, label 1 represents ONOO-(test b T-FAP is added 10 μM, and test NA-FAP is added 10 μM, tests NR-FAP
It is added 4 μM), 2 represent blank control, and 3 represent 100 μM of ClO-, 4 represent 100 μM of H2O2, 5 represent 100 μM·OH, 6 represent 100 μM
O2 ·-, 7 represent 100 μM1O2, 8 represent 100 μM of NO, and 9 represent 100 μM of TBHP, and 10 represent 5mM GSH, and 11 represent 100 μM of Hcy,
12 represent 5mMCys, and 13 represent 100 μM of vitamin Cs, and 14 represent 100 μM of H2S, 15 represent 100 μM of HSO3 -, 16 represent 100 μM
SO4 2-, 17 represent 100 μM of PO4 3-, 18 represent 100 μM of NO2 -, 19 represent 100 μM of NO3 -, 20 represent 100 μM of AcO-, 21 represent
100μM HCO3 -, 22 represent 100 μM of CO3 2-, 23 represent 100 μM of Na+, 24 represent 100 μM of K+, 25 represent 100 μM of Mg2+, 26
Represent 100 μM of Cu2+, 27 represent 100 μM of Zn2+, 28 represent 100 μM of Ca2+, 29 represent 100 μM of Fe2+, 30 represent 100 μM of Fe3+。
As shown in Figure 4, other potential interference substances cannot cause the change in fluorescence of probe, including active oxygen/active nitrogen (H2O2,
TBHP, NO,1O2, O2 ·-, ClO-,·OH), active sulfur (GSH, Cys, Hcy, H2S), anion (HSO3 -, SO4 2-, PO4 3-, NO2 -,
NO3 -, AcO-, HCO3 -And CO3 2-), cation (Na+, K+, Mg2+, Cu2+, Zn2+, Ca2+, Fe2+And Fe3+) and vitamin C.It compares
Under, three kinds of probes are only in ONOO-Apparent fluorescence enhancement is shown in the presence of (corresponding label 1).In conclusion this implementation
Probe BT-FAP, NA-FAP and NR-FAP of example preparation are to ONOO-Specificity with height, is fully available in physiological environment
ONOO-Fluorescent visual research.
(4) probe dyes imaging experiment to living cells:
As shown in figure 5, the present invention further demonstrates three kinds of probes BT-FAP, NA-FAP and NR- of the preparation of embodiment 1
FAP may be implemented on a cellular level to ONOO-Detection.Control group: figure a, d and g be respectively probe BT-FAP, NA-FAP and
Colored graph of the NR-FAP to HepG2 cell.Experimental group: HepG2 cell first uses ONOO in figure b, e and h-Releasing agent SIN-1 is incubated for
Then 30min is incubated for 20min with three kinds of probes respectively again, then carry out confocal fluorescent imaging.Removing group: in figure c, f and i,
First with a kind of uric acid (known ONOO-Scavenger) pretreatment 3 hours of HepG2 cell, then use ONOO-Releasing agent SIN-1
It is incubated for 30min, is then incubated for 20min with three kinds of probes respectively, finally carries out confocal fluorescent imaging.The result shows that with compareing
Group is compared, and the fluorescence intensity of experimental group cell obviously increases, and after being pre-processed with uric acid compared with experimental group, cell fluorescence intensity is bright
Aobvious decline.Therefore, probe BT-FAP, NA-FAP and NR-FAP can be used for detecting ONOO in living cells-The fluctuation situation of content.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for those skilled in the art
For member, various changes and changes are possible in this application.Within the spirit and principles of this application, it is made it is any modification,
Equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
1. the fluorescence probe of the detection peroxynitrite based on formamide recognition group, it is characterised in that: respectively BT-FAP,
NA-FAP or NR-FAP, in which:
The structural formula of the BT-FAP are as follows:
Or；The structural formula of the NA-FAP are as follows:
Or；The structural formula of the NR-FAP are as follows:
2. the preparation side of the fluorescence probe of the detection peroxynitrite based on formamide recognition group as described in claim 1
Method, it is characterised in that:
Reaction equation is respectively as follows:
Wherein, compound 1 indicates that benzothiazole-amino naphthalenes, compound 2 indicate that naphthalimide, compound 3 indicate benzo phenoxazine
Piperazine；
Reaction step are as follows: the compound 1,2 or 3 in above-mentioned reaction equation is dissolved in formic acid respectively, after mixing evenly, is obtained mixed
Close liquid, react setting time to get.
3. preparation method as claimed in claim 2, it is characterised in that: the ratio of the compound 1,2 or 3 and formic acid are as follows:
0.2-1.5mM:0.5-5mL.
4. preparation method as claimed in claim 2, it is characterised in that: the reaction temperature is 60-100 DEG C, and the reaction time is
3-6h；Preferably, the temperature of the reaction is 75-85 DEG C, and the time of reaction is 4.5-5h.
5. such as the described in any item preparation methods of claim 2-4, it is characterised in that: further include revolving reaction product solution
The step of doing, purifying；Preferably, reaction product is purified using silica gel column chromatography.
6. preparation method as claimed in claim 5, it is characterised in that: the eluant, eluent of the silica gel column chromatography be methylene chloride and
The mixed liquor of methanol, and compound 1,2 or 3 purifies the body of methylene chloride and methanol used with three reaction products of formic acid respectively
Product ratio is followed successively by 80-130:1 respectively, 75-125:1,40-60:1；It is respectively preferably 100:1,100:1,50:1.
7. the fluorescence probe of the detection peroxynitrite based on formamide recognition group is in living cells as described in claim 1
In to ONOO-Application in imaging.
8. application as described in claim 1, it is characterised in that: the application is imaging side of the fluorescence probe in liver cancer cells
Method includes the following steps: cultured liver cancer cells ONOO-Releasing agent is incubated with the dimethyl sulfoxide solution of probe again after being incubated for
It educates, after being incubated for setting time, removes the dimethyl sulfoxide solution of probe, and cleaned with PBS, then carry out laser confocal imaging,
To obtain the final product.
9. application as claimed in claim 8, it is characterised in that: in the imaging method, ONOO-Releasing agent is SIN-1；It is preferred that
, in the imaging method, the time of incubation is 20-40min.
10. the fluorescence probe and/or such as of the detection peroxynitrite based on formamide recognition group as described in claim 1
It the described in any item methods of claim 2-6 and/or applies in field of biological detection as claim 7-9 is described in any item
Application.
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