CN108164494A - A kind of two-photon fluorescence probe for detecting peroxynitrite and its preparation method and application - Google Patents
A kind of two-photon fluorescence probe for detecting peroxynitrite and its preparation method and application Download PDFInfo
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- CN108164494A CN108164494A CN201810140844.4A CN201810140844A CN108164494A CN 108164494 A CN108164494 A CN 108164494A CN 201810140844 A CN201810140844 A CN 201810140844A CN 108164494 A CN108164494 A CN 108164494A
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- photon fluorescence
- onoo
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- 239000000523 sample Substances 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 title claims abstract 3
- 238000003384 imaging method Methods 0.000 claims abstract description 19
- 210000004413 cardiac myocyte Anatomy 0.000 claims abstract description 14
- 230000006698 induction Effects 0.000 claims abstract description 4
- 230000002107 myocardial effect Effects 0.000 claims abstract description 3
- 230000000451 tissue damage Effects 0.000 claims abstract 2
- 231100000827 tissue damage Toxicity 0.000 claims abstract 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 37
- 210000004027 cell Anatomy 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 210000001519 tissue Anatomy 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 7
- 229940125904 compound 1 Drugs 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical group O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
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- 206010002091 Anaesthesia Diseases 0.000 claims description 3
- 230000037005 anaesthesia Effects 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 210000004165 myocardium Anatomy 0.000 claims description 3
- 238000000482 two photon fluorescence microscopy Methods 0.000 claims description 3
- 210000003462 vein Anatomy 0.000 claims description 3
- 230000002861 ventricular Effects 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- 241001529936 Murinae Species 0.000 claims description 2
- 229940009456 adriamycin Drugs 0.000 claims description 2
- 238000010226 confocal imaging Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 229940045799 anthracyclines and related substance Drugs 0.000 claims 1
- 230000003115 biocidal effect Effects 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 230000004044 response Effects 0.000 abstract description 7
- 239000003817 anthracycline antibiotic agent Substances 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 3
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical class C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 150000004799 α-ketoamides Chemical group 0.000 abstract description 2
- 238000012545 processing Methods 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 6
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 6
- 229940116269 uric acid Drugs 0.000 description 6
- 230000000747 cardiac effect Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 210000003470 mitochondria Anatomy 0.000 description 4
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 4
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 230000003387 muscular Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 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
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000012632 fluorescent imaging Methods 0.000 description 2
- 210000005003 heart tissue Anatomy 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
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- 230000035515 penetration Effects 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- NCGICGYLBXGBGN-UHFFFAOYSA-N 3-morpholin-4-yl-1-oxa-3-azonia-2-azanidacyclopent-3-en-5-imine;hydrochloride Chemical compound Cl.[N-]1OC(=N)C=[N+]1N1CCOCC1 NCGICGYLBXGBGN-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
- 238000012449 Kunming mouse Methods 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
- 150000001263 acyl chlorides Chemical class 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 150000001454 anthracenes Chemical class 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229910052786 argon Inorganic materials 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
- 201000011510 cancer Diseases 0.000 description 1
- 230000003683 cardiac damage Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
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- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000010186 staining Methods 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
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000000007 visual 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
- 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
- C07D311/78—Ring systems having three or more relevant rings
-
- 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
- G01N21/6486—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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- 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
Abstract
The invention discloses a kind of two-photon fluorescence probes for detecting peroxynitrite and preparation method and application, and structural formula is：The two-photon fluorescence probe is named as TPNIR FP, the two-photon fluorescence probe is using Nile red derivative as fluorogen, using alpha ketoamide functional group as ONOO‑Reactive group, with ONOO‑Energy quick response (<10s), and high sensitivity, selectivity are good.Can apply to anthracycline antibiotic induction cardiac muscle cell and myocardial tissue damage in ONOO‑Imaging research in.
Description
Technical field
The invention belongs to synthesize and detection technique field, and in particular to a kind of two-photon fluorescence for detecting peroxynitrite
Probe and preparation method and application.
Background technology
In vivo, peroxynitrite (ONOO-) by Superoxide radicalanion and nitric oxide reaction generation, energy
Contemporary table oxidative stress and nitrification stress level.As one of highest endogenous toxin of reactivity, ONOO-It can lead to
Peroxidating or nitrification biomolecule, regulating cell signal path, inducing cell apoptosis or necrosis, and then lead to tissue organ function
Obstacle.Research shows that ONOO-Morbidity with a variety of diseases and progress are closely related, including neurodegenerative disease, diabetes,
Cancer etc..In addition, in the pathogenesis of the drug-induced cardiac toxic of anthracene nucleus, ONOO-Play very crucial effect.So
And ONOO-Half-life short and Css in physiological conditions is extremely low so that the ONOO in detection living cells and living tissue-
Become very difficult.
Due to its high-resolution and lossless Flaw characterization, the fluorescence analysis based on probe can be real in cell and animal model
When visualize molecular events, the solution for the above problem provides effective analysis method.The hair of novel fluorescence imaging sensor
Exhibition is greatly promoted the progress of cell biology and diagnosis imaging etc..And the two-photon fluorescence probe of near infrared emission
It is more applicable in living tissue imaging since tissue penetration is deep, background fluorescence interference is small.But two-phpton property is good at present
And with near-infrared fluorescent transmitting, to ONOO-Sensitive fluorescence probe is less.
Invention content
In order to solve above-mentioned technical problem in the prior art, the object of the present invention is to provide a kind of detection peroxide is sub-
Two-photon fluorescence probe of nitrate anion and its preparation method and application.The two-photon fluorescence probe is named as TPNIR-FP, it should
Two-photon fluorescence probe is using Nile red derivative as fluorogen, using alpha-keto amide functional group as ONOO-Reactive group, with ONOO-
Energy quick response (<10s), and high sensitivity, selectivity are good.It can apply to cardiac muscle cell and the cardiac muscle of anthracycline antibiotic induction
ONOO in tissue damage-Imaging research in.
In order to solve the above technical problems, the technical scheme is that：
A kind of two-photon fluorescence probe for detecting peroxynitrite, structural formula are：
The preparation method of above-mentioned two-photon fluorescence probe, includes the following steps：
Reaction equation is：
1) by compound TPNIR-NH2It is dissolved in atent solvent, and adds in triethylamine thereto, after stirring evenly, obtain mixed
Close liquid；
2) dichloromethane solution of compound 1 is added drop-wise to dropwise in the mixed liquor of step 1), after reacting setting time, i.e.,
.
Atent solvent for not with the solvent of acyl chloride reaction.
Preferably, in step 1), the atent solvent be dichloromethane, tetrahydrofuran or dioxane, further preferably
For anhydrous methylene chloride.
Preferably, compound TPNIR-NH2, compound 1 and triethylamine the ratio between the amount of substance be 1:2-10:2-5.
Preferably, in step 2), the temperature of reaction is 0-35 DEG C, and the time of reaction is 1-3h.
It is further preferred that in step 2), the temperature of reaction is 25-30 DEG C, and the time of reaction is 1.5h.
Preferably, the step of reaction product solution is spin-dried for, is purified is further included in step 2).
It is further preferred that in step 2), reaction product is purified using silica gel column chromatography.
Still more preferably, mixed liquor of the eluant, eluent of the silica gel column chromatography for dichloromethane and methanol, dichloromethane
The volume ratio of alkane and methanol is 150-250:1；Preferably 200:1.
Application of the above-mentioned two-photon fluorescence probe in living cells and imaging of tissue；Especially in anthracycline antibiotic induction
ONOO in cardiac muscle cell and myocardial tissue damage-Imaging in application.
Imaging method of the above-mentioned two-photon fluorescence probe in cardiac muscle cell, includes the following steps：By cultured cardiac muscle
Cell is incubated after being stimulated with stimulant with the dimethyl sulfoxide solution of TPNIR-FP again, after being incubated setting time, removes TPNIR-FP
Dimethyl sulfoxide solution, and cleaned with PBS, then carry out laser confocal imaging.
Preferably, the stimulant is adriamycin and Epi-ADM.
Preferably, the time of incubation is 20-40min.
Imaging method of the above-mentioned two-photon fluorescence probe in murine myocardium, includes the following steps：
The drug of setting concentration is injected in mouse peritoneal, then in the tail vein injection probe solution of mouse, certain time
Afterwards, heart will be extractd after mouse anesthesia, takes left ventricular tissues slice for two-photon fluorescence imaging.
The beneficial effects of the invention are as follows：
1) probe molecule maximum emission wavelength is located near infrared region (~630nm), has moderate Stokes
Displacement (~50nm), significantly reduces self-absorption, improves imaging accuracy and tissue penetration depths.
2) probe is to ONOO-It can quick response (reaction finishes in 10s).
3) probe is to ONOO-With highly sensitive and selectivity.
4) probe coloring in living cells is good, and dyeing time is shorter (30min), and staining efficiency is higher.
5) the accurate targetted mitochondria of probe energy, instruction cardiac muscle cell ONOO under anthracycline antibiotic stimulation-The raising of concentration.
6) probe can realize ONOO in the cardiac muscular tissue of drug-induced damage-Concentration difference imaging.
7) probe synthesis step is relatively easy, yield is higher, easy purifying.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not form the improper restriction to the application for explaining the application.
Fig. 1 is ONOO of the novel fluorescence probe of the present invention to various concentration-Fluorescence response spectrogram；Abscissa is wavelength
(nm), ordinate is fluorescent emission intensity.
Fig. 2 is the ONOO of fluorescence intensity and various concentration of the novel fluorescence probe of the present invention at 630nm-Linear pass
System；Abscissa is ONOO-Concentration (μM), ordinate are the fluorescent emission intensity at probe 630nm.
Fig. 3 is the real-time change figure of fluorescence intensity of the novel fluorescence probe of the present invention at 630nm at any time；Abscissa is
Different time (s), ordinate are the fluorescent emission intensity at probe 630nm.
Fig. 4 is the fluorescence intensity situation at novel fluorescence probe 630nm of the present invention in the presence of variety classes substance.It is horizontal
Coordinate is variety classes substance, and ordinate is the fluorescent emission intensity at probe 630nm.
Fig. 5 is novel fluorescence probe of the present invention and fluorescence of the commercialization mitochondrial dye in human liver cancer cell (HepG2)
Colored graph.
Fig. 6 is fluorescence imaging figure of the novel fluorescence probe of the present invention in cardiac muscle cell (H9c2).
Fig. 7 is fluorescence imaging figure of the novel fluorescence probe of the present invention in cardiac muscular tissue.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.It is 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 singulative
It is also 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 combination thereof.
The present invention has existing raw material midbody TPNIR-NH before utilizing2With compound 1, by single step reaction it is simple,
It efficiently synthesizes, finally by the isolated probe TPNIR-FP of silica gel chromatographic column.Compound TPNIR-NH2With 1 reference literature
Shang,H.；Chen,H.；Tang,Y.；Ma,Y.；Lin, W.Biosens.Bioelectron.2017,95,81-86 and Xie,
X.；Yang,X.；Wu,T.；Li,Y.；Li,M.；Tan,Q.；Wang,X.；Tang,B.Anal.Chem.2016,88,8019-
8025 synthesis.
Embodiment：The synthesis of fluorescence probe
Under protection of argon gas, by compound TPNIR-NH2(160mg, 0.5mmol) is dissolved in 10mL anhydrous methylene chlorides,
And triethylamine (58 μ L, 2.0mmol) is added in, 5.0min is stirred at room temperature.Then, compound 1 (1.06g, 5.0mmol) is dissolved in
5.0mL dichloromethane is added dropwise in above-mentioned mixed liquor, continues that 1.5h is stirred at room temperature.After completion of the reaction, concentration is spin-dried for solvent,
Solid is carried out column chromatography for separation, and (eluant, eluent is dichloromethane：Methanol 200:1, V/V) atropurpureus solid 174mg (yields are obtained
70%).
Nuclear-magnetism and mass spectral characteristi：
1H NMR(400MHz,DMSO-d6):δ 11.42 (s, 1H), 8.69 (s, 1H), 8.40 (d, J=8.9Hz, 2H),
8.34 (d, J=8.9Hz, 2H), 8.25 (d, J=9.4Hz, 1H), 7.95 (s, 2H), 7.93 (s, 1H), 7.47 (dd, J=9.4,
2.2Hz, 1H), 7.30 (d, J=1.9Hz, 1H), 3.70 (q, J=7.0Hz, 4H), 3.08 (br s, 4H), 1.26 (t, J=
7.0Hz,6H).
13C NMR(100MHz,DMSO-d6):δ186.62,161.81,161.69,158.23,155.47,150.52,
148.46,143.31,142.96,137.45,131.96,131.79,131.02,126.98,123.82,122.31,120.97,
119.58,119.18,118.69,118.26,95.65,45.53,26.54,24.45.
HRMS(ESI):calculated for C29H26N3O5 +(M+)496.1867,found 496.1865.
Effect experiment：
Probe is to ONOO-Fluorescence response experiment：
TPNIR-FP (storing solution 1.0mM, 200 μM) is added in after PBS (pH 7.4) is buffered and is diluted with water, and add in difference
The ONOO of concentration-, it is 2.0 μM to make probe ultimate density, PBS a concentration of 50mM, ONOO-A concentration of 0-10 μM.As shown in Figure 1, work as
When being excited with 570nm, probe is in the basic unstressed configuration transmittings of 630nm or so, the ONOO with various concentration-It is (right successively from bottom to top
The ONOO answered-A concentration of 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM) be incubated after, fluorescence is notable
Enhancing, and 630nm fluorescence intensities and ONOO-Concentration is presented good linearly related (Fig. 2).Detection is calculated according to 3 σ of formula/k
It is limited to 34nM so that TPNIR-FP has endogenous trace ONOO in detection biosystem-Ability.
Then, probe TPNIR-FP is had studied to ONOO-Dynamic response.As shown in figure 3, probe (2.0 μM) itself
Fluorescence is weaker at 630nm, as 10 μM of ONOO of addition-Later, fluorescence enhances rapidly, and vertex is reached in 10s, shows probe energy
Enough quick response ONOO-, can capture that reactivity in physiological environment is high, ONOO of half-life short-。
Then, the selectivity of probe is had studied.In Fig. 4, label 1 represents 10 μM of ONOO-, 2 represent blank control, and 3 represent
100μM H2O2, 4 represent 100 μM of t-BuOOH, and 5 represent 100 μM of NO, and 6 represent 100 μM1O2, 7 represent 100 μM of O2 ·-, 8 generations
100 μM of ClO of table-, 9 represent 100 μM of OH, and 10 represent 5mM GSH, and 11 represent 100 μM of Cys, and 12 represent 100 μM of Hcy, and 13
Represent 100 μM of H2S, 14 represent 100 μM of NO3 -, 15 represent 100 μM of NO2 -, 16 represent 100 μM of SO4 2-, 17 represent 100 μM
SO3 2-, 18 represent 100 μM of CO3 2-, 19 represent 100 μM of PO4 3-, 20 represent 100 μM of Na+, 21 represent 100 μM of K+, 22 represent
100μM Ca2+, 23 represent 100 μM of Zn2+, 24 represent 100 μM of Cu2+, 25 represent 100 μM of Fe2+, 26 represent 100 μM of Fe3+,
27 represent 100 μM of vitamin Cs.As shown in Figure 4, other potential interference substances cannot cause the change in fluorescence of probe, including work
Property oxygen/active nitrogen (H2O2,t-BuOOH,NO,1O2,O2 ·-,ClO-, andOH), active sulfur (GSH, Cys, Hcy, and H2S)、
Anion (NO3 -,NO2 -,SO4 2-,SO3 2-,CO3 2-,and PO4 3-), cation (Na+,K+,Ca2+,Zn2+,Cu2+,Fe2+,and
Fe3+) and vitamin C.In contrast, TPNIR-FP is only in ONOO-Apparent fluorescence is shown in the presence of (corresponding label 1) to increase
By force.In conclusion probe TPNIR-FP is to ONOO-Specificity with height, available for ONOO in physiological environment-Fluorescence can
It is studied depending on changing.
Probe dyes imaging experiment to living cells and cardiac muscular tissue：
Due to containing lipophilic cation in structure so that probe tendency is gathered in mitochondria.Therefore, using common location reality
The subcellular area distribution situation for having investigated TPNIR-FP is tested, as shown in figure 5, figure a is using commercialization mitochondrial dye Mito-
The fluorescent staining figure of Tracker Green, figure b are the fluorescent staining figures using TPNIR-FP, and figure c is figure a with scheming being superimposed for b
Figure, figure d are the scatterplot associated diagrams of stacking chart.HepG2 cells first use ONOO-Producing agent SIN-1 stimulates 30min, then with line grain
Body commercialization dyestuff Mito-Tracker Green and probe TPNIR-FP are incubated 30min jointly, then carry out confocal fluorescent
Imaging.The result shows that the green fluorescence of Mito-Tracker Green can overlap well with the red fluorescence of TPNIR-FP, altogether
Orientation factor is up to 0.94, illustrates TPNIR-FP energy targetted mitochondrias, and detect ONOO-.Since mitochondria is ONOO-Origin and
The main cell device to play a role, therefore, probe TPNIR-FP are ONOO in imaging cells-Superior molecular tool.
H9c2 cardiac muscle cell ONOO in drug-induced damage has been monitored followed by TPNIR-FP-The variation of concentration.Ah
Mycin (Dox) and Epi-ADM (Epi) are used for inducing cardiomyocytes toxicity as representative anthracycline antibiotic.H9c2 cardiac muscle cell
Distinct methods processing is carried out, then dyes 30min with probe TPNIR-FP again, carries out confocal fluorescent imaging.As shown in fig. 6, a
The blank control group of PBS processing is used for cell, cell is using 10 μM of Dox processing in b, and cell is used at 20 μM of Dox in c
It managing, cell is handled first with 100 μM of uric acid in d, then with 20 μM of Dox processing, the average fluorescent strength output figure that e is figure a to d,
F is the blank control group that cell uses PBS processing, and cell is using 10 μM of Epi processing in g, and cell is used at 20 μM of Epi in h
It managing, cell is handled first with 100 μM of uric acid in i, then with 20 μM of Epi processing, the average fluorescent strength output figure that j is f to i, with
Blank control group is compared, and the cell of Dox and Epi processing groups sends out substantially brighter red fluorescence, and as drug dose increases,
Fluorescence probe is stronger, which can be by ONOO-Scavenger uric acid is inhibited.The above results show the heart of Dox or Epi processing
Myocyte experienced ONOO-Outburst, and the ONOO raised-Concentration and drug dose positive correlation, probe TPNIR-FP can well may be used
Depending on changing this process.
Later, using probe TPNIR-FP, the fluorescent visual in drug-induced mouse heart damage model is realized
ONOO-Level variation.Kunming mice is randomized into blank control group (PBS), low dose group (25mg/kg), high dose group
(50mg/kg) and removing group (100mg/kg Uric acid 50mg/kg).Mouse peritoneal injection respective concentration drug (0,
The Dox or Epi of 25,50, and 50mg/kg), tail vein injection probe TPNIR-FP (50 μM, 100 μ L) later.For clear
Except the mouse of group, it is being administered with before probe load, uric acid (100mg/kg) is injected intraperitoneally in advance.All mouse are plucked after anesthesia
Except heart, left ventricular tissues slice is taken for two-photon fluorescence imaging.As shown in fig. 7, compared with blank control group, drug-treated
The heart tissue of group mouse send out with the positively related red fluorescence of drug dose, and the fluorescence can be inhibited by uric acid.It is above-mentioned
The phenomenon that change in fluorescence result in heart tissue in H9c2 cardiac muscle cell with observing is highly consistent, further confirms Dox
With endogenous ONOO during Epi induced cardiac damages-Up-regulation, probe TPNIR-FP can visualize as good imaging tool
This result.
The foregoing is merely the preferred embodiments of the application, are not limited to the application, for the skill of this field
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of two-photon fluorescence probe for detecting peroxynitrite, it is characterised in that：Its structural formula is：
2. the preparation method of two-photon fluorescence probe described in claim 1, it is characterised in that：Include the following steps：
Reaction equation is：
1) by compound TPNIR-NH2It is dissolved in atent solvent, and adds in triethylamine thereto, after stirring evenly, obtain mixed liquor；
2) dichloromethane solution of compound 1 is added drop-wise to dropwise in the mixed liquor of step 1), react setting time after to get.
3. preparation method according to claim 2, it is characterised in that：In step 1), the atent solvent for dichloromethane,
Tetrahydrofuran or dioxane, further preferably anhydrous methylene chloride.
4. preparation method according to claim 2, it is characterised in that：Compound TPNIR-NH2, compound 1 and triethylamine
The ratio between amount of substance is 1:2-10:2-5.
5. preparation method according to claim 2, it is characterised in that：It is further included in step 2) and carries out reaction product solution
The step of being spin-dried for, purifying；
Preferably, in step 2), reaction product is purified using silica gel column chromatography；
Preferably, the eluant, eluent of the silica gel column chromatography is the body of dichloromethane and the mixed liquor of methanol, dichloromethane and methanol
Product is than being 150-250:1；Preferably 200:1.
6. application of the two-photon fluorescence probe described in claim 1 in living cells and imaging of tissue；Especially in anthracycline antibiosis
Element induction cardiac muscle cell and myocardial tissue damage in ONOO-Imaging in application.
7. imaging method of the two-photon fluorescence probe described in claim 1 in cardiac muscle cell, it is characterised in that：Including as follows
Step：It is incubated again with the dimethyl sulfoxide solution of TPNIR-FP after cultured cardiac muscle cell is stimulated with stimulant, is incubated setting
After time, the dimethyl sulfoxide solution of TPNIR-FP is removed, and cleaned with PBS, then carry out laser confocal imaging.
8. imaging method according to claim 7, it is characterised in that：The stimulant is adriamycin and Epi-ADM.
9. imaging method according to claim 7, it is characterised in that：The time of incubation is 20-40min.
10. imaging method of the two-photon fluorescence probe described in claim 1 in murine myocardium, it is characterised in that：Packet
Include following steps：
The drug of setting concentration is injected in mouse peritoneal, it, after a certain period of time, will then in the tail vein injection probe solution of mouse
Heart is extractd after mouse anesthesia, takes left ventricular tissues slice for two-photon fluorescence imaging.
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Cited By (7)
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| CN108623611A (en) * | 2018-06-22 | 2018-10-09 | 北京工业大学 | A kind of synthesis and application of the fluorescence probe of detection hydrogen peroxide |
| CN109608414A (en) * | 2018-12-26 | 2019-04-12 | 山东师范大学 | Detect the fluorescence probe and its preparation method and application of peroxynitrite |
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| CN110590752A (en) * | 2019-09-25 | 2019-12-20 | 山东师范大学 | Compound based on anthracycline structure and preparation method and application thereof |
| CN110964022A (en) * | 2019-12-24 | 2020-04-07 | 济南大学 | Fluorescent probe for detecting peroxynitrite ions and preparation method and application thereof |
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| CN108623611A (en) * | 2018-06-22 | 2018-10-09 | 北京工业大学 | A kind of synthesis and application of the fluorescence probe of detection hydrogen peroxide |
| CN108623611B (en) * | 2018-06-22 | 2020-09-25 | 北京工业大学 | Synthesis and application of fluorescent probe for detecting hydrogen peroxide |
| CN109608414A (en) * | 2018-12-26 | 2019-04-12 | 山东师范大学 | Detect the fluorescence probe and its preparation method and application of peroxynitrite |
| CN109608414B (en) * | 2018-12-26 | 2020-09-29 | 山东师范大学 | Fluorescent probe for detecting peroxynitrite and preparation method and application thereof |
| CN109897627A (en) * | 2019-03-20 | 2019-06-18 | 山西大学 | A kind of near infrared fluorescent probe and its preparation method and application of quick detection ONOO- |
| CN110452687A (en) * | 2019-06-19 | 2019-11-15 | 长沙理工大学 | A kind of sodium dithionite colorimetric fluorescence probe and its preparation method and application |
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| CN110590752A (en) * | 2019-09-25 | 2019-12-20 | 山东师范大学 | Compound based on anthracycline structure and preparation method and application thereof |
| CN110590752B (en) * | 2019-09-25 | 2020-09-25 | 山东师范大学 | Compound based on anthracycline structure and preparation method and application thereof |
| CN110964022A (en) * | 2019-12-24 | 2020-04-07 | 济南大学 | Fluorescent probe for detecting peroxynitrite ions and preparation method and application thereof |
| CN112457286A (en) * | 2020-12-02 | 2021-03-09 | 青岛科技大学 | Application of compound containing oxyanion in preparation of fluorescent molecular probe for detecting nitroso peroxide ion |
| CN112457286B (en) * | 2020-12-02 | 2022-03-04 | 青岛科技大学 | Application of compound containing oxyanion in preparation of fluorescent molecular probe for detecting nitroso peroxide ion |
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