CN110483573A - A kind of Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe and its preparation method and application - Google Patents
A kind of Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe and its preparation method and application Download PDFInfo
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- CN110483573A CN110483573A CN201910861886.1A CN201910861886A CN110483573A CN 110483573 A CN110483573 A CN 110483573A CN 201910861886 A CN201910861886 A CN 201910861886A CN 110483573 A CN110483573 A CN 110483573A
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- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 239000000523 sample Substances 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 28
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 claims abstract description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 33
- 210000003470 mitochondria Anatomy 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 238000004440 column chromatography Methods 0.000 claims description 12
- 239000012043 crude product Substances 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 9
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 229940125904 compound 1 Drugs 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 4
- PECAOKZHORDWAI-UHFFFAOYSA-N 3,6-diiodo-9h-carbazole Chemical compound C1=C(I)C=C2C3=CC(I)=CC=C3NC2=C1 PECAOKZHORDWAI-UHFFFAOYSA-N 0.000 claims description 3
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 claims description 3
- BGMHQBQFJYJLBP-UHFFFAOYSA-N 4-ethynylbenzaldehyde Chemical compound O=CC1=CC=C(C#C)C=C1 BGMHQBQFJYJLBP-UHFFFAOYSA-N 0.000 claims description 3
- 229910021595 Copper(I) iodide Inorganic materials 0.000 claims description 3
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 claims description 3
- 229940126214 compound 3 Drugs 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- LSXDOTMGLUJQCM-UHFFFAOYSA-M copper(i) iodide Chemical compound I[Cu] LSXDOTMGLUJQCM-UHFFFAOYSA-M 0.000 claims description 3
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 3
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims 2
- 238000004090 dissolution Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 26
- 238000010521 absorption reaction Methods 0.000 abstract description 14
- 238000003384 imaging method Methods 0.000 abstract description 12
- 230000002438 mitochondrial effect Effects 0.000 abstract description 7
- 230000004044 response Effects 0.000 abstract description 6
- 239000007850 fluorescent dye Substances 0.000 abstract description 5
- 238000000482 two photon fluorescence microscopy Methods 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 3
- 231100000263 cytotoxicity test Toxicity 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 230000031857 establishment of mitochondrion localization Effects 0.000 abstract description 2
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 239000003480 eluent Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000007334 copolymerization reaction Methods 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- -1 hypochlorite anion Chemical class 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000003896 Myeloperoxidases Human genes 0.000 description 2
- 108090000235 Myeloperoxidases Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 229940125782 compound 2 Drugs 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- UWSONZCNXUSTKW-UHFFFAOYSA-N 4,5-Dimethylthiazole Chemical compound CC=1N=CSC=1C UWSONZCNXUSTKW-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 125000002777 acetyl group Chemical class [H]C([H])([H])C(*)=O 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- XTEGARKTQYYJKE-UHFFFAOYSA-N chloric acid Chemical group OCl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-N 0.000 description 1
- 229940005991 chloric acid Drugs 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Inorganic materials Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011155 quantitative monitoring Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000006557 surface reaction Methods 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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- 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 discloses a kind of Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probes and its preparation method and application, wherein Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe, is so that using carbazole as parent, structural formula is as follows:When fluorescence probe of the present invention is applied to HClO detection in aqueous solution, selectivity is high, fast response time, fluorescence probe ratio (I525nm/I465nm) and the concentration of HClO between have a good linear relationship, detection limit is down to 35nM.In addition, the fluorescent probe molecule also has excellent two-photon absorption performance.Cytotoxicity test shows the probe for cell almost without what toxic side effect.The experiment of two-photon confocal fluorescent micro-imaging shows that the probe has good mitochondria positioning ability and cell-permeant, two-photon fluorescence imaging and quantitative detection suitable for HClO in cell mitochondrial.
Description
Technical field
The present invention relates to a kind of Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe and its preparation method and application,
To realize the HClO in two photon imaging quantitative detection cell mitochondrial, there is two-photon absorption function admirable, cytotoxicity is low,
The advantages that permeable membrane and good biocompatibility, selectivity and photostability are high.
Background technique
Recently, reactive oxygen species are since to cause medical researchers with internal a variety of being associated with for bioprocess more next for it
More concerns.As highly reactive form of oxygen species, endogenous HClO is mainly the mistake being catalyzed by internal myeloperoxidase (MPO)
Reaction between hydrogen oxide and chloride ion generates.Endogenous HClO becomes one pair in organism due to its Strong oxdiative ability
Sword sword.The HClO of normal physiologic levels can be effectively suppressed internal microorganism and pathogen and adjust Apoptosis.However, excessive
The generation of HClO then leads to cell and tissue damage, and may be related with certain diseases such as diseases associated with inflammation or cancer.In body fluid
In, hypochlorous acid and hypochlorite anion are in balance in the level of micro-molar concentration, when body is in morbid state,
Millimolar concentration may be increased to.Therefore, the real-time monitoring of internal HClO level has important meaning for the research of health science
Justice.
Mitochondria not only provides energy for human life activity, also participates in the physiology course of Various Complex.Its exception with
The diseases such as cancer, diabetes, Alzheimer's disease are related.As the key consumer of intracellular oxygen, mitochondria is intracellular work
The main source of property oxygen species (ROS).The one kind of HClO as reactive oxygen species, maintains concentration pair appropriate in mitochondria
Guarantee intracellular multiple functions is normally highly important.Therefore it in subcellsular level, is monitored especially in mitochondria secondary
Chloric acid is especially significant and value.Targetted mitochondria class fluorescence probe is mainly based upon mitochondria and has negative film potential,
And then it is positively charged using triphenylphosphine or quaternary ammonium salt etc. is introduced per se with the fluorogen of positive charge or in fluorogen
Group positions mitochondria.
Fluorescence probe method is commonly used as effective because it has the advantages that selectivity is high, high sensitivity, real-time monitoring
Bioanalytical tools.It is more quick to environmental factor and instrument parameter with the fluorescence probe of single fluorescence intensity signal in response
Sense, is more suitable for quantitative analysis due to internal calibrations ability using the Ratio-type probe of two kinds of fluorescence intensity ratios.Currently,
The Ratiometric fluorescent probe that many can be used for internal HClO imaging successfully developed, but wherein most of is in single photon
Excitation is lower to realize in-vivo imaging.For bio-imaging, short wavelength excites the light injury that will lead to sample, shallowly penetrates and comes
The problems such as from the autofluorescence of inherent biomolecule, and the application of two photon imaging then greatly compensates for these defects.Thus
The important topic that two-photon fluorescence probe has been used as researcher to study.
Carbazole compound is because having stronger electron donation, big conjugated system, good rigid plane, stable
Optical property and be easy to chemical modification introduce functional group and be frequently applied the parent for fluorescence probe.As a kind of classics
Fluorogen reported by many documents using carbazole by the single photon fluorescence probe of parent, but glimmering with the two-photon that it is designed
Light probe is then relatively rare.
Summary of the invention
The present invention is intended to provide a kind of Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe and preparation method thereof and
Purposes the technical problem to be solved is that a kind of suitable fluorescence probe structure is obtained by MOLECULE DESIGN there is two-photon to inhale
The advantages that receipts function admirable, cytotoxicity is low, permeable membrane and good biocompatibility, and selectivity and photostability are high, to realize water
The quantitative detection of HClO and the two-photon fluorescence imaging in mitochondria in solution and cell mitochondrial.
The Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe of the present invention, is to be abbreviated as using carbazole as parent
MCL, structural formula are as follows:
The preparation method of the Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe of the present invention, includes the following steps:
Step 1: the synthesis of compound 1
Potassium hydroxide (2.0g, 35.8mmol), potassium iodide (0.4g, 2.39mmol) are added in acetone soln (200mL)
It is heated 1 hour with Isosorbide-5-Nitrae-dibromobutane (7.73g, 35.8mmol) and at 60 DEG C, is then slowly added to 3,6- diiodocarbazol
(10g, 23.9mmol) continues to be heated to reflux 12 hours；It cools down and is spin-dried for after reaction, crude product is obtained after washing, is passed through
Column chromatography (petroleum ether: methylene chloride=10:1 is as eluant, eluent) purification, obtains intermediate 1,6.8g, yield 51.4%.
Step 2: the synthesis of compound 2
Under conditions of nitrogen protection, by compound 1 (2g, 3.6mmol), 4- ethynylbenzaldehyde (1.4g,
10.8mmol), bis-triphenylphosphipalladium palladium dichloride (0.0102g, 0.014mmol), cuprous iodide (0.0054g, 0.028mmol) and
Triethylamine (7mL) is dissolved in tetrahydrofuran (10mL), reacts 12 hours at 30 DEG C；It cools down and is spin-dried for after reaction, obtain
It to crude product, is purified by column chromatography (petroleum ether: methylene chloride=4:1 is as eluant, eluent), obtains intermediate 2,1.3g, produced
Rate is 64.5%.
Step 3: the synthesis of compound 3
Under conditions of nitrogen protection, compound 2 (1g, 1.8mmol) is added in acetonitrile (10mL), reacts 1 at 80 DEG C
Hour, triphenylphosphine (2.8g, 10.8mmol) is added, heating reaction 36 hours is continued；It cools down and is spin-dried for after reaction, obtain
It to crude product, is purified by column chromatography (methylene chloride: methanol=40:1 is as eluant, eluent), obtains intermediate 3,0.83g, produced
Rate is 56%.
Step 4: the synthesis of target product MCL
Under conditions of nitrogen protection, by compound 3 (0.83g, 1.0112mmol), methanesulfonic acid (20 μ L) and 2- sulfydryl second
Alcohol (0.237g, 3.0334mmol) is dissolved in methylene chloride (20mL), is reacted 12 hours at 25 DEG C；It cools down after reaction
And be spin-dried for, crude product is obtained, is purified by column chromatography (methylene chloride: methanol=40:1 is as eluant, eluent), it is solid to obtain white
Body MCL, 0.34g, yield 36%.
The synthesis process of two-photon fluorescence probe MCL of the present invention is as follows:
The purposes of two-photon fluorescence probe of the present invention, as detection examination when HClO in quantitative detection cell Mitochondria
Agent uses, and detection method is as follows:
Fluorescence probe MCL of the present invention is dissolved in the mother liquor for producing 2mM in DMSO, with to obtain 5mL, takes 15 μ L mother liquors in containing
In the sample cell of the PBS buffer solution of 3mL pH=7.4, to prepare 10 μM of detection liquid.It tests it and different equivalent HClO is added
Ultra-violet absorption spectrum data afterwards.Detection reagent has absorption peak at 308nm and 340nm respectively, with the increasing of the equivalent of HClO
Add, the absorption peak that MCL is located at 308nm and 340nm gradually decreases, and the absorption at 380nm gradually rises, when HClO reaches
After 5 times of equivalents, absorption curve no longer changes.With being continuously added for HClO (0-60 μM), it can be observed that fluorescence emission maximum
Peak by 465nm gradually red shift to 525nm.After HClO reaches 5 times of equivalents, fluorescence curve no longer changes, and illustrates to have reached full
And equivalent.After the HClO of different equivalents is separately added into 10 μM of detection reagents, fluorescence ratio (I525nm/I465nm) can be in a few seconds
Reach maximum value.When the HClO of 0-10 μM of concentration range is added separately to 10 μM of detection reagents, ratio fluorescent (I525nm/
I465nm) and the concentration of HClO between have good linear relationship (R=3 δ/k), detection limit is down to 35nM.By its of 10 times of equivalents
It analyzes substrate and is added separately to after acting on 20min in 10 μM of detection reagents, and the fluorescence spectrum detected within the scope of 370-650nm becomes
Change, it can be seen that probe MCL only shows apparent change in fluorescence to HClO, indicates there is specificity response.In different pH value
It is investigated in buffer solution to 10 μM of detection reagents and its to the fluorescence spectrum of HClO response, this is the experimental results showed that probe
The MCL and its ratio fluorescent (I for responding HClO525nm/I465nm) insensitive to pH value in the range of pH=6~9, it is suitable for weak base
Property mitochondria in HClO detection.In addition, by this probe molecule and mitochondria red quotient dye carried out the common location of mitochondria at
As the two-photon co-focusing imaging with HClO in mitochondria.
Two-photon fluorescence probe structure of the present invention is simple, is readily synthesized.Probe is sensitive to the response quickly of HClO, and shows
Specificity responds out.After HClO is added, two acetals in probe structure can be responded to site and be oxidized to two aldehyde radicals (figure
1), the cloud density distribution of reaction front and back changes, and fluorescence and UV absorption property also change correspondingly.Two-photon copolymerization is burnt glimmering
The experiment of light micro-imaging shows that the probe is good to HeLa cell permeability, can be with mitochondria (the positioning system in effective position cell
Number is the 0.93) two-photon fluorescence imaging suitable for HClO in cell mitochondrial.
Detailed description of the invention
Fig. 1 is fluorescent probe molecule MCL of the present invention and HClO reaction mechanism figure.
Fig. 2 is addition HClO (0-60 μM) (a) uv absorption spectra in 10 μM of probes；(b) fluorescence emission spectrogram of compound.
Fig. 3 is the fluorescence emission peak intensity rate (I that HClO (10 μM, 20 μM, 50 μM) are added in 10 μM of probes525nm/
I465nm) with the relational graph of time.
Fig. 4 is that the fluorescence emission peak intensity rate (I after HClO (0-10 μM) is added in 10 μM of probes525nm/I465nm) with it is dense
The linear relationship chart of degree.
Fig. 5 is the fluorescence selectivity spectrogram that other analysis substrates of 10 times of equivalents are added in 10 μM of probes.
Fig. 6 is the fluorescence emission peak intensity rate (I being added before and after HClO (50 μM) in 10 μM of probes525nm/I465nm) and pH
Relational graph.
Fig. 7 is effective two photon absorption cross section figure that probe MCL is added before and after HClO, and illustration is that opposite two-photon fluorescence is strong
Spend (Iout) and input power (Iin) logarithmic relationship figure.
Fig. 8 is the HeLa cell survival rate under the action of probe molecule of various concentration (0 μM, 10 μM, 20 μM, 30 μM)
Figure.
Fig. 9 is 10 μM of probes and 1 μM of MitoTracker red while dye HeLa cell mitochondrial locating verification copolymerization altogether
Burnt fluorescence imaging figure.
Figure 10 is that the two-photon of the exogenous HClO (0-40 μM) of addition various concentration in HeLa cell is total to focused cell
Image, concentration and probe concentration are 10 μM, and blue channel fluorescent emission capture range is 420-470nm, and green channel fluorescent emission is received
Integrate range as 500-540nm, excitation wavelength 740nm.
Specific embodiment
Below by embodiment, the present invention will be further described.
Embodiment 1: the synthesis of compound 1
Potassium hydroxide (2.0g, 35.8mmol) is added in acetone soln (200mL), potassium iodide (0.4g, 2.39mmol)
It is heated 1 hour with Isosorbide-5-Nitrae-dibromobutane (7.73g, 35.8mmol) and at 60 DEG C.Be slowly added to 3,6- diiodocarbazol (10g,
23.9mmol), continue to be heated to reflux 12 hours.It is spin-dried for after cooling, crude product is obtained after washing.By column chromatography (petroleum ether:
Methylene chloride=10:1 is as eluant, eluent) purification, obtain intermediate 1,6.8g, yield 51.4%.1H NMR(400MHz,
CDCl3, ppm) and δ 8.34 (d, J=1.5Hz, 2H), 7.74 (d, J=1.6Hz, 1H), 7.71 (d, J=1.6Hz, 1H), 7.19
(s, 1H), 7.17 (s, 1H), 4.29 (t, J=7.0Hz, 2H), 3.37 (t, J=6.4Hz, 2H), 2.06-1.98 (m, 2H),
1.86(m,2H).13C NMR(100MHz,CDCl3,ppm)δ139.38,134.69,129.48,124.08,110.76,81.93,
42.37,32.82,30.02,27.44.
Embodiment 2: the synthesis of compound 2
Under conditions of nitrogen protection, by compound 1 (2g, 3.6mmol), 4- ethynylbenzaldehyde (1.4g,
10.8mmol), bis-triphenylphosphipalladium palladium dichloride (0.0102g, 0.014mmol), cuprous iodide (0.0054g, 0.028mmol) and
Triethylamine (7mL) is dissolved in tetrahydrofuran (10mL), reacts 12 hours at 30 DEG C.It is spin-dried for after cooling, obtains crude product.It is logical
Column chromatography (petroleum ether: methylene chloride=4:1 is as eluant, eluent) purification is crossed, intermediate 2,1.3g, yield 64.5% are obtained.1H
NMR(400MHz,CDCl3, ppm) and δ 10.04 (s, 2H), 8.33 (s, 2H), 7.89 (t, J=7.4Hz, 4H), 7.71 (m, 6H),
7.42 (d, J=8.5Hz, 2H), 4.38 (m, 2H), 3.42 (t, J=6.3Hz, 1H), 3.19 (t, J=6.6Hz, 1H), 2.15-
2.01(m,2H),1.92(m,2H).13C NMR(100MHz,CDCl3,ppm)δ191.47,140.67,135.13,133.12,
131.92,130.18,130.14,129.67,129.59,124.63,122.62,113.56,109.14,94.83,87.58,
32.84,30.74,27.59,5.54.
Embodiment 3: the synthesis of compound 3
Under conditions of nitrogen protection, compound 2 (1g, 1.8mmol) is added in acetonitrile (10mL), reacts 1 at 80 DEG C
Hour, triphenylphosphine (2.8g, 10.8mmol) is added, heating 36 hours is continued.It is spin-dried for after cooling, obtains crude product.Pass through
Column chromatography (methylene chloride: methanol=40:1 is as eluant, eluent) purification, obtains intermediate 3,0.83g, yield 56%.1H
NMR(400MHz,DMSO-d6, ppm) δ 10.05 (s, 2H), 8.53 (s, 2H), 7.98 (d, J=8.0Hz, 4H), 7.87 (m,
3H), 7.79 (d, J=8.0Hz, 4H), 7.76-7.68 (m, 16H), 4.51 (t, J=6.9Hz, 2H), 3.62 (t, J=
15.1Hz,2H),2.00–1.93(m,2H),1.62(m,2H).13C NMR(100MHz,CDCl3,ppm)δ191.43,140.73,
135.13,135.08,135.05,133.60,133.50,132.15,132.05,131.88,130.50,130.37,130.20,
130.08,129.67,128.57,128.45,124.24,122.31,118.10,117.24,113.34,110.05,94.88,
87.59,42.55,29.19,22.66,20.15.
Embodiment 4: the synthesis of target product MCL
Under conditions of nitrogen protection, by compound 3 (0.83g, 1.0112mmol), methanesulfonic acid (20 μ L), 2- sulfydryl second
Alcohol (0.237g, 3.0334mmol) is dissolved in methylene chloride (20mL), is reacted 12 hours at 25 DEG C.It is spin-dried for, obtains after cooling
To crude product.It is purified by column chromatography (methylene chloride: methanol=40:1 is as eluant, eluent), obtains white solid MCL,
0.34g, yield 36%.1H NMR(400MHz,DMSO-d6,ppm)δ8.43(s,2H),7.84(m,3H),7.67(m,14H),
7.62-7.59 (m, 2H), 7.54 (d, J=8.2Hz, 4H), 7.46 (d, J=8.2Hz, 4H), 6.09 (s, 2H), 4.51-4.43
(m, 4H), 3.87 (m, 2H), 3.58 (t, J=14.6Hz, 2H), 3.20 (m, 4H), 1.97-1.88 (m, 2H), 1.58 (m, 2H)
.13C NMR(100MHz,DMSO-d6,ppm)δ140.13,139.74,134.86,133.48,131.13,130.17,129.59,
126.91,124.27,122.85,121.80,118.69,117.84,112.85,110.21,91.14,87.52,85.55,
71.76,41.69,33.53,20.10,19.57.
Embodiment 5: the spectrum test of fluorescent probe molecule
Fluorescence probe MCL of the present invention is dissolved in the mother liquor for producing 2mM in DMSO, with to obtain 5mL, takes 15 μ L mother liquors in containing
In the sample cell of the PBS buffer solution of 3mL pH=7.4, to prepare 10 μM of detection liquid.It is tested containing different equivalent HClO
Under ultra-violet absorption spectrum data.Detection reagent has absorption peak at 308nm and 340nm respectively, with the increasing of the equivalent of HClO
Add, the absorption peak that MCL is located at 308nm and 340nm gradually decreases, and the absorption at 380nm gradually rises, when HClO reaches
After 5 times of equivalents, absorption curve no longer changes (Fig. 2 a).With being continuously added for HClO (0-60 μM), it can be observed that fluorescence is most
Big emission peak by 465nm gradually red shift to 525nm.After HClO reaches 5 times of equivalents, fluorescence curve no longer changes, and explanation reaches
Saturation equivalent (Fig. 2 b) is arrived.After the HClO of different equivalents is separately added into 10 μM of detection reagents, fluorescence ratio (I525nm/
I465nm) maximum value (Fig. 3) can be reached in a few seconds.The HClO of 0-10 μM of concentration range is added separately to 10 μM of detection reagents
When, ratio fluorescent (I525nm/I465nm) and the concentration of HClO between have good linear relationship (R=3 δ/k), detection limit down to
35nM (Fig. 4).Other analysis substrates of 10 times of equivalents are added separately to after acting on 20min in 10 μM of detection reagents, detection
Fluorescence spectrum variation within the scope of 370-650nm, it can be seen that probe MCL only shows apparent change in fluorescence to HClO, indicates
(Fig. 5) is responded with specificity.To 10 μM of detection reagents and its to the fluorescence of HClO response in the buffer solution of different pH value
Spectrum is investigated, this experimental results showed that probe MCL and its respond HClO ratio fluorescent (I525nm/I465nm) pH=6~
In 9 ranges (Fig. 6) insensitive to pH value, it is suitable for HClO detection in weakly alkaline mitochondria.
Embodiment 6: the two-photon performance test of fluorescent probe molecule
Using two-photoninduced fluorescence measuring technique, we measure probe MCL and its respond the two-photon suction after HClO
Receive performance.Maximum effectively two-photon absorption action section value is presented in probe MCL at 720nm, is 50GM.5 times of equivalents are added
After HClO, maximum effectively two-photon absorption action section value is presented in reaction product at 760nm, is 60GM (Fig. 7).Change incident
The energy of exciting light, input power (Iin=0.3~0.8mW) and relative fluorescence output energy (Iout) at logarithmic relationship, slope point
Not Wei 1.97 and 1.99, meet the rule (illustration in Fig. 7) of two-phpton property.It should be experiments have shown that probe MCL be inhaled with two-photon
Property is received, can be applied to the two-photon fluorescence imaging of intracellular HClO detection.
Embodiment 7: cell culture and cytotoxicity test
Cell culture: by HeLa cell in addition 90%DMEM (sugar and amino acid) and 10%FCS (fetal calf serum)
Culture adds 1% mycillin mixed liquor to prevent germ contamination, and 37 DEG C of constant temperature contain 5%CO2Insulating box culture.
Cytotoxicity: according to document report, pass through MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromine
Salt) it tests and carrys out test cell toxicity.First HeLa cell is cultivated 24 hours in 96- orifice plate before test, probe is being added
Fresh DMEM is first changed to before, then is separately added into the probe MCL (0,10,20 and 30 μM) of various concentration, treated cell
It is cultivated 24 hours under the conditions of 5% carbon dioxide content, 37 DEG C.Then, then into cell 5mg/mL MTT (40 μ L/ are added
), well continue culture 4 hours (37 DEG C, 5%CO2).Culture solution is drawn in DMSO (150 hole μ L/), records the suction at 570nm
Luminosity.According to the formula of cell survival degree: cell survival rate %=OD570(sample)/OD570(control group) × 100, finally carefully
Born of the same parents' survival rate (Fig. 8).Test result shows that the bio-toxicity of MCL is smaller, is appropriate for cell imaging.
Embodiment 8: mitochondria positioning test
HeLa cell is put in laser copolymerization by DEME (invitrogen) culture solution culture, imaging the previous day, HeLa cell
In burnt ware, then 10 μM of MCL is added into HeLa cell and is placed in 37 DEG C, containing 5%CO2Cell incubator in be incubated for it is 0.5 small
When, after washing 3 times with neutral PBS buffer solution, then 1 μM of commercialization mitochondrial stain is added into culture dish
MitoTracker red (MTR) solution simultaneously continues to be incubated for 0.5 hour, after washing 3 times with neutral PBS buffer solution later, into
Row two-photon fluorescence co-focusing imaging.Setting probe molecule MCL is blue channel (λem=420-470nm, λex=740nm)；If
Setting commercialization mitochondrial stain MitoTracker red (MTR) is red channel (λem=580-600nm, λex=579nm).
The result shows that the fluorescent image overlapping of the two is good, and the Pearson common location coefficient of MCL and MTR is calculated as 0.93, explanation
MCL can target the mitochondria (Fig. 9) in living cells well.
Embodiment 9: intracellular HClO two-photon fluorescence imaging
With 5 groups of probe culture HeLa cell 30 minutes, the HClO (0,5,10,20 and 40 μM) of various concentration is then used respectively
Culture carried out two photon imaging after 30 minutes.Blue channel (λ is setem=420-470nm) and green channel (λem=500-
540nm) it is imaged and is observed (Figure 10).In blank control group, it can be observed that blue channel and green channel are all shown
Significant fluorescence out, and blue-fluorescence is better than green fluorescence, both fluorescence are attributable to probe MCL itself.It is also possible to
It is clearly observed the bright fluorescence of intracellular filament, this is the representative configuration of mitochondria.With the increase of HClO concentration,
The intensity of blue-fluorescence and green fluorescence is gradually reduced, and blue-fluorescence declines faster, and the increase of HClO concentration can be observed
Lead to the variation of pseudocolour picture.These results clearly demonstrate that probe MCL can be by two photon imaging technology Quantitative Monitoring
HClO is horizontal in living cells mitochondria and its fluctuates.
Claims (3)
1. a kind of Mitochondrially targeted hypochlorous acid Ratio-type two-photon fluorescence probe, is using carbazole as parent, it is characterised in that its
Structural formula is as follows:
2. a kind of preparation method of two-photon fluorescence probe described in claim 1, it is characterised in that include the following steps:
Step 1: the synthesis of compound 1
Potassium hydroxide, potassium iodide and Isosorbide-5-Nitrae-dibromobutane are added in acetone soln and is heated 1 hour at 60 DEG C, then slowly
3,6- diiodocarbazol is added, continues heating reflux reaction；It cools down and is spin-dried for after reaction, crude product is obtained after washing, is passed through
Column chromatography purification, obtains intermediate 1；
Step 2: the synthesis of compound 2
Under conditions of nitrogen protection, by compound 1,4- ethynylbenzaldehyde, bis-triphenylphosphipalladium palladium dichloride, cuprous iodide and
Triethylamine is dissolved in tetrahydrofuran, is reacted at 30 DEG C；It cools down and is spin-dried for after reaction, obtain crude product, pass through
Column chromatography purification, obtains intermediate 2；
Step 3: the synthesis of compound 3
Under conditions of nitrogen protection, it is added at 2,80 DEG C of compound and reacts 1 hour into acetonitrile, add triphenylphosphine, after
Continuous heating reaction；It cools down and is spin-dried for after reaction, obtain crude product, purified by column chromatography, obtain intermediate 3；
Step 4: the synthesis of target product MCL
Under conditions of nitrogen protection, in methylene chloride by the dissolution of compound 3, methanesulfonic acid and 2 mercapto ethanol, at 25 DEG C
It is reacted；It cools down and is spin-dried for after reaction, obtain crude product, purified by column chromatography, obtain white solid MCL.
3. a kind of purposes of two-photon fluorescence probe described in claim 1, it is characterised in that:
It is used when HClO in quantitative detection cell Mitochondria as detection reagent.
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