CN105001856B - One class is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles - Google Patents
One class is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 81
- 230000003859 lipid peroxidation Effects 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 43
- 210000003463 organelle Anatomy 0.000 title claims abstract description 27
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Substances C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003054 catalyst Substances 0.000 claims abstract description 13
- -1 triphenylphosphine cation Chemical class 0.000 claims abstract description 12
- 239000012024 dehydrating agents Substances 0.000 claims abstract description 10
- 150000003053 piperidines Chemical class 0.000 claims abstract description 10
- 239000002808 molecular sieve Substances 0.000 claims abstract description 9
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims abstract description 9
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims abstract description 8
- FXURYRWDOBBQLX-UHFFFAOYSA-N N1C=CC=C1.[B].[F] Chemical group N1C=CC=C1.[B].[F] FXURYRWDOBBQLX-UHFFFAOYSA-N 0.000 claims abstract description 4
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 150000007980 azole derivatives Chemical class 0.000 claims description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 claims description 13
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 claims description 13
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 claims description 13
- 229930016911 cinnamic acid Natural products 0.000 claims description 13
- 235000013985 cinnamic acid Nutrition 0.000 claims description 13
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 10
- 229910052796 boron Inorganic materials 0.000 claims description 9
- 229910052731 fluorine Inorganic materials 0.000 claims description 9
- 239000011737 fluorine Substances 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 7
- 239000007789 gas Substances 0.000 claims description 6
- 150000003512 tertiary amines Chemical class 0.000 claims description 6
- 229910052786 argon Inorganic materials 0.000 claims description 5
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 5
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 4
- 150000003233 pyrroles Chemical class 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical group CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims description 4
- 150000001412 amines Chemical class 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical compound CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 claims description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 2
- 150000001299 aldehydes Chemical class 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 229910001914 chlorine tetroxide Inorganic materials 0.000 claims description 2
- 230000018044 dehydration Effects 0.000 claims description 2
- 238000006297 dehydration reaction Methods 0.000 claims description 2
- XXBDWLFCJWSEKW-UHFFFAOYSA-N dimethylbenzylamine Chemical compound CN(C)CC1=CC=CC=C1 XXBDWLFCJWSEKW-UHFFFAOYSA-N 0.000 claims description 2
- 238000002189 fluorescence spectrum Methods 0.000 claims description 2
- 238000005342 ion exchange Methods 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Chemical compound [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- NBBJYMSMWIIQGU-UHFFFAOYSA-N propionic aldehyde Natural products CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 claims description 2
- 150000003335 secondary amines Chemical class 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 125000002077 trioxo-lambda(7)-bromanyloxy group Chemical group *O[Br](=O)(=O)=O 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 51
- 238000006243 chemical reaction Methods 0.000 abstract description 21
- 239000007787 solid Substances 0.000 abstract description 10
- 210000000170 cell membrane Anatomy 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 9
- 210000003470 mitochondria Anatomy 0.000 abstract description 9
- 210000003712 lysosome Anatomy 0.000 abstract description 8
- 230000001868 lysosomic effect Effects 0.000 abstract description 8
- 230000008685 targeting Effects 0.000 abstract description 8
- 239000000376 reactant Substances 0.000 abstract description 7
- 239000007850 fluorescent dye Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 150000002632 lipids Chemical class 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 230000002596 correlated effect Effects 0.000 abstract description 2
- 230000007774 longterm Effects 0.000 abstract description 2
- 241000522254 Cassia Species 0.000 abstract 1
- 239000012847 fine chemical Substances 0.000 abstract 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 abstract 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 abstract 1
- 230000003834 intracellular effect Effects 0.000 abstract 1
- 230000035479 physiological effects, processes and functions Effects 0.000 abstract 1
- 230000036299 sexual function Effects 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 25
- 238000004043 dyeing Methods 0.000 description 11
- 238000000799 fluorescence microscopy Methods 0.000 description 10
- 230000006837 decompression Effects 0.000 description 9
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- 238000003384 imaging method Methods 0.000 description 6
- 239000003480 eluent Substances 0.000 description 5
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- 238000000926 separation method Methods 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Substances CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
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- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940006460 bromide ion Drugs 0.000 description 1
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- 108010037721 cytase Proteins 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N diethylenediamine Natural products C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- YAYTWOCUDHQKEC-UHFFFAOYSA-N formaldehyde;triphenylphosphane Chemical compound O=C.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 YAYTWOCUDHQKEC-UHFFFAOYSA-N 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
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- 230000001590 oxidative effect Effects 0.000 description 1
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- 230000001717 pathogenic effect Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- FJVZDOGVDJCCCR-UHFFFAOYSA-M potassium periodate Chemical compound [K+].[O-]I(=O)(=O)=O FJVZDOGVDJCCCR-UHFFFAOYSA-M 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
One class is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles, belongs to field of fine chemical.The probe is fluorine boron azole derivatives and Chinese cassia tree aldehyde reaction, with piperidines as catalyst, 4 molecular sieves as dehydrating agent, gained reactant obtains fluorescent probe molecule by quaternized.Contain triphenylphosphine cation, morpholine, quaternary ammonium salt cationic isoreactivity group in probe respectively, different activities group makes probe have the subcellular organelle targeting of selectivity, is optionally positioned at the subcellular organelles such as mitochondria, lysosome, cell membrane.The probe has no toxic side effect, it is adaptable to monitor different physiology course sub-cellular device inner lipid peroxidating processes by fluorescence microscope or Fluorescein activated cell sorter.The probe solid state is stable in properties, it is easy to long-term to preserve, and directly perceived, succinct and with low cost detection reagent is provided to probe into intracellular and lipid peroxidation correlated process, and subcellular organelle targeting sexual function makes detection provide more microcosmic structural information.
Description
Technical field
It is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles the present invention relates to a class, it belongs to essence
Thin chemical technology field.
Background technology
Lipid peroxidation (Lipid peroxidation, LPO) by clearly report be numerous diseases main pathogenesis,
Such as cancer, atherosclerosis, the nervous system disease, etc..The essence of LPO is the type free base based on unrighted acid
Chain reaction, it is typically to be triggered by active oxygen radical (reactive oxygen species, ROS), and it is to body
Pathogenic damage often beyond ROS to the Oxidative demage of intercellular membrane structure.LPO comes to the pernicious result that body brings
Chain reaction is persistently carried out, and chain reaction can constantly discharge active material, such as MDA, such material can trigger various destructions
Property reaction, damage body activity.Therefore, prevention of the LPO processes for specified disease in research organism or previous tretament
It is significant.
Fluorescence microscopy is suitable for carrying out living cells in lossless dynamic prison as a kind of monitoring means in real time directly perceived
Survey.Although existing part report monitors the generation of LPO using fluorescence imaging means, however reported with subcellular organelle targeting
Property LPO probes but seldom, be mainly based upon commercialization LPO probes C11-BODIPY581/591, and its synthesis and preparation process ratio
It is cumbersome, cause its relatively costly, it is expensive, it is unfavorable for widely using, the only one LPO probe of targetted mitochondria
MitoPerOx is also based on C11-BODIPY581/591The structure for carrying out derives, and synthesis is complicated.For more other subcellular organelles
The LPO probes of targeting there is no report.According to the demand of research associated conditions, we are devoted to design has different subcellular organelles
The LPO probes of targeting, for the Clinics and Practices of LPO relevant diseases provide more microcosmic information and research tool.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a series of for targetting different subcellular organelles and it can be detected
Fluorescence probe of middle lipid peroxidation process and preparation method thereof, line fluorescent probe preparation cost is low, selectivity targeting is different
Subcellular organelle, to lipid peroxidation PROCESS SENSITIVE and can by ratio fluorescent be imaged relative quantification monitoring lipid peroxidation enter
Journey.
The technical solution adopted by the present invention is：For targetting different subcellular organelles and wherein lipid peroxidation process can be detected
Fluorescence probe, with fluorine boron pyrroles as parent, with following general structure：
R in formula1、R2 = H、C1-C10Straight or branched alkyl；R3=H or, wherein：R4 =
, X- = Cl-、Br-、I-、SO4 2-、ClO4 -、
BrO4 -、IO4 - 。
One class is used for the preparation method of the fluorescence probe for monitoring lipid peroxidation process in different subcellular organelles, the R3 =
The fluorescence probe preparation method of H is：
It is raw material with 1 mole of the azole derivatives of fluorine boron two and 1 mole of cinnamic acid, with piperidines as catalyst, 4 molecules
It is dehydrating agent to sieve, and the azole derivatives of fluorine boron two are 1 with the mol ratio of catalyst:0.01,130 DEG C in toluene, in argon gas
Or stirred under the protection of nitrogen gas, 8-24h is reacted, cinnamic acid is generated containing altogether with the active methylene group dehydration of the pyrroles of fluorine boron two
The fluorescence probe of yoke double bond.
One class is used for the preparation method of the fluorescence probe for monitoring lipid peroxidation process in different subcellular organelles, the R3 =Fluorescence probe preparation method comprise the following steps：
（1）The preparation of the azole derivatives M of fluorine boron two
It is raw material with 1 mole of the azole derivatives of fluorine boron two containing 3- chloropropyls and 1 mole of cinnamic acid, is with piperidines
Catalyst, 4 molecular sieves are dehydrating agent, and the azole derivatives of fluorine boron two are 1 with the mol ratio of catalyst:0.01, in toluene
130 DEG C, stirred under the protection of argon gas or nitrogen gas, react 8-24h, cinnamic acid takes off with the active methylene group of the pyrroles of fluorine boron two
Water generates the azole derivatives M of fluorine boron two containing conjugated double bond；
（2）Probe molecule is obtained by following two routes：
Route one
The azole derivatives M of fluorine boron two is dissolved in acetone, chloroform or DMSO equal solvents with corresponding swollen amine, is added and intermediate
M mol ratios are 1:1 potassium carbonate and KI reacts 3-10h at 30-130 DEG C, obtains fluorescent probe molecule；The secondary amine choosing
From dimethylamine, morpholine, methyl piperazine or benzyl diethylenediamine；
Route two
The azole derivatives M of fluorine boron two with corresponding tertiary amine or triphenylphosphine derivates is dissolved in acetone, chloroform or DMSO etc. molten
In agent, it is 1 to add with intermediate M mol ratios:1 KI reacts 3-24h at 30-130 DEG C, obtains probe molecule, described
Fluorescence probe stirs 24h in methyl alcohol with the sodium salt containing different anions or sylvite, makes to carry out ion exchange, obtains containing not
With the fluorescence probe of counter ion；The tertiary amine is selected from trimethylamine, dimethyl benzyl amine or dimethylamino propionic aldehyde, the triphenylphosphine
Derivative is selected from triphenylphosphine or aldehyde radical triphenylphosphine.
The fluorescence probe targets the change being positioned in different subcellular organelles and by fluorescence spectrum and indicates Asia respectively
The lipid peroxidation process of organelle.The fluorescence probe is used for targeting positioning and its inside of different subcellular organelles in living cells
The detection of lipid peroxidation process.The living cells that can be labeled is Hella cell lines, MCF-7 cell lines, RAW264.7 cells
Strain, 7702 cell lines etc..
The beneficial effects of the invention are as follows：It is a series of for targetting different subcellular organelles and can detect that wherein lipid peroxidation enters
The fluorescence probe of journey is reacted under nitrogen or argon as substrate with fluorine boron pyrroles with cinnamic acid, with piperidines as catalyst, 4
Molecular sieve is dehydrating agent；Gained reactant obtains fluorescent probe molecule with the reaction of swollen amine, tertiary amine or triphenylphosphine again.Plurality of probes
Molecule launches fluorescence in the long-wavelength region of 590nm, sensitive to active oxygen radical, that is, participate in the lipid peroxidation process in living cells
Afterwards, conjugated double bond is destroyed, and its launch wavelength is changed into the short wavelength of 520nm or so, by short wavelength (about 520nm) and long wavelength
（About 590nm）The ratio value changes of the fluorescent emission intensity at two, can detect the level of lipid peroxidation in living cells.Meanwhile,
Different probe is due to containing different activities group（That is R4）, there is different subcellular organelle targetings, i.e. plurality of probes can divide for it
Jian Ce not different subcellular organelles in living cells（Mitochondria, lysosome or cell membrane）Interior lipid peroxidation process.The probe sheet
Body is stable in properties, it is easy to long-term to preserve, and has sensitive response to lipid peroxidation process, and can be indicated by ratio fluorescent imaging
The degree of lipid peroxidation, provides to probe into the lipid peroxidation correlated process in living cells in microstructure and intuitively detects
Reagent.
Brief description of the drawings
Fig. 1 is fluorescence probe A in CH2Cl2The spectrum change after lipid peroxidation process is participated in solution.
Fig. 2 is fluorescence probe A to the fluorescence imaging after the fat drips dyeing in MCF-7 cells.
Fig. 3 is that fluorescence probe A carries out fluorescence imaging detection to the lipid peroxidation process in fat drips in MCF-7 cells.
Fig. 4 is the spectrum change after fluorescence probe B-1 participates in lipid peroxidation process in the solution.
Fig. 5 is fluorescence probe B-1 to the fluorescence imaging after lysosome dyeing in RAW264.7 cells.
Fig. 6 is the fluorescence imaging that fluorescence probe B-1 is carried out to lyase body lipid peroxidating process in RAW264.7 cells
Detection.
Fig. 7 is the fluorescence imaging after fluorescence probe C-1 is dyeed to MCF-7 cells Mitochondria.
Fig. 8 is the fluorescence imaging inspection that fluorescence probe C-1 is carried out to MCF-7 cell Mitochondria inner lipid peroxidating processes
Survey.
Fig. 9 is fluorescence probe C-2 to the fluorescence imaging after cell membrane dyeing in Hella cells.
Figure 10 is the fluorescence imaging inspection that fluorescence probe C-2 is carried out to lipid peroxidation process on cell membrane in Hella cells
Survey.
Specific embodiment
Embodiment 1
Tetramethyl fluorine boron pyrroles 500mg, the cinnamic acid 129mL, the piperidines for adding 500mL is added to be in two mouthfuls of reaction bulbs
Catalyst, 4 molecular sieves are dehydrating agent, and 120 DEG C of stirring reaction 8h in toluene, reactant decompression is spin-dried for, after removing toluene, silicon
Glue post separation（Eluent is dichloromethane：N-hexane=1:4）Obtain violet solid 155mg, yield 23%.
Embodiment 2
Tetramethyl fluorine boron pyrroles 500mg, the cinnamic acid 94mL, the piperidines for adding 500mL is added to be in two mouthfuls of reaction bulbs
Catalyst, 4 molecular sieves are dehydrating agent, and 120 DEG C of stirring reaction 8h in toluene, reactant decompression is spin-dried for, after removing toluene, silicon
Glue post separation（Eluent is dichloromethane：N-hexane=1:4）Obtain violet solid 194mg, yield 31%.
It is dissolved in product 100mg is obtained in upper step in DMSO, adds excess morpholine 100mL, 24mg K2CO3With 30mg KI,
60 DEG C of reaction 12h, reactant is washed and by CH2Cl2Extraction, retains organic phase, and after anhydrous magnesium sulfate is dried, decompression is spin-dried for, silica gel
Post separation（Eluent is dichloromethane：Methyl alcohol=20:1）Obtain violet solid 81mg, yield 75%.
Embodiment 3
Tetramethyl fluorine boron pyrroles 500mg, the cinnamic acid 88mL, the piperidines for adding 500mL is added to be in two mouthfuls of reaction bulbs
Catalyst, 4 molecular sieves are dehydrating agent, and 120 DEG C of stirring reaction 8h in toluene, reactant decompression is spin-dried for, after removing toluene, silicon
Glue post separation（Eluent is dichloromethane：N-hexane=1:4）Obtain violet solid 217mg, yield 35%.
It is dissolved in product 100mg is obtained in upper step in acetone, adds excess tertiary amine 50mg, KI 30mg, 60 DEG C of reaction 12h,
Concentration of reaction solution, and ether is added, there is darkviolet solid to separate out, as target product.Fluorescence probe is dissolved in methyl alcohol, is added
, be spin-dried for for reaction solution decompression after 50 DEG C of stirring 24h by the potassium metaperiodate of 1000mg, is dissolved in a small amount of dichloromethane, is filtered to remove inorganic
Salt, retains filtrate, and decompression again is spin-dried for, and obtains 141mg darkviolet solids, and as counter ion is the fluorescence probe of periodate,
Two step gross production rates 88%.
Embodiment 4
Tetramethyl fluorine boron pyrroles 500mg, the cinnamic acid 83mL, the piperidines for adding 500mL is added to be in two mouthfuls of reaction bulbs
Catalyst, 4 molecular sieves are dehydrating agent, and 120 DEG C of stirring reaction 8h in toluene, reactant decompression is spin-dried for, after removing toluene, silicon
Glue post separation（Eluent is dichloromethane：N-hexane=1:4）Obtain violet solid 196mg, yield 32%.
It is dissolved in product 100mg is obtained in upper step in acetone, adds excess triphenylphosphine formaldehyde 200mg, KI 30mg, 60
DEG C reaction 12h, concentration of reaction solution, and add ether has darkviolet solid to separate out, as target product.Fluorescence probe is dissolved in
In methyl alcohol, the KBr of 500mg is added, be spin-dried for reaction solution decompression after 50 DEG C of stirring 24h, be dissolved in a small amount of dichloromethane, be filtered to remove
Inorganic salts, retain filtrate, and decompression again is spin-dried for, and obtain 140mg darkviolet solids, and as counter ion is the fluorescence spy of bromide ion
Pin, two step gross production rates 91%.
Embodiment 5
Fluorescence probe A is used for the detection of lipid peroxidation process in fat drips in living cells.Hella cell lines, MCF-7 cells
Fat drips in the cells such as strain, RAW264.7 cell lines can be marked by probe, and detect level of lipid peroxidation therein.
Fig. 1 is in solution (CH2Cl2) in by chemicals simulate lipid peroxidation process when probe spectrum change, with
The extension of lipid peroxidation time, fluorescence intensity of the probe at 520nm gradually strengthens, and the fluorescence at 590nm gradually subtracts
It is weak.Wherein illustration is that peak intensity ratio changes with time at two, with the intensification of level of lipid peroxidation, peak intensity and length at shortwave
Peak intensity ratio also strengthens at ripple, i.e., rate value can reflect the degree of lipid peroxidation.
Fig. 2 be the fluorescence washed after twice to the min of MCF-7 cell dyeings 30 and using PBS using the fluorescence probe A of 1mM into
Picture.The nm of excitation wavelength 488, acquisition range 560-630 nm, cell imaging fat drips are clear-cut, without bias light interference.
Fig. 3 is the interior generation lipid peroxidation process of cell fat drips after dyeing, same to use 488nm laser excitations, while
In the interval collection short wavelength emissions light of 500-560nm（a）With the interval collection longwave transmissions of 560-630nm（b）, wherein short wavelength emissions table
Bright probe take part in the lipid peroxidation process in fat drips；Can intuitively be reflected with the ratio image of long wave passage by shortwave passage
Level of lipid peroxidation in cell in fat drips（c）.
Embodiment 6
Fluorescence probe B-1 is used for the detection of lipid peroxidation process in lysosome in living cells.Hella cell lines, MCF-7
Lysosome in the cells such as cell line, RAW264.7 cell lines can be marked by probe, and detect lipid peroxidation journey therein
Degree.
Fig. 4 is the spectrum of probe when simulating lipid peroxidation process by chemicals in solution (water/methyl alcohol=2/1)
Change, with the extension of lipid peroxidation time, fluorescence intensity of the probe at 520nm gradually strengthens, the fluorescence at 590nm
Gradually weaken.Wherein illustration is that peak intensity ratio changes with time at two, with the intensification of level of lipid peroxidation, peak at shortwave
By force with long wave at peak intensity ratio also strengthen, i.e., rate value can reflect the degree of lipid peroxidation.
Fig. 5 be using 1mM fluorescence probe B-1 to the min of RAW264.7 cell dyeings 5 and using PBS wash after twice it is glimmering
Photoimaging.The nm of excitation wavelength 488, acquisition range 560-630 nm, cell imaging lysosome is clear-cut, without bias light interference.
Fig. 6 is the interior generation lipid peroxidation process of cytase body after dyeing, same to use 488nm laser excitations, together
When in the interval collection short wavelength emissions light of 500-560nm（a）With the interval collection longwave transmissions of 560-630nm（b）, wherein short wavelength emissions
Show that probe take part in the lipid peroxidation process in lysosome；Can be directly perceived with the ratio image of long wave passage by shortwave passage
Level of lipid peroxidation in reflection cell in lysosome（c）.
Embodiment 7
Fluorescence probe C-1 is used for the detection of lipid peroxidation process in living cells Mitochondria.Hella cell lines, MCF-7
Mitochondria in the cells such as cell line, RAW264.7 cell lines can be marked by probe, and detect lipid peroxidation journey therein
Degree.
Fig. 7 is the fluorescence washed after twice to the min of MCF-7 cell dyeings 5 and using PBS using the fluorescence probe C-1 of 1mM
Imaging.The nm of excitation wavelength 488, acquisition range 560-630 nm, cell imaging mitochondria is clear-cut, without bias light interference.
Fig. 8 is the interior generation lipid peroxidation process of cell mitochondrial after dyeing, same to use 488nm laser excitations, together
When in the interval collection short wavelength emissions light of 500-560nm（a）With the interval collection longwave transmissions of 560-630nm（b）, wherein short wavelength emissions
Show that probe take part in Intramitochondrial lipid peroxidation process；Can be directly perceived with the ratio image of long wave passage by shortwave passage
Level of lipid peroxidation in reflection cell Mitochondria（c）.
Embodiment 8
Fluorescence probe C-2 is used for the detection of lipid peroxidation process on cell membrane in living cells.Hella cell lines, MCF-7
Cell membrane in the cells such as cell line, RAW264.7 cell lines can be marked by probe, and detect lipid peroxidation journey therein
Degree.
Fig. 9 is the fluorescence washed after twice to the min of Hella cell dyeings 5 and using PBS using the fluorescence probe C-2 of 1mM
Imaging.The nm of excitation wavelength 488, acquisition range 560-630 nm, cell imaging cell membrane is clear-cut, without bias light interference.
Figure 10 is that lipid peroxidation process occurs on cell film after dyeing, same to use 488nm laser excitations, together
When in the interval collection short wavelength emissions light of 500-560nm（a）With the interval collection longwave transmissions of 560-630nm（b）, wherein short wavelength emissions
Show that probe take part in the lipid peroxidation process on cell membrane；Can be directly perceived with the ratio image of long wave passage by shortwave passage
Level of lipid peroxidation on reflection cell in cell membrane（c）.
Claims (4)
1. a class is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles, with fluorine boron pyrroles as parent, its
It is characterised by, the probe has following general structure：
R in formula1、R2 = H、C1-C10Straight or branched alkyl；R3=H or, wherein：R4 =
Or, X- = Cl-、Br-、I-、ClO4 -、BrO4 -Or IO4 -。
2. a class according to claim 1 is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles
Preparation method, it is characterised in that the R3The fluorescence probe preparation method of=H is：
It is raw material with 1 mole of the azole derivatives of fluorine boron two and 1 mole of cinnamic acid, with piperidines as catalyst, 4 molecular sieves are
Dehydrating agent, the azole derivatives of fluorine boron two are 1 with the mol ratio of catalyst:0.01,130 DEG C in toluene, in argon gas or nitrogen
Stirred under the protection of gas gas, react 8-24h, cinnamic acid contains with the active methylene group dehydration generation of the azole derivatives of fluorine boron two
The fluorescence probe of conjugated double bond.
3. a class according to claim 1 is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles
Preparation method, it is characterised in that the R3 = Fluorescence probe preparation method comprise the following steps：
（1）The preparation of the azole derivatives M of fluorine boron two
It is raw material with 1 mole of the azole derivatives of fluorine boron two containing 3- chloropropyls and 1 mole of cinnamic acid, is catalysis with piperidines
Agent, 4 molecular sieves are dehydrating agent, and the azole derivatives of fluorine boron two are 1 with the mol ratio of catalyst:0.01,130 in toluene
DEG C, stirred under the protection of argon gas or nitrogen gas, 8-24h is reacted, cinnamic acid is dehydrated with the active methylene group of the pyrroles of fluorine boron two
Fluorine boron two azole derivatives M of the generation containing conjugated double bond；
（2）Probe molecule is obtained by following route one or the synthesis of route two：
Route one：
The azole derivatives M of fluorine boron two is dissolved in acetone, chloroform or DMSO solvents with swollen amine, is separately added into and is spread out with the pyrroles of fluorine boron two
Biological M mol ratios are 1:1 potassium carbonate and KI reacts 3-10h at 30-130 DEG C, obtains fluorescence probe；The secondary amine choosing
From dimethylamine, morpholine, methyl piperazine or 4- chloromethyl benzyl diethylenediamines；
Route two：
The azole derivatives M of fluorine boron two and tertiary amine or triphenylphosphine derivates are dissolved in acetone, chloroform or DMSO solvents, add with
The azole derivatives M mol ratios of fluorine boron two are 1:1 KI reacts 3-24h at 30-130 DEG C, obtains fluorescence probe, described glimmering
Light probe stirs 24h in methyl alcohol with the sodium salt containing different anions or sylvite, makes to carry out ion exchange, obtains containing different
The fluorescence probe of counter ion；The tertiary amine is selected from trimethylamine, 4- chloromethyls benzyl dimethylamine or dimethylamino propionic aldehyde, the triphen
Base phosphine-derivatives are selected from triphenylphosphine or aldehyde radical triphenylphosphine.
4. a class according to claim 1 is used to monitor the fluorescence probe of lipid peroxidation process in different subcellular organelles
Using, it is characterised in that：The fluorescence probe targets the change for being positioned in different subcellular organelles and passing through fluorescence spectrum respectively
Change the lipid peroxidation process for indicating subcellular organelle.
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