CN109134545A - A kind of Mitochondrially targeted fluorescence probe and the preparation method and application thereof - Google Patents
A kind of Mitochondrially targeted fluorescence probe and the preparation method and application thereof Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims description 11
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 108
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 15
- 230000008685 targeting Effects 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 150000001875 compounds Chemical class 0.000 claims description 23
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 230000035484 reaction time Effects 0.000 claims description 14
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical group CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 claims description 10
- VGCXGMAHQTYDJK-UHFFFAOYSA-N Chloroacetyl chloride Chemical compound ClCC(Cl)=O VGCXGMAHQTYDJK-UHFFFAOYSA-N 0.000 claims description 6
- 229910015900 BF3 Inorganic materials 0.000 claims description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical group C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 5
- 239000007850 fluorescent dye Substances 0.000 claims description 5
- YXYAHRAFINYJPL-UHFFFAOYSA-N 4-methyl-1h-pyrrole-2-carboxylic acid Chemical compound CC1=CNC(C(O)=O)=C1 YXYAHRAFINYJPL-UHFFFAOYSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 239000003054 catalyst Substances 0.000 claims description 4
- 239000012024 dehydrating agents Substances 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000000908 ammonium hydroxide Substances 0.000 claims description 3
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims 2
- KAESVJOAVNADME-UHFFFAOYSA-N 1H-pyrrole Natural products C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims 1
- 206010011224 Cough Diseases 0.000 claims 1
- 235000019253 formic acid Nutrition 0.000 claims 1
- RIOQSEWOXXDEQQ-UHFFFAOYSA-O triphenylphosphanium Chemical compound C1=CC=CC=C1[PH+](C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-O 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 10
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- 230000002438 mitochondrial effect Effects 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
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- 238000001727 in vivo Methods 0.000 description 3
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- 238000003786 synthesis reaction Methods 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000031857 establishment of mitochondrion localization Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- GLLVXRTWJXVQAF-ZDUSSCGKSA-N (2S)-2-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methylamino]benzoyl]amino]-4-methylidenepentanedioic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CC(=C)C(O)=O)C(O)=O)C=C1 GLLVXRTWJXVQAF-ZDUSSCGKSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
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- 101001005618 Homo sapiens Dimethylglycine dehydrogenase, mitochondrial Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- FFDGPVCHZBVARC-UHFFFAOYSA-N N,N-dimethylglycine Chemical compound CN(C)CC(O)=O FFDGPVCHZBVARC-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 206010056677 Nerve degeneration Diseases 0.000 description 1
- 102100023363 Sarcosine dehydrogenase, mitochondrial Human genes 0.000 description 1
- 101150028021 Sardh gene Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 238000004177 carbon cycle Methods 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
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- 230000008859 change Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 108700003601 dimethylglycine Proteins 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
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- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- BXGTVNLGPMZLAZ-UHFFFAOYSA-N n'-ethylmethanediimine;hydrochloride Chemical compound Cl.CCN=C=N BXGTVNLGPMZLAZ-UHFFFAOYSA-N 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
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- 230000035699 permeability Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002213 purine nucleotide Substances 0.000 description 1
- 125000000561 purinyl group Chemical class N1=C(N=C2N=CNC2=C1)* 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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- 210000004881 tumor cell Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- 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
- C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- 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
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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Abstract
The invention discloses a kind of BODIPY fluorescence probes of the formaldehyde in targeting detection mitochondria, the probe molecule probe is targeting group as fluorogen, with triphenyl phosphonium base using BODIPY, have the advantages that be quick on the draw, detect that limit is low, specific good, good biocompatibility, can be applied to the formaldehyde in targeting detection mitochondria.
Description
Technical field
The present invention relates to a kind of fluorescence probes for detecting formaldehyde, and in particular to a kind of fluorescence probe of targetted mitochondria and its
Preparation method and application.
Background technique
Formaldehyde present in environment has had a large amount of reports to the harm of human health, but the endogenous generated in vivo
Formaldehyde and its caused related disease research are relatively fewer.Formaldehyde is prevalent in each system of human body, especially in
Pivot nervous system, the circulatory system, digestion and urinary system etc..Formaldehyde can with some important biomolecule molecules, as protein, DNA,
RNA and neurotransmitter etc. are chemically reacted, and influence its structure and function.It may be to cause first that synthesis is unbalance with katabolism
Aldehyde is accumulated in vivo and excessive reason.Studies have shown that endogenous formaldehyde and aging, neuron degeneration, tumour cell division
Proliferation etc., especially to neurodegenerative disease closely related (" endogenous formaldehyde and its related mankind's major disease ", Tong Zhiqian
Deng natural science progress, 11 phases in 2008).
Normal concentration of formaldehyde is able to maintain that body metabolism balances, and formaldehyde and the raising of relevant active carbonyl group substance can draw
Rise many diseases, including cancer, diabetes, chronic liver disease, neurodegenerative disease and cardiovascular disease etc. (" endogenous formaldehyde with
The progress of alzheimer disease mechanism ", Li Weiwei, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 20 phases in 2012).Formaldehyde is made in vivo
For a kind of signal of interest factor, intracorporal carbon cycle process is participated in, one of them typical example is exactly that formaldehyde has participated in leaf
The mitochondria single carbon cyclic process that acid is adjusted.In this process, dimethylglycine is in cyclophorase DMGDH and SARDH
Effect is lower to be generated glycine and releases the formaldehyde of two molecules, and the formaldehyde of generation can generate 5,10- with the folic acid of mitochondrial internal
Methylene folic acid, for further participating in the single carbon cyclic process in cytoplasm and nucleus, final production goes out to be used to tie up
The substances such as purine nucleotides, thymidylic acid, methionine, the serine of cell normal physiological function are held, and cell single carbon circulation is different
Toward with certain diseases such as dysplasia, cancer occur for frequentation.Some researches show that excessive formaldehyde can trigger mitochondria half
Guang aspartase apoptotic process makes cell death.And formaldehyde is in human body, the amount fluctuation of especially mitochondrial internal is raw for human body
The influence of reason and pathology still not clear enough (" the chemical reaction progress that formaldehyde participates in organism ", Han Yan, Wuhan engineering
College journal, 01 phase in 2018).Therefore, exploitation is readily synthesized, has good selectivity, can be used for quickly detecting in mitochondria
The fluorescence probe of formaldehyde is of great significance.
Summary of the invention
In order to overcome the deficiencies of the prior art, the present invention is intended to provide a kind of BODIPY of the formaldehyde in targeting detection mitochondria
Fluorescence probe, which has the advantages that be quick on the draw, low, the specific good, good biocompatibility that detects limit.
Another object of the present invention is to provide a kind of BODIPY fluorescence probes of the formaldehyde in targeting detection mitochondria
Synthetic method.
To achieve the above object, it is provided by the invention targeting detection mitochondria in formaldehyde BODIPY fluorescence probe with
BODIPY is parent nucleus, take triphenyl phosphonium base as targeting group, and structural formula is as follows:
A kind of preparation method of above-mentioned BODIPY fluorescence probe, which is characterized in that the method preparation route is by following anti-
It answers shown in formula (1)-(4).
Wherein, in reaction equation (1), reaction condition includes, 4- methyl -2- pyrrole carboxylic acid and chloracetyl chloride shown in formula a
Molar ratio is 1.5~3:1, preferably 2~2.2:1；Reaction temperature is room temperature, and the reaction time is 10~120min.
In reaction equation (2), formula b compound reacts in the presence of triethylamine with boron trifluoride ether solution, reaction temperature control
For system at 25-30 DEG C, the reaction time is 2-12 hours.
In reaction equation (3), formula c compound reacts in the presence of dehydrating agent/catalyst with formula d compound, and reaction temperature is
Room temperature, reaction time are 12-24 hours.
In reaction equation (4), in the molten tetrahydrofuran solution of formula e compound, ammonium hydroxide (1~2h) is slowly added dropwise, is stirred at room temperature anti-
It answers 6~24 hours, obtains fluorescent probe molecule shown in formula f.
Preferably, in reaction equation (1), the molar ratio of 4- methyl -2- pyrrole carboxylic acid and chloracetyl chloride shown in formula a is preferably
2~2.2:1；Reaction time is preferably 30min.
In reaction equation (2), formula b compound reacts in the presence of triethylamine with boron trifluoride ether solution, reaction temperature control
For system at 25 DEG C, the reaction time is preferably 4 hours.
In reaction equation (3), the dehydrating agent is dicyclohexylcarbodiimide (DCC) or 1- (3- dimethylamino-propyl) -3-
Ethyl-carbodiimide hydrochloride (EDCI)；Catalyst be 4-dimethylaminopyridine (DMAP) or I-hydroxybenzotriazole (HOBT),
Reaction time is preferably 12 hours.
In reaction equation (4), it is preferably 12 hours that the reaction time, which is stirred at room temperature,.
A kind of purposes of the content of formaldehyde of above-mentioned fluorescent probe molecule in detection solution, physiological environment and/or cell.
Fluorescent probe molecule detection mechanism of the invention is as follows:
(1) it when BODIPY fluorescence probe of the present invention is applied to detection formaldehyde, since formaldehyde and amino react, leads
Cause probe molecule that decaying phenomenon has occurred under excited state, fluorescence signal weakens or quenching, so as to selectively examine
Survey formaldehyde.(2) for the influence using reaction front and back to entire compound fluorescence intensity as the detection signal of identification, test fluorescence is strong
Degree obtains linear fit curve, to quantitatively detect the concentration to formaldehyde in sample with the variation of concentration of formaldehyde.(3) due to
Proton can be pumped out between mitochondrial outer membrane by the electron transport chain on mitochondrial inner membrane from mitochondrial matrix, thereby result in line
Potential difference on Mitochondria Membrane, by the way that probe molecule to be connected to the triphenyl phosphonium cation of new lipid, by mitochondrial inner membrane
Probe molecule targeting is gathered in mitochondrial matrix by potential difference, to realize the detection of formaldehyde in mitochondria.
Compared with prior art, the present invention has the following beneficial effects:
(1) probe molecule structure novel of the invention, using BODIPY as parent, using amino as detection moiety, and by
Lipophilic cation triphenyl phosphonium group is introduced in Molecular Design, probe molecule can be targeted and is gathered in mitochondria,
And detection of the realization to formaldehyde in mitochondria.
(2) probe molecule of the present invention can detecte the content of formaldehyde of nanomolar concentrations, and detection limit is low, not have to cell itself
Toxic side effect, cell permeability is good, and macromolecule water-solubility is good, can be prepared into solution and be tested, avoid organic solvent
It uses, reduces tissue damage, biocompatibility with higher.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
The preparation of 1 formula b compound of embodiment
Into 25ml round-bottomed flask, it is added the chloracetyl chloride of the 4- methyl -2- pyrrole carboxylic acid and 1mmol of 2.1mmol, then plus
Enter 2mL methylene chloride, room temperature magnetic agitation 30 minutes, detect fully reacting through TLC, be concentrated under reduced pressure, silica gel column chromatography separates (second
Acetoacetic ester/petroleum ether is eluting solvent), obtain formula b compound represented.Yield (89%, in terms of chloracetyl chloride).
The preparation of 2 formula c compound of embodiment
Into 25ml round-bottomed flask, the formula b compound of 1mmol is added, the triethylamine and 2mL dichloro of 2mmol is then added
0.5mL boron trifluoride ether is slowly added dropwise in methane under agitation, and control reaction temperature is 25 DEG C, reacts 6 hours through TLC
Detection formula b compound fully reacting is concentrated under reduced pressure and removes solvent, and residue is separated (ethyl acetate/petroleum through silica gel column chromatography
Ether is eluting solvent), obtain formula c compound represented.Yield (85%).
The synthesis of 3 formula d compound of embodiment
Into 25ml round-bottomed flask, the formula c compound of 1mmol and the formula d compound of 2.1mmol are dissolved in methylene chloride,
Methylene chloride (each 1mL) solution of DCC (1.5mmol) and DMAP (0.1mmol) is added dropwise respectively again, reaction is stirred at room temperature overnight
(about 12 hours) are concentrated under reduced pressure remove solvent after the reaction was completed, and residue is separated (ethyl acetate/petroleum through silica gel column chromatography
Ether is eluting solvent), obtain formula e compound represented.Yield (93%).
The synthesis of 4 fluorescent probe molecule compound f of embodiment
Into 25mL round-bottomed flask, the formula e compound of 1mmol is added, 4mmol DMSO stirring and dissolving is added, then in room
It is slowly added dropwise under temperature ammonium hydroxide (0.4mL, concentration 28%), reacts at room temperature 8 hours, remove solvent through being concentrated under reduced pressure after the reaction was completed,
Residue is separated into (ethyl acetate/petroleum ether is eluting solvent) through silica gel column chromatography, obtains fluorescence probe shown in formula f point
Son.Yield (84%).1H NMR(400MHz,DMSO-d6)δ8.90-9.23(1H),8.69-8.84(2H),8.17-8.33
(1H),7.61(1H),7.29-7.43(30H),7.10(1H),6.93(1H),5.42(1H),3.18-3.32(6H),1.8
(4H),1.3(4H)。
Probe molecular fluorescence Strength Changes under the different concentration of formaldehyde of embodiment 5
It is separately added into the PBS buffer solution (pH=7.4,5%DMSO) that concentration and probe concentration shown in formula f is 5 μM different dense
(0-250 μM, concentration of formaldehyde: 0,10,20,40,60,80,100,120,150,200,250), then use pH 7.4 to the formaldehyde of degree
PBS buffer solution constant volume, act on and carry out fluorescence spectrum test (λ ex=440nm) after a hour, it is strong to detect fluorescence in each system
It spends (at maximum emission peak λ em=528nm).
The result shows that fluorescence intensity reduces at system 528nm with the increase of OCHO concentration.Linear fit curve it is linear
Regression constant is 0.9962, shows that probe can quantitatively measure the concentration of OCHO.
6 probe molecule mitochondria positioning of embodiment detects aldehyde test
It (1) is 3 × 10 by density5The HeLa cell inoculation of a/mL is covered with coverslip (22mm × 22mm) to sterilizing
In 35mm culture dish, culture is adherent to cell in CO2 incubator (37 DEG C, 5%CO2).
(2) 100 μM of formaldehyde are added into cell culture fluid, after being incubated for 20min, are added to Tissue Culture Dish for 5 μM of probe
In, it is incubated for 40min in cell incubator, mitochondria positioning dyestuff (MitoTracker Deep Red) is added to cell for 5 μM
10min is incubated in culture dish；
(3) sample is rinsed cell 3 times with PBS buffer solution, is imaged under fluorescence microscope after sample preparation.After tested and calculate,
Obtain Pearson correlation coefficient R=0.9335.Show that probe molecule of the invention can be located at mitochondria detection formaldehyde.
Embodiment described above is merely a preferred embodiment of the present invention, and the simultaneously exhaustion of the feasible implementation of non-present invention.For
It is any apparent to made by it under the premise of without departing substantially from the principle of the invention and spirit for those skilled in the art
Change, should all be contemplated as falling within claims of the invention.
Claims (4)
1. a kind of fluorescence probe of Mitochondrially targeted detection formaldehyde, which is characterized in that have the following structure:
2. the preparation method of fluorescence probe according to claim 1, which is characterized in that preparation route is by following reaction formula
(1) shown in-(4):
Wherein, in reaction equation (1), the molar ratio of 4- methyl -2- pyrrole carboxylic acid and chloracetyl chloride shown in formula a is 1.5~3:1,
It is preferred that 2~2.2:1, reaction temperature is room temperature, and the reaction time is 10~120min；
In reaction equation (2), formula b compound reacts in the presence of triethylamine with boron trifluoride ether solution, and reaction temperature control exists
25-30 DEG C, the reaction time is 2-12 hours；
In reaction equation (3), formula c compound reacts in the presence of dehydrating agent/catalyst with formula d compound, and reaction temperature is room temperature,
Reaction time is 12-24 hours；
In reaction equation (4), in the molten tetrahydrofuran solution of formula e compound, ammonium hydroxide (1~2h is added) is slowly added dropwise, is stirred at room temperature anti-
It answers 6~24 hours, obtains fluorescent probe molecule shown in formula f.
3. preparation method according to claim 2, which is characterized in that in reaction equation (1), 4- methyl -2- pyrrole shown in formula a
The molar ratio for coughing up formic acid and chloracetyl chloride is 2~2.2:1；Reaction time is 30min；
In reaction equation (2), formula b compound reacts in the presence of triethylamine with boron trifluoride ether solution, and reaction temperature control exists
25 DEG C, the reaction time is 4 hours；
In reaction equation (3), the dehydrating agent is dicyclohexylcarbodiimide (DCC), and catalyst is 4-dimethylaminopyridine
(DMAP), the reaction time is 12 hours.
In reaction equation (4), it is 12 hours that the reaction time, which is stirred at room temperature,.
4. the purposes that fluorescence probe according to claim 1 is applied to formaldehyde in targeting detection mitochondria.
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