CN105623647B - A kind of fluorescence probe for detecting intracellular CO and its preparation method and application - Google Patents
A kind of fluorescence probe for detecting intracellular CO and its preparation method and application Download PDFInfo
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- CN105623647B CN105623647B CN201610069351.7A CN201610069351A CN105623647B CN 105623647 B CN105623647 B CN 105623647B CN 201610069351 A CN201610069351 A CN 201610069351A CN 105623647 B CN105623647 B CN 105623647B
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- 239000000523 sample Substances 0.000 title claims abstract description 50
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 23
- OUYLXVQKVBXUGW-UHFFFAOYSA-N 2,3-dimethyl-1h-pyrrole Chemical compound CC=1C=CNC=1C OUYLXVQKVBXUGW-UHFFFAOYSA-N 0.000 claims abstract description 19
- UJHSIDUUJPTLDY-UHFFFAOYSA-N (2-nitrophenyl)-phenylmethanone Chemical compound [O-][N+](=O)C1=CC=CC=C1C(=O)C1=CC=CC=C1 UJHSIDUUJPTLDY-UHFFFAOYSA-N 0.000 claims abstract description 18
- SKDHHIUENRGTHK-UHFFFAOYSA-N 4-nitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC=C(C(Cl)=O)C=C1 SKDHHIUENRGTHK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- -1 nitrobenzophenone fluorine boron Chemical compound 0.000 claims abstract description 15
- 230000004044 response Effects 0.000 claims abstract description 8
- 150000003233 pyrroles Chemical class 0.000 claims abstract description 6
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims abstract description 4
- 125000003754 ethoxycarbonyl group Chemical group C(=O)(OCC)* 0.000 claims abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims abstract description 3
- 150000002940 palladium Chemical class 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 32
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 26
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 8
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 239000003208 petroleum Substances 0.000 claims description 8
- 238000003384 imaging method Methods 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 5
- 229910015900 BF3 Inorganic materials 0.000 claims description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 4
- 229910002666 PdCl2 Inorganic materials 0.000 claims description 4
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 claims description 3
- 229910052796 boron Inorganic materials 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000004624 confocal microscopy Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 235000015097 nutrients Nutrition 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
- 238000010792 warming Methods 0.000 claims description 3
- ZYMCBJWUWHHVRX-UHFFFAOYSA-N (4-nitrophenyl)-phenylmethanone Chemical class C1=CC([N+](=O)[O-])=CC=C1C(=O)C1=CC=CC=C1 ZYMCBJWUWHHVRX-UHFFFAOYSA-N 0.000 claims description 2
- 150000000469 3,5-xylenols Chemical class 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 2
- 235000010288 sodium nitrite Nutrition 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- AYUUKEMEFZPHQQ-UHFFFAOYSA-N 2-chloro-3-nitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC=CC(C(Cl)=O)=C1Cl AYUUKEMEFZPHQQ-UHFFFAOYSA-N 0.000 claims 1
- QTWJRLJHJPIABL-UHFFFAOYSA-N 2-methylphenol;3-methylphenol;4-methylphenol Chemical compound CC1=CC=C(O)C=C1.CC1=CC=CC(O)=C1.CC1=CC=CC=C1O QTWJRLJHJPIABL-UHFFFAOYSA-N 0.000 claims 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims 1
- 229910052763 palladium Inorganic materials 0.000 abstract description 5
- 238000010189 synthetic method Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- TUAMRELNJMMDMT-UHFFFAOYSA-N 3,5-xylenol Chemical compound CC1=CC(C)=CC(O)=C1 TUAMRELNJMMDMT-UHFFFAOYSA-N 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 15
- JYHHJVKGDCZCCL-UHFFFAOYSA-J carbon monoxide;dichlororuthenium Chemical compound [O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].Cl[Ru]Cl.Cl[Ru]Cl JYHHJVKGDCZCCL-UHFFFAOYSA-J 0.000 description 9
- 238000005160 1H NMR spectroscopy Methods 0.000 description 8
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 8
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 8
- 230000005311 nuclear magnetism Effects 0.000 description 8
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 6
- 230000003595 spectral effect Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- MFFMQGGZCLEMCI-UHFFFAOYSA-N 2,4-dimethyl-1h-pyrrole Chemical group CC1=CNC(C)=C1 MFFMQGGZCLEMCI-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 229910002091 carbon monoxide Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 206010011224 Cough Diseases 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 101150003085 Pdcl gene Proteins 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000010410 reperfusion Effects 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- QPVRKFOKCKORDP-UHFFFAOYSA-N 1,3-dimethylcyclohexa-2,4-dien-1-ol Chemical class CC1=CC(C)(O)CC=C1 QPVRKFOKCKORDP-UHFFFAOYSA-N 0.000 description 1
- WTMJHBZSSSDBFQ-UHFFFAOYSA-N 2,3,4-trimethyl-1h-pyrrole Chemical class CC1=CNC(C)=C1C WTMJHBZSSSDBFQ-UHFFFAOYSA-N 0.000 description 1
- BWWHTIHDQBHTHP-UHFFFAOYSA-N 2-nitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC=CC=C1C(Cl)=O BWWHTIHDQBHTHP-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical class ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical group OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- WBJINCZRORDGAQ-UHFFFAOYSA-N formic acid ethyl ester Natural products CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011246 intracellular protein detection Methods 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- CSJDCSCTVDEHRN-UHFFFAOYSA-N methane;molecular oxygen Chemical compound C.O=O CSJDCSCTVDEHRN-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000006501 nitrophenyl group Chemical group 0.000 description 1
- 150000002941 palladium compounds Chemical class 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000002341 toxic gas Substances 0.000 description 1
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
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Abstract
The invention discloses a kind of intracellular CO of detection fluorescence probe and its preparation method and application, a kind of fluorescence probe for detecting intracellular CO, structural formula are as follows：Wherein, R H, CH3、‑CH2CH3Or COOEt.Synthesizing new ring palladium base group of the present inventionCan be faster sensitiveer to CO response, it not only increases the selectivity to CO after being combined with BODIPY, and realizes faster more sensitively detection CO.Its preparation method is paranitrobenzoyl chloride, dimethyl pyrrole and BFEE, or paranitrobenzoyl chloride, dimethyl pyrrole derivative and the pyrroles (nitrobenzophenone BODIPY) of BFEE reaction generation nitrobenzophenone fluorine boron two, then the nitro in nitrobenzophenone BODIPY is reduced into amino, add nitrite and 3,5 xylenol idol nitridation reactions, it is eventually adding palladium salt progress ring palladium to react to obtain target product, that is, detects intracellular CO fluorescence probe (ACP CO).Synthetic method of the present invention is simple, suitable for industrialized production.
Description
Technical field
The present invention relates to a kind of fluorescence probe, more particularly to a kind of BODIPY classes fluorescence probe, its synthetic method and its
Intracellular CO application is detected, belongs to detection technique field.
Background technology
For many years, carbon monoxide (carbon monoxide, CO) forms carbon oxygen blood because it is easily combined with hemoglobin
Lactoferrin, hemoglobin is lost ability and the effect of oxygen carrying, cause tissue to suffocate, it is dead when serious.All the time, CO is by people
Be considered a kind of toxic gas.In the 1970s, it is found that body can produce CO with endogenous, it is mainly in vivo
Produced by heme oxidase enzymolysis ferroheme.CO and other gas signal small molecules NO, H2S is the same, in the life of body regulation
All played an important role in terms of reason and pathology.CO major physiological effect be for myocardial ischaemia and Reperfu- sion when tissue damage
Protection is provided.Compared to tradition CO gases, CO release molecule (CORMs) quilts of transition-metal-carbonyl compound are sucked directly to body
The potential drug of tissue damage when being considered clinically to prevent myocardial ischaemia and Reperfu- sion.Endogenous gas signaling molecule because
It is rapid etc. special with persistently producing, propagating.Current CO detection techniques, such as palladium sulfate-ammonium molybdate colorimetric formula method, electrochemistry
Method, gas chromatography, infrared ray Carbon Monoxide Detection method etc., generally require to delay processing or the tissue destroyed or produce cell to split
Solve product.
In recent years, it is more rare for detecting internal CO fluorescence probe.Such as using BODIPY as fluorescent parent (Brian
W.Michel,Alexander R.Lippert,and Christopher J.Chang,J.Am.Chem.Soc.2012,134,
15668-15671) probe, and using two-photon dyestuff coumarin derivative as fluorescent parent (K.Zheng, W.Lin, L.Tan,
H.Chena and H.Cuia, Chem.Sci., 2014,5,3439-3448.) fluorescence probe.The response of both the above probe
Group is all the ring palladium compound formed with benzylamine, although selectivity is good, reaction time length (1h or 30min).
The content of the invention
The defects of to overcome prior art, the invention provides a kind of fluorescence probe for detecting intracellular CO and its preparation side
Method and application, such fluorescence probe structure novelty, high sensitivity, good light stability.
To achieve the above object, the technical scheme is that：
A kind of fluorescence probe for detecting intracellular CO, structural formula are as follows：
Wherein, R H ,-CH3、-CH2CH3Or-COOEt.
Synthesizing new ring palladium base group of the present inventionCan be faster sensitiveer to CO response, its with
BODIPY not only increases the selectivity to CO after combining, and realizes faster more sensitively detection CO.
A kind of preparation method for the fluorescence probe for detecting intracellular CO, by paranitrobenzoyl chloride, dimethyl pyrrole and three
It is fluorinated borate ether, or paranitrobenzoyl chloride, dimethyl pyrrole derivative and BFEE reaction generation nitrobenzophenone fluorine
The pyrroles (nitrobenzophenone BODIPY) of boron two, is then reduced into amino by the nitro in nitrobenzophenone BODIPY, adds nitrous acid
Salt and MX idol nitridation reaction, it is eventually adding palladium salt progress ring palladium and reacts to obtain target product, be i.e. detection is thin
Intracellular CO fluorescence probe (ACP-CO).
Synthetic method of the present invention is simple, suitable for industrialized production.
Preferably, the dimethyl pyrrole is 2,4- dimethyl pyrroles.
Its course of reaction is as follows：
Wherein, R H ,-CH3、-CH2CH3Or-COOEt.
Preferably, step is as follows：
(1) by paranitrobenzoyl chloride and dimethyl pyrrole, or paranitrobenzoyl chloride and dimethyl pyrrole derivative it is molten
Solution is cooled down in solvent 1 after being heated to reflux, and adds solvent 2, and 5,5- bis- fluoro- 1 is obtained after adding BFEE heating instead,
3,7,9- tetramethyls -10- (4- nitrobenzophenones) -5H-4 λ4,5λ4- two pyrroles [1,2-c:2', 1'-f] [1,3,2] diaza boron,
That is nitrobenzophenone BODIPY；
(2) nitrobenzophenone BODIPY and ammonium formate are dissolved in solvent, amino is produced after then adding Pd/C stirring reactions
Phenyl BODIPY；
(3) aminophenyl BODIPY and concentrated hydrochloric acid are dissolved in solvent, add nitrite solution, after stirring, add 3,
Azo BODIPY is obtained after the aqueous slkali stirring of 5- xylenols；
(4) azo BODIPY dyestuffs are dissolved in solvent, then add PdCl2, after being stirred overnight at room temperature, produce detection
Intracellular CO fluorescence probe.
It is further preferred that step (1) concretely comprises the following steps：By paranitrobenzoyl chloride and dimethyl pyrrole, or
Paranitrobenzoyl chloride and dimethyl pyrrole derivative are dissolved in CH2Cl2In, be heated to reflux 1-2h, after cooling add triethylamine,
Toluene, question response add BFEE after 10-20 minutes, are warming up to 50-60 DEG C of reaction 2-4 hour, are spin-dried for, use chromatographic column
Separation, gained solid is nitrobenzophenone BODIPY；
Described nitrobenzoyl chloride, dimethyl pyrrole or dimethyl pyrrole derivative, triethylamine, toluene, boron trifluoride
Ether and CH2Cl2Amount ratio be 7-10：7-20：3.5-5.5：30-50：3-6：20-50(g：g：mL：mL：mL：mL)；Chromatogram
Post separation eluant, eluent ratio is VPetroleum ether：VDichloromethane=4-6：1.The conversion ratio of raw material is improved, while improves nitrobenzophenone BODIPY's
Yield and purity.
It is further preferred that step (2) concretely comprise the following steps：Nitrobenzophenone BODIPY, ammonium formate are dissolved in
CH2Cl2, Pd/C is then added, stirring at normal temperature is filtered after 1-3 hours, and filtrate is spin-dried for obtaining into aminophenyl BODIPY；Described nitre
Base benzene BODIPY, ammonium formate, Pd/C, CH2Cl2Usage ratio be：5~10：50~70：1~5:10-30(g：g：g：mL).Carry
The conversion ratio of high raw material, while improve aminophenyl BODIPY yield and purity.
It is further preferred that step (3) concretely comprise the following steps：Aminophenyl BODIPY, concentrated hydrochloric acid are dissolved in DMF
In, sodium nitrite in aqueous solution is added, 3-4h is stirred at 0-5 DEG C, is then slowly dropped to MX
In NaOH solution, 4-5h is stirred at 0-5 DEG C, the filter residue chromatogram post separation after filtering, obtains yellow solid azo BODIPY；
Described aminophenyl BODIPY, concentrated hydrochloric acid, DMF, natrium nitrosum, 3,5- xylenols, NaOH, the dosage of water
Than for 1-3：3-5：10-15：3-5：3-5：4-8：20-30(g：mL：mL：g：g：g：mL).Chromatogram post separation eluant, eluent ratio is
VDichloromethane：VPetroleum ether=1-3：2.The conversion ratio of raw material is improved, while improves azo BODIPY yield and purity.
It is further preferred that the solvent in the step (4) is CH3OH；Described azo BODIPY, CH3OH、PdCl2's
Usage ratio is 5~10：10~30：10~20 (g：mL：g).The conversion ratio of raw material is improved, while improves the intracellular CO's of detection
The yield and purity of fluorescence probe.
A kind of application of the intracellular CO of above-mentioned detection fluorescence probe in intracellular CO is detected.
A kind of method for detecting intracellular CO, step are：
(1) the intracellular CO of above-mentioned detection fluorescence probe is dissolved in physiological saline, cushioning liquid or nutrient solution, matched somebody with somebody
It is stand-by that storing solution storage is made；
(2) storing solution in step (1) is added into the appropriate buffer solution containing cell tissue to be tested.
CO method, step are in a kind of detection living cells：
(1) 5-10 μM of the cushioning liquid of the fluorescence probe containing the intracellular CO of above-mentioned detection is added into cultured cell to put
It is to be incubated 8-12 minutes in incubator；
(2) cell is washed away into unnecessary probe with physiology PBS, then carries out Laser scanning confocal microscopy, such as
Fruit is detection endogenous material, be just it is direct carry out co-focusing imaging, be just to have added thing to be detected and if additional detection
After matter, 8-12 minutes are incubated again, are then imaged.
Compared with prior art, the beneficial effects of the invention are as follows：
1. synthesis is simple, detection speed is fast, need not be incubated for a long time in detection process；
2. selectivity is high, other bioactive small molecules do not disturb；
3. Detection wavelength 512nm is in visible region, small to cellular damage.The favourable intracellular detection for carrying out CO.
Brief description of the drawings
Fig. 1 is the probe ACP-CO fluorescence intensities of the present invention and the relation of CORM-2 change in concentration；Abscissa is wavelength
(nm), ordinate is fluorescence intensity；The figure is the fluorescence spectrum at 512nm；
The selectivity relative to active small molecular common in organism that Fig. 2 is the probe ACP-CO of the present invention is tested；
Fig. 3 is the probe ACP-CO relative intensity of fluorescence and the working curve of CORM-2 concentration of the present invention；Abscissa is
CORM-2 concentration, ordinate are relative intensity of fluorescence；
The probe ACP-CO that Fig. 4 is the present invention is copolymerized micro-imaging to the laser of human liver cancer cell (HepG2)；Scheme a and figure b
For cell imaging figure, figure c is the above two stacking chart, and figure d is cell light field figure.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples.
Embodiment 1
(1) paranitrobenzoyl chloride (7g), 2,4- dimethyl pyrroles (7g) are dissolved into CH2Cl2In (20mL), it is heated to reflux
1h.Triethylamine (3.5g), toluene (30ml) are added after cooling.Question response adds BFEE (3g) after 15 minutes, heating
React 3 hours, be spin-dried for, with chromatogram post separation (eluant, eluent to 50 degree：VPetroleum ether：VDichloromethane=5：1), it is spin-dried for obtaining orange solids nitro
Phenyl BODIPY.
Nuclear-magnetism and mass spectral characteristi：
1HNMR(400MHz,CDCl3):δ=1.36 (s, 6H), 2.57 (s, 6H), 6.02 (s, 2H), 7.54 (d, J=8Hz,
2H), 8.39 (d, J=8Hz, 2H)
ESI-MS:calculated for[M-H]-=368.1, found 368.1.
(2) nitrobenzophenone BODIPY (5g), ammonium formate (50g) are dissolved in CH2Cl2(10mL), then adds Pd/C12
(1g), stirring at normal temperature 2 hours, filtering, filtrate are spin-dried for obtaining aminophenyl BODIPY.
Nuclear-magnetism and mass spectral characteristi：
1HNMR(400MHz,CDCl3):δ=1.49 (s, 6H), 2.54 (s, 6H), 3.89 (s, 2H), 5.97 (s, 2H),
6.79 (d, J=4Hz, 2H), 7.01 (d, J=8Hz, 2H)
ESI-MS:calculated for[M+H]+=340.2, found 340.2.
(3) aminophenyl BODIPY (1g), dense HCl (3mL) are dissolved in DMF (10mL), add natrium nitrosum (3g) water
Solution (10mL), stirs 3h at 0-5 DEG C, is then slowly dropped to MX (3g) NaOH (4g) solution
In, 4h is stirred at 0-5 DEG C, is filtered, filter residue dissolving is spin-dried for, chromatogram post separation (eluant, eluent：VDichloromethane：VPetroleum ether=1：1), obtain yellow
Color solid azo BODIPY.
Nuclear-magnetism and mass spectral characteristi：
1HNMR(400MHz,DMSO-d6):δ=1.44 (s, 6H), 2.47 (s, 12H), 6.21 (s, 2H), 6.63 (s, 2H),
7.56 (d, J=8Hz, 2H), 7.95 (d, J=8Hz, 2H), 10.07 (s, 1H)
ESI-MS:calculated for[M-H]-=471.2, found 471.2.
(4) azo BODIPY (5g) is dissolved in CH3In OH (10mL), PdCl is then added2(10g), was stirred at room temperature
At night, filter to obtain yellow solid.
Nuclear-magnetism characterizes：
1HNMR(400MHz,DMSO-d6):δ=1.50 (s, 6H), 2.20 (s, 6H), 2.47 (s, 6H), 6.23 (s, 2H),
6.60 (s, 2H), 7.35 (d, J=8Hz, 1H), 7.56 (s, 1H), 8.12 (d, J=8Hz, 1H), 9.75 (s, 1H).
Embodiment 2
(1) paranitrobenzoyl chloride (0.14g), 2,4- dimethyl pyrroles (0.14g) are dissolved into CH2Cl2In (2mL), heating
Flow back 1h.Triethylamine (0.35g), toluene (3ml) are added after cooling.Question response adds BFEE after 15 minutes
(0.3g), it is warming up to 50 degree and reacts 3 hours, be spin-dried for, with chromatogram post separation (eluant, eluent：VPetroleum ether：VDichloromethane=5：1), it is spin-dried for orange
Color solid nitrobenzophenone BODIPY.
Nuclear-magnetism and mass spectral characteristi：
1HNMR(400MHz,CDCl3):δ=1.36 (s, 6H), 2.57 (s, 6H), 6.02 (s, 2H), 7.54 (d, J=8Hz,
2H), 8.39 (d, J=8Hz, 2H)
ESI-MS:calculated for[M-H]-=368.1, found 368.1.
(2) nitrobenzophenone BODIPY (10g), ammonium formate (100g) are dissolved in CH2Cl2(20mL), then adds Pd/C12
(2g), stirring at normal temperature 2 hours, filtering, filtrate are spin-dried for obtaining aminophenyl BODIPY.
Nuclear-magnetism and mass spectral characteristi：
1HNMR(400MHz,CDCl3):δ=1.49 (s, 6H), 2.54 (s, 6H), 3.89 (s, 2H), 5.97 (s, 2H),
6.79 (d, J=4Hz, 2H), 7.01 (d, J=8Hz, 2H)
ESI-MS:calculated for[M+H]+=340.2, found 340.2.
(3) aminophenyl BODIPY (2g), dense HCl (6mL) are dissolved in DMF (20mL), add natrium nitrosum (6g) water
Solution (10mL), stirs 3h at 0-5 DEG C, is then slowly dropped to MX (6g) NaOH (8g) solution
In, 4h is stirred at 0-5 DEG C, is filtered, filter residue dissolving is spin-dried for, chromatogram post separation (eluant, eluent：VDichloromethane：VPetroleum ether=1：1), obtain yellow
Color solid azo BODIPY.
Nuclear-magnetism and mass spectral characteristi：
1HNMR(400MHz,DMSO-d6):δ=1.44 (s, 6H), 2.47 (s, 12H), 6.21 (s, 2H), 6.63 (s, 2H),
7.56 (d, J=8Hz, 2H), 7.95 (d, J=8Hz, 2H), 10.07 (s, 1H)
ESI-MS:calculated for[M-H]-=471.2, found 471.2.
(4) azo BODIPY (15g) is dissolved in CH3In OH (30mL), PdCl is then added2(30g), was stirred at room temperature
At night, filter to obtain yellow solid.
Nuclear-magnetism characterizes：
1HNMR(400MHz,DMSO-d6):δ=1.50 (s, 6H), 2.20 (s, 6H), 2.47 (s, 6H), 6.23 (s, 2H),
6.60 (s, 2H), 7.35 (d, J=8Hz, 1H), 7.56 (s, 1H), 8.12 (d, J=8Hz, 1H), 9.75 (s, 1H).
Embodiment 3
The present invention is same as Example 1, and difference is：2,4- dimethyl pyrroles are changed to 2,3,4- trimethyl pyrroles
Cough up.
Embodiment 4
The present invention is same as Example 1, and difference is：2,4- dimethyl pyrroles are changed to 2,4- dimethyl -3- second
Base pyrroles.
Embodiment 5
The present invention is same as Example 1, and difference is：2,4- dimethyl pyrroles are changed to 2,4- dimethyl -3- pyrroles
Cough up Ethyl formate.
Effect test：
1. respectively with the probe ACP-CO of 5.0 μM of embodiments 1 to 0,10,20,30,40,50,60,70,80 and 90 μM
CROM-2 carries out fluorescence response test, obtains probe ACP-CO fluorescence intensity and the relation of CORM-2 change in concentration；Test bar
Part：Excitation/emission wavelength=495/512nm, 50mM PBS/DMSO (pH 7.4,7:3, v/v), 20min is incubated at 37 DEG C.It is horizontal
Coordinate is wavelength (nm), and ordinate is fluorescence intensity.The excitation wavelength of probe is 495nm, launch wavelength 512nm, and test is imitated
Fruit is shown such as Fig. 1, it can be seen that fluorescence gradually strengthens with the increase of CORM-2 concentration, fluorescence 512nm at.This can be illustrated
Probe can be used for CO detection, and its incubation time is only 20min, and the time is short, and detection speed is fast.
2. the probe ACP-CO of the embodiment of the present invention 1 selectivity experiment.By 5.0 μM of probe ACP-CO respectively with difference
Bioactive substance such as active oxygen .OH, HS-、O1、NO、H2O2、ClO-With sulfydryl small molecule Hcy, Cys, GSH and reproducibility
Material Vc, Fe2+It is incubated, its fluorescence intensity and probe is incubated CORM-2 contrasts.Test result such as Fig. 2, it may be said that the bright present invention
Probe ACP-CO selectivity it is high, other bioactive small molecules do not disturb.
3. the working curve of the probe ACP-CO of the embodiment of the present invention 1 relative intensity of fluorescence and CORM-2 concentration, such as
Fig. 3；Abscissa is CORM-2 concentration, and ordinate is relative intensity of fluorescence.
4. the probe ACP-CO of the embodiment of the present invention 1 is copolymerized micro-imaging to the laser of human liver cancer cell (HepG2)；
HepG2 cells are by the DMEM nutrient solution cultures of high sugar, and before imaging, cell attachment is on cover glass, then adding 5 μM of probes
PBS, 37 DEG C are incubated 10 minutes, add 200 μM of CORM-2, then carry out Laser scanning confocal microscopy.Its result is such as
Fig. 4, the cell imaging figure that figure a is the nucleus dyestuff DAPI that 405nm lasers excite lower collection 430-480nm, can be observed
It is colored to nucleus, cell keeps activity well.Figure b be 488nm lasers excite lower collection 500-550nm cell into
As figure, probe ACP-CO its in cytoplasm have obvious dyeing.It is stacking charts of the figure a with scheming b to scheme c, and figure d is cell light field figure,
To show that cell still keeps good cell viability in whole experiment process.
Above-mentioned although the embodiment of the present invention is described with reference to accompanying drawing, not to invention protection domain
Limitation, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not required to
Pay various modifications that creative work can make or deformation is still within the scope of the present invention.
Claims (10)
1. a kind of fluorescence probe for detecting intracellular CO, it is characterized in that, structural formula is as follows：
Wherein, R H ,-CH3、-CH2CH3Or-COOEt.
2. a kind of preparation method for the fluorescence probe for detecting intracellular CO as claimed in claim 1, it is characterized in that, will be to nitro
Chlorobenzoyl chloride, dimethyl pyrrole and BFEE, or paranitrobenzoyl chloride, dimethyl pyrrole derivative and boron trifluoride
The pyrroles of ether reaction generation nitrobenzophenone fluorine boron two is nitrobenzophenone BODIPY, then by the nitro in nitrobenzophenone BODIPY also
Original adds nitrite and MX idol nitridation reaction, is eventually adding palladium salt and carries out the reaction of ring palladiumization into amino
Target product is obtained, that is, detects intracellular CO fluorescence probe.
3. a kind of preparation method of fluorescence probe for detecting intracellular CO as claimed in claim 2, it is characterized in that, step is such as
Under：
(1) paranitrobenzoyl chloride and dimethyl pyrrole, or paranitrobenzoyl chloride and dimethyl pyrrole derivative are dissolved in
In solvent 1, cooled down after being heated to reflux, add solvent 2,5,5- bis- fluoro- 1,3,7 is obtained after adding BFEE heating instead,
9- tetramethyls -10- (4- nitrobenzophenones) -5H-4 λ4,5λ4- two pyrroles [1,2-c:2', 1'-f] [1,3,2] diaza boron, i.e. nitre
Base phenyl BODIPY；
(2) nitrobenzophenone BODIPY and ammonium formate are dissolved in solvent, aminophenyl is produced after then adding Pd/C stirring reactions
BODIPY；
(3) aminophenyl BODIPY and concentrated hydrochloric acid are dissolved in solvent, add nitrite solution, after stirring, add 3,5- bis-
Azo BODIPY is obtained after the aqueous slkali stirring of methylphenol；
(4) azo BODIPY dyestuffs are dissolved in solvent, then add PdCl2, after being stirred overnight at room temperature, produce detection cell
Interior CO fluorescence probe.
4. a kind of preparation method of fluorescence probe for detecting intracellular CO as claimed in claim 3, it is characterized in that, the step
(1) concretely comprise the following steps：Paranitrobenzoyl chloride and dimethyl pyrrole, or paranitrobenzoyl chloride and dimethyl pyrrole are derived
Thing solution is in CH2Cl2In, 1-2h is heated to reflux, triethylamine, toluene are added after cooling, question response adds borontrifluoride after 10-20 minutes
Borate ether, 50-60 DEG C of reaction 2-4 hour is warming up to, is spin-dried for, with chromatogram post separation, gained solid is nitrobenzophenone BODIPY；
Described paranitrobenzoyl chloride, dimethyl pyrrole or dimethyl pyrrole derivative, triethylamine, toluene, boron trifluoride second
Ether and CH2Cl2Amount ratio be 7-10：7-20：3.5-5.5：30-50：3-6：20-50, g：g：mL：mL：mL：mL；Chromatographic column point
It is V from eluant, eluent ratioPetroleum ether：VDichloromethane=4-6：1.
5. a kind of preparation method of fluorescence probe for detecting intracellular CO as claimed in claim 3, it is characterized in that, the step
(2) concretely comprise the following steps：Nitrobenzophenone BODIPY, ammonium formate are dissolved in CH2Cl2, then add Pd/C, stirring at normal temperature 1-3 hours
After filter, filtrate is spin-dried for obtaining aminophenyl BODIPY；Described nitrobenzophenone BODIPY, ammonium formate, Pd/C, CH2Cl2Use
Amount ratio is：5~10：50~70：1~5:10~30, g：g：g：mL.
6. a kind of preparation method of fluorescence probe for detecting intracellular CO as claimed in claim 3, it is characterized in that, the step
(3) concretely comprise the following steps：Aminophenyl BODIPY, concentrated hydrochloric acid are dissolved in DMF, sodium nitrite in aqueous solution is added, at 0-5 DEG C
3-4h is stirred, is then slowly dropped in the NaOH solution of MX, 4-5h is stirred at 0-5 DEG C, is filtered
Filter residue chromatogram post separation afterwards, obtains yellow solid azo BODIPY；
Described aminophenyl BODIPY, concentrated hydrochloric acid, DMF, natrium nitrosum, 3,5- xylenols, NaOH, the amount ratio of water are
1-3：3-5：10-15：3-5：3-5：4-8：20-30, g：mL：mL：g：g：g：mL；Chromatogram post separation eluant, eluent ratio is VDichloromethane：
VPetroleum ether=1-3：2.
7. a kind of preparation method of fluorescence probe for detecting intracellular CO as claimed in claim 3, it is characterized in that, the step
(4) solvent in is CH3OH；Described azo BODIPY, CH3OH、PdCl2Usage ratio be 5~10：10~30：10~
20, g：mL：g.
A kind of 8. application of fluorescence probe for detecting intracellular CO as claimed in claim 1 in intracellular CO is detected.
9. a kind of method for detecting intracellular CO, it is characterized in that, step is：
(1) fluorescence probe of the intracellular CO of detection described in claim 1 is dissolved in physiological saline, buffer solution or nutrient solution
In, it is stand-by to be configured to storing solution storage；
(2) storing solution in step (1) is added into the appropriate buffer solution containing cell tissue to be tested.
10. a kind of method for detecting CO in living cells, it is characterized in that, step is：
(1) 5-10 μM of addition of cushioning liquid of the fluorescence probe containing the intracellular CO of detection described in claim 1 is cultivated
Cell be put in incubator be incubated 8-12 minutes；
(2) cell is washed away into unnecessary probe with physiology PBS, then carries out Laser scanning confocal microscopy, if
Detect endogenous material, then be it is direct carry out co-focusing imaging, be then to have added material to be detected and if additional detection
Afterwards, 8-12 minutes are incubated again, are then imaged.
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