CN104610960B - A kind of fluorescent probe of detection cysteine and preparation method thereof and using method - Google Patents
A kind of fluorescent probe of detection cysteine and preparation method thereof and using method Download PDFInfo
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 235000018417 cysteine Nutrition 0.000 title claims abstract description 66
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 10
- 230000004044 response Effects 0.000 claims abstract description 35
- 239000000523 sample Substances 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 15
- 150000003233 pyrroles Chemical class 0.000 claims abstract description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 42
- 150000001875 compounds Chemical class 0.000 claims description 34
- 238000006243 chemical reaction Methods 0.000 claims description 25
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 claims description 18
- 239000012086 standard solution Substances 0.000 claims description 17
- 239000003960 organic solvent Substances 0.000 claims description 16
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
- 239000001257 hydrogen Substances 0.000 claims description 13
- 229910052739 hydrogen Inorganic materials 0.000 claims description 13
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical class ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims description 12
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 12
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 9
- 150000003851 azoles Chemical class 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 8
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical class ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 6
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- 230000005284 excitation Effects 0.000 claims description 6
- 238000002189 fluorescence spectrum Methods 0.000 claims description 6
- 229910052731 fluorine Inorganic materials 0.000 claims description 6
- 239000011737 fluorine Substances 0.000 claims description 6
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 6
- MFFMQGGZCLEMCI-UHFFFAOYSA-N 2,4-dimethyl-1h-pyrrole Chemical class CC1=CNC(C)=C1 MFFMQGGZCLEMCI-UHFFFAOYSA-N 0.000 claims description 5
- -1 2- thiophenyl Chemical group 0.000 claims description 5
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical compound OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 claims description 5
- LULAYUGMBFYYEX-UHFFFAOYSA-N metachloroperbenzoic acid Natural products OC(=O)C1=CC=CC(Cl)=C1 LULAYUGMBFYYEX-UHFFFAOYSA-N 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 239000008366 buffered solution Substances 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- TVCXVUHHCUYLGX-UHFFFAOYSA-N 2-Methylpyrrole Chemical class CC1=CC=CN1 TVCXVUHHCUYLGX-UHFFFAOYSA-N 0.000 claims description 3
- 150000007529 inorganic bases Chemical class 0.000 claims description 3
- 150000007530 organic bases Chemical class 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 235000011181 potassium carbonates Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 235000011118 potassium hydroxide Nutrition 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 3
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims 1
- 229960004365 benzoic acid Drugs 0.000 claims 1
- 150000002978 peroxides Chemical class 0.000 claims 1
- KMZJRCPGGAQHGC-UHFFFAOYSA-N trisodium boric acid borate Chemical compound [Na+].[Na+].[Na+].OB(O)O.[O-]B([O-])[O-] KMZJRCPGGAQHGC-UHFFFAOYSA-N 0.000 claims 1
- 125000005605 benzo group Chemical group 0.000 abstract description 15
- 230000003834 intracellular effect Effects 0.000 abstract description 7
- UKFBSHNQPVYKIX-UHFFFAOYSA-N 7$l^{4}-thiabicyclo[4.1.0]hepta-1,3,5-triene 7-oxide Chemical group C1=CC=C2S(=O)C2=C1 UKFBSHNQPVYKIX-UHFFFAOYSA-N 0.000 abstract description 5
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 abstract description 4
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 abstract description 4
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 abstract description 3
- 239000000975 dye Substances 0.000 abstract 1
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 40
- 239000007787 solid Substances 0.000 description 16
- 238000005160 1H NMR spectroscopy Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 12
- 239000002904 solvent Substances 0.000 description 12
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 10
- 238000003149 assay kit Methods 0.000 description 10
- 238000004440 column chromatography Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 8
- 238000003756 stirring Methods 0.000 description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
- 238000007445 Chromatographic isolation Methods 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- 239000007795 chemical reaction product Substances 0.000 description 5
- 238000011097 chromatography purification Methods 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 229960003180 glutathione Drugs 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 3
- WDCYWAQPCXBPJA-UHFFFAOYSA-N 1,3-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC([N+]([O-])=O)=C1 WDCYWAQPCXBPJA-UHFFFAOYSA-N 0.000 description 3
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 125000004185 ester group Chemical group 0.000 description 3
- 238000002073 fluorescence micrograph Methods 0.000 description 3
- 239000010977 jade Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- PAPNRQCYSFBWDI-UHFFFAOYSA-N DMP Natural products CC1=CC=C(C)N1 PAPNRQCYSFBWDI-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000003969 glutathione Nutrition 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- YAWNFGAHEUCLNJ-UHFFFAOYSA-N [B].OB(O)O Chemical compound [B].OB(O)O YAWNFGAHEUCLNJ-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- HRYZWHHZPQKTII-UHFFFAOYSA-N chloroethane Chemical compound CCCl HRYZWHHZPQKTII-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229960003750 ethyl chloride Drugs 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- JEUXZUSUYIHGNL-UHFFFAOYSA-N n,n-diethylethanamine;hydrate Chemical compound O.CCN(CC)CC JEUXZUSUYIHGNL-UHFFFAOYSA-N 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- OENLEHTYJXMVBG-UHFFFAOYSA-N pyridine;hydrate Chemical compound [OH-].C1=CC=[NH+]C=C1 OENLEHTYJXMVBG-UHFFFAOYSA-N 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000007843 reactive sulfur species Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
Abstract
The invention discloses a kind of fluorescent probe of detection cysteine and preparation method thereof and using method.Benzo BODIPY of the present invention using launch wavelength near infrared region is fluorogen, and in 3 introducing benzene sulfoxide moieties for being easy to modify.Under conditions of it there is cysteine, benzene sulfoxide moiety is replaced by cysteine leaves away, and then obtains the benzo BODIPY of cysteine replacement." ratiometer type " the cysteine probe and its dedicated test test kit of two pyrroles's methine class dyestuff of fluorine boron that the present invention is provided has good response to cysteine solution, the detection to intracellular cysteine can be realized, with easy to operate, it is with low cost, response is sensitive, it is easy to the advantages of promotion and application.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of to use as cysteine fluorescence probe material
Two pyrroles's methine of fluorine boron-benzene sulfoxide derivant and preparation method thereof and using method.
Background technology
Cysteine (Cysteine, Cys) belongs to intracellular work with glutathion (GSH) and homocysteine (Hcy)
Property sulfur species (Intracellular reactive sulfur species, RSS), take part in cellular redox reaction
Deng multi-signal conductive process, play an important role in the physiology and pathological process of life entity.And work as intracellular half Guang
A series of physiology such as hepatic injury, cardiovascular disease, nervous system degenerative disease will be caused when propylhomoserin is in abnormal levels to ask
Topic (referring to N.J.Pace and E.Weerapana, Diverse Functional Roles of Reactive Cysteines,
ACS Chem.Biol.,2013,8：283-296).Therefore, effective detection or monitoring biological sample or half Guang in environmental sample
Propylhomoserin has become the study hotspot of association area in recent years.
Fluorescence detection due to its outstanding detection sensitivity and selectivity, and can realize to biological sample it is real-time,
Line is detected and is subject to the extensive concern of researcher.Two pyrroles's methine class (BODIPY) fluorescence molecule of fluorine boron has good light because of which
The particular advantages such as stability, narrow absorption and launch wavelength, high molar extinction coefficient and quantum yield and become the method most
One of important fluorescent parent, is widely used in the fluoroscopic examination of various testing molecules.
The small-molecule fluorescent probe for detecting cysteine developed at present is based primarily upon sulfydryl and 2,4- dinitros
What the specific chemical between benzenesulfonamido- or 2,4- dinitro benzene semi-annular jade pendant acyl ester groups reacted and designed.When there is cysteine
Under conditions of, 2, the 4- dinitro benzenes sulfonamido or 2,4- dinitro benzene semi-annular jade pendant acyl ester group in probe molecule can be by cysteine
In sulfydryl replace, original 2,4- dinitro benzenes sulfonamido or 2,4- dinitro benzene semi-annular jade pendant acyl ester group are left away and cause probe
The photoluminescent property of molecule changes, so as to realize that the specificity to cysteine sets.
However, the cysteine probe great majority reported are subject to the biological same aminoacid containing sulfydryl in vivo, such as：It is high
The interference of cysteine (Hcy) and glutathion (GSH) is (referring to J.Bouffard, Y.Kim, T.M.Swager etc., A Highly
Selective Fluorescent Probe for Thiol Bioimaging, Org.Lett., 2008,10：37-40).This
Outward, the cysteine probe great majority that these have been reported be fluorescence " on/off " or " on-off " type (referring to Y.Kim, M.Choi,
S.Seo etc., A Selective Fluorescent Probe for Cysteine and Its Imaging in Live
Cells, RSC Adv., 2014,4：64183-64186), it is vulnerable to detection environment, the condition such as temperature, concentration and probe concentration when such as detecting
Impact, it is difficult to realize the specific detection to complicated biology cysteine in vivo.Therefore need it is a kind of it is novel, with good
Good biological stability and cysteine fluorescent probe that " ratiometer type " is detected can be realized.
The content of the invention
In order to overcome drawbacks described above of the prior art, the present invention is intended to provide one kind is from two pyrroles's methine of fluorine boron and benzene
Sulfoxide for detecting fluorescent probe of cysteine and preparation method thereof and using method.
It is fluorogen that the core of the present invention is the benzo BODIPY using launch wavelength near infrared region, and is being easy to
The 3- positions of modification introduce benzene sulfoxide moiety.Under conditions of it there is cysteine, benzene sulfoxide moiety is replaced by cysteine leaves away,
And then obtain the benzo BODIPY (now the sulfydryl on cysteine is connected to the 3- positions of benzo BODIPY) of cysteine replacement.
As the fluorescence intensity for reacting former and later two compounds is more or less the same but fluorescence emission wavelengths difference, therefore, by such scheme,
Preferable " ratiometer type " fluorescence response is obtained, the sensitivity of detection is substantially increased.
First, in order to achieve the above object, the invention provides fluorine boron two pyrroles methine-benzene of the one kind as shown in formula (I)
Sulfoxide derivant.
In formula (I), R1Any one of for hydrogen, or methyl；R2For in hydrogen, or methyl, or ethyl, or isopropyl, or fluorine
Any one.
Compound shown in formula (I) is specially compound (BOD-C) shown in formula (II),
The preparation method of above-mentioned probe comprises the following steps：
(1) under phosphorus oxychloride catalysis, the chloro- iso-indoles -1- aldehyde of 3- shown in formula (III) is with azoles organic molten
Reaction in agent obtains 3- chlorobenzenes the BODIPY replaced shown in formula (IV).
In formula (IV), R1Any one of for hydrogen, or methyl.
(2) under an inert atmosphere, in the presence of a base, compound shown in formula (IV) in organic solvent with formula (V) shownization
Compound reaction obtains the 3- (4-R replaced shown in formula (VI)2- thiophenyl)-benzo BODIPY.
In formula (V), R2Any one of for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine；In formula (VI), R1For
Any one of hydrogen, or methyl；R2Any one of for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine.
(3) under an inert atmosphere, the 3- (4-R under an inert atmosphere, replacing shown in formula (VI)2- thiophenyl)-benzo
BODIPY reacts compound shown in the formula of obtaining final product (I) in organic solvent with metachloroperbenzoic acid.
Above-mentioned preparation method, organic solvent described in step (1) are dichloromethane, acetonitrile, 1,2- dichloroethanes or tetrahydrochysene
Furan；
The azoles are pyrroles, 2- methylpyrroles or 2,4- dimethyl pyrroles；
Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) is 1～0.1 with the mol ratio of the azoles:1；
Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) is 0.5～5 with the mol ratio of phosphorus oxychloride:1；
The reaction temperature is 0～80 degree；Response time is 1～48 hour；
As preferential：Organic solvent described in step (1) is dichloromethane；The azoles are 2,4- diformazans
Base pyrroles；Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) is 0.5 with the mol ratio of the azoles：1；Formula (III) institute
Show that the chloro- iso-indoles -1- aldehyde of 3- is 1 with the mol ratio of phosphorus oxychloride：1；The reaction temperature is 25 degree；Response time is little for 24
When.
Above-mentioned preparation method, alkali described in step (2) are 1～5 with the mol ratio of compound shown in formula (V):1；
The alkali is organic base or inorganic base；
The organic base is triethylamine, pyridine or diisopropyl ethyl amine；The inorganic base is potassium carbonate, sodium carbonate, hydrogen
Sodium oxide, potassium hydroxide, sodium bicarbonate or potassium bicarbonate；
The mol ratio of replacement phenylmercaptan. and compound shown in formula (IV) shown in formula (V) is 1～20:1；
The reaction temperature is 0～40 degree, and the response time is 0.1～2 hour；
The reaction dissolvent of step 2 is organic molten；Described organic solvent be dichloromethane, acetonitrile, 1,2- dichloroethanes,
Tetrahydrofuran or DMF；
As preferred：Alkali described in step (2) is 2.5 with the mol ratio of compound shown in formula (V)：1；Take shown in formula (V)
For phenylmercaptan. and compound shown in formula (IV) mol ratio be 10：1；Reaction temperature is 25 degree；Response time is 1 hour；It is described
Organic solvent be dichloromethane.
Above-mentioned preparation method, metachloroperbenzoic acid described in step (3) and the 3- (4-R replaced shown in formula (VI)2- benzene
Thiophenol group)-benzo BODIPY mol ratio be 1～5:1；
The organic solvent is chloroform, dichloromethane, acetonitrile, DMF, DMSO or 1,2- dichloroethanes；
The reaction temperature is 0～50 degree, and the response time is 1～24 hour；
As preferred：Metachloroperbenzoic acid described in step (3) and the 3- (4-R replaced shown in formula (VI)2- phenylmercaptan.
Base)-benzo BODIPY mol ratio be 1.5：1；The organic solvent is dichloromethane；The reaction temperature is 25 degree；Reaction
Time is 4 hours.
Invention further provides a kind of test kit of detection cysteine, including compound and solvent shown in formula (I).
The concentration of compound shown in formula (I) is 0.001mM～100mM, specially 1mM.
The solvent is water, ethanol, in dimethyl sulfoxide any one.
Shown in formula (I), compound or mentioned reagent box are applied to the detection of cysteine in water solution.
The content of the cysteine in water solution is detected especially by following steps：
(1) compound shown in the formula (I) of same concentrations is added in the buffer salt of variable concentrations cysteine, configuration is at least
The standard solution containing compound shown in formula (I) of 3 kinds of different cysteine contents.
Shown buffer solution is being phosphate buffered solution, Tris-HCl buffer solution, HEPES buffer solution, boric acid-boron
Any one of sour sodium buffer solution, specifically being phosphate buffered solution；
The pH value of shown standard solution is 5～12, specifically being 7.2；
In shown standard solution, the concentration of compound shown in formula (I) is 1nM～10 μM；
In shown standard solution, the content of cysteine is 0.1nM～1mM；
(2) fluorescence emission spectrum of the standard solution is determined respectively, and excitation wavelength is 530nm, with semicystinol concentration
For abscissa, with I584/I552Or I552/I584For vertical coordinate, standard curve is set up.
I584Represent the standard solution in the fluorescence emission peak intensity level that wavelength is at 584nm；
I552Represent the standard solution in the fluorescence emission peak intensity level that wavelength is at 552nm；
(3) compound shown in formula (I) is added in testing sample, control its concentration with formula (I) institute in the standard solution
Show that the concentration of compound is equal；Determine its fluorescence emission spectrum under exciting light of the excitation wavelength for 530nm, i.e., it is bent according to standard
Line computation draws the cysteine content of testing sample.
In above-mentioned steps (2) or step (3), fluorescence intensity is detected on luminoscope.
The present invention has following features：
1) fluorescent probe that the present invention is provided is red solid, with good structure and optical stability.
2) fluorescent probe that the present invention is provided, concentration sensitive of its solution to cysteine, with semicystinol concentration
Increase, under uviol lamp, observe that the fluorescence of its aqueous solution is changed into bright green from orange-yellow.
3) fluorescent probe that the present invention is provided, its launch wavelength is 552nm and 584nm, is dual wavelength response, can disappear significantly
Except impact of the testing conditions difference to result during detection, the sensitivity of detection is improved.
4) fluorescent probe that the present invention is provided is linear to semicystinol concentration, for the accurate survey of cysteine
Amount.
" ratiometer type " the cysteine probe and its test kit of the BODIPY class dyestuffs that the present invention is provided is to cysteine
Solution has good response, can realize the detection to intracellular cysteine, with easy to operate, with low cost, response
It is sensitive, it is easy to the advantages of promotion and application.
Description of the drawings
Fig. 1 is the synthetic route of fluorescent probe BOD-C prepared by embodiment 1.
Fig. 2 is color response figure of the BOD-C test kits of the preparation of embodiment 6 to aqueous cystein solution.
Fig. 3 is fluorescence response figure of the BOD-C test kits of the preparation of embodiment 6 to different aqueous cystein solutions.
Fig. 4 is the ratio of fluorescent emission intensity of the BOD-C test kits of the preparation of embodiment 6 under wavelength 552nm and 584nm
I552/I584With semicystinol concentration relation curve.
Fig. 5 is fluorescence response figure of the BOD-C test kits of the preparation of embodiment 6 to common coexisting ion or biological micromolecule.
Fig. 6 is fluorescence imaging figure of the BOD-C test kits of the preparation of embodiment 6 to intracellular cysteine；Wherein, (a) it is
Cell fluorescence image not plus before BOD-C；B () is the cell fluorescence image after adding BOD-C；C () is addition BOD-C
With cell fluorescence image after cysteine.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, are obtained from commercial channels.
As shown in figure 1, embodiment 1, the preparation of fluorescent probe BOD-C
Step a)：Under an inert atmosphere, 0.5g 3- chloro- iso-indoles -1- aldehyde and 0.23mL 2,4- dimethyl pyrrole are added
Enter in 10mL anhydrous methylene chlorides, add 0.25mL phosphorus oxychloride, 3.9mL is added after stirring 30 minutes under ice bath anhydrous
Triethylamine and 3.9mL boron trifluoride diethyl etherate, are stirred at room temperature 24 hours.After question response is complete, solvent is spin-dried for, Jing column chromatography purification is obtained
To intermediate 3- chlorobenzenes BODIPY0.74g (yield is 87%), red solid.
1H NMR(400MHz,CDCl3) δ 7.69 (d, J=8.2Hz, 1H), 7.65 (d, J=8.2Hz, 1H), 7.43 (t, J
=7.6Hz, 1H), 7.30-7.23 (m, 2H), 7.19 (s, 1H), 5.95 (s, 1H), 2.48 (s, 3H), 2.20 (s, 3H).
Step b)：Under an inert atmosphere, by 0.5g 3- chlorobenzenes BODIPY, 0.25g to methylbenzene phenyl-sulfhydrate and 1.0mL without
Water triethylamine is dissolved in 10mL anhydrous methylene chlorides, is stirred at room temperature 10 minutes.After reaction completely, solvent is spin-dried for, Jing column chromatographies are pure
Change obtains 3- (4- methylphenyl-sulfanyls) benzo BODIPY 0.59g (yield is 87%), red solid.
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=7.9Hz, 1H), 7.44 (d, J=7.6Hz, 2H), 7.27 (t, J
=7.3Hz, 1H), 7.18 (s, 1H), 7.10 (d, J=7.5Hz, 2H), 6.91 (t, J=7.5Hz, 1H), 6.74 (d, J=
8.2Hz,1H),5.93(s,1H),2.48(s,3H),2.30(s,3H),2.20(s,3H)；13C NMR(100MHz,CDCl3)δ
151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,
124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c)：Under an inert atmosphere, 0.15g 3- (4- methylphenyl-sulfanyls) benzo BODIPY is dissolved in into 10mL dichloromethanes
In alkane, 0.10g metachloroperbenzoic acids are added in system in three batches, reacted 4 hours under 0 degree.After question response is complete, it is spin-dried for anti-
Liquid, Jing column chromatographic isolation and purifications is answered to obtain end product BOD-C0.08g (yield 52%), red solid.
1H NMR(400MHz,CDCl3) δ 8.19 (d, J=8.4Hz, 1H), 7.80 (d, J=8.2Hz, 2H), 7.68 (d, J
=8.2Hz, 1H), 7.38 (s, 1H), 7.31 (t, J=7.6Hz, 1H), 7.20 (d, J=6.0Hz, 3H), 7.15 (t, J=
7.7Hz,1H),6.08(s,1H),2.55(s,3H),2.27(s,3H),2.25(s,3H)；HRMS(ESI-TOF):m/z
409.1360[M+H]+,calc’d.409.1357。
The preparation of embodiment 2, fluorescent probe BOD-C
Step a)：Under an inert atmosphere, 0.5g 3- chloro- iso-indoles -1- aldehyde and 0.54mL 2- methylpyrroles are added to
In 10mL anhydrous acetonitriles, add 1.25mL phosphorus oxychloride, under ice bath stir 30 minutes after add 3.9mL anhydrous triethylamines and
3.9mL boron trifluoride diethyl etherate, stirs 48 hours under 0 degree.After question response is complete, solvent is spin-dried for, during Jing column chromatography purification is obtained
Mesosome 3- chlorobenzenes BODIPY 0.65g (yield is 76%), red solid.
1H NMR(400MHz,CDCl3) δ 7.69 (d, J=8.2Hz, 1H), 7.65 (d, J=8.2Hz, 1H), 7.43 (t, J
=7.6Hz, 1H), 7.30-7.23 (m, 2H), 7.19 (s, 1H), 5.95 (s, 1H), 2.48 (s, 3H), 2.20 (s, 3H).
Step b)：Under an inert atmosphere, by 0.5g 3- chlorobenzenes BODIPY, 1.25g to methylbenzene phenyl-sulfhydrate and 1.3mL without
Water pyridine is dissolved in 10mL anhydrous acetonitriles, is stirred 6 minutes at 40 degree.After reaction completely, solvent is spin-dried for, Jing column chromatography purification is obtained
3- (4- methylphenyl-sulfanyls) benzo BODIPY 0.50g (yield is 74%), red solid.
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=7.9Hz, 1H), 7.44 (d, J=7.6Hz, 2H), 7.27 (t, J
=7.3Hz, 1H), 7.18 (s, 1H), 7.10 (d, J=7.5Hz, 2H), 6.91 (t, J=7.5Hz, 1H), 6.74 (d, J=
8.2Hz,1H),5.93(s,1H),2.48(s,3H),2.30(s,3H),2.20(s,3H)；13C NMR(100MHz,CDCl3)δ
151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,
124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c)：Under an inert atmosphere, 0.15g 3- (4- methylphenyl-sulfanyls) benzo BODIPY is dissolved in 10mL chloroforms,
0.10g metachloroperbenzoic acids are added in system in three batches, is reacted 1 hour under 50 degree.After question response is complete, reaction is spin-dried for
Liquid, Jing column chromatographic isolation and purifications obtain end product BOD-C35mg (yield 23%), red solid.
1H NMR(400MHz,CDCl3) δ 8.19 (d, J=8.4Hz, 1H), 7.80 (d, J=8.2Hz, 2H), 7.68 (d, J
=8.2Hz, 1H), 7.38 (s, 1H), 7.31 (t, J=7.6Hz, 1H), 7.20 (d, J=6.0Hz, 3H), 7.15 (t, J=
7.7Hz,1H),6.08(s,1H),2.55(s,3H),2.27(s,3H),2.25(s,3H)；HRMS(ESI-TOF):m/
z409.1360[M+H]+,calc’d.409.1357。
The preparation of embodiment 3, fluorescent probe BOD-C
Step a)：Under an inert atmosphere, 0.5g 3- chloro- iso-indoles -1- aldehyde and 2.3mL pyrroles are added to into 10mL anhydrous
In tetrahydrofuran, add 0.13mL phosphorus oxychloride, under ice bath stir 30 minutes after add 3.9mL anhydrous triethylamines and
3.9mL boron trifluoride diethyl etherate, stirs 1 hour at 80 degree.After question response is complete, solvent is spin-dried for, Jing column chromatography purification obtains centre
Body 3- chlorobenzenes BODIPY 0.36g (yield is 42%), red solid.
1H NMR(400MHz,CDCl3) δ 7.69 (d, J=8.2Hz, 1H), 7.65 (d, J=8.2Hz, 1H), 7.43 (t, J
=7.6Hz, 1H), 7.30-7.23 (m, 2H), 7.19 (s, 1H), 5.95 (s, 1H), 2.48 (s, 3H), 2.20 (s, 3H).
Step b)：Under an inert atmosphere, by 0.5g 3- chlorobenzenes BODIPY, 0.13g is anhydrous to methylbenzene phenyl-sulfhydrate and 2.8g
Potassium carbonate is dissolved in 10mL anhydrous tetrahydro furans, is stirred 60 minutes at 0 degree.After reaction completely, solvent, Jing column chromatography purification are spin-dried for
Obtain 3- (4- methylphenyl-sulfanyls) benzo BODIPY 0.42g (yield is 61%), red solid.
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=7.9Hz, 1H), 7.44 (d, J=7.6Hz, 2H), 7.27 (t, J
=7.3Hz, 1H), 7.18 (s, 1H), 7.10 (d, J=7.5Hz, 2H), 6.91 (t, J=7.5Hz, 1H), 6.74 (d, J=
8.2Hz,1H),5.93(s,1H),2.48(s,3H),2.30(s,3H),2.20(s,3H)；13C NMR(100MHz,CDCl3)δ
151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,
124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c)：Under an inert atmosphere, 0.15g 3- (4- methylphenyl-sulfanyls) benzo BODIPY is dissolved in 10mL acetonitriles,
0.35g metachloroperbenzoic acids are added in system in three batches, is reacted 24 hours under 0 degree.After question response is complete, reaction is spin-dried for
Liquid, Jing column chromatographic isolation and purifications obtain end product BOD-C 55mg (yield 36%), red solid.
1H NMR(400MHz,CDCl3) δ 8.19 (d, J=8.4Hz, 1H), 7.80 (d, J=8.2Hz, 2H), 7.68 (d, J
=8.2Hz, 1H), 7.38 (s, 1H), 7.31 (t, J=7.6Hz, 1H), 7.20 (d, J=6.0Hz, 3H), 7.15 (t, J=
7.7Hz,1H),6.08(s,1H),2.55(s,3H),2.27(s,3H),2.25(s,3H)；HRMS(ESI-TOF):m/
z409.1360[M+H]+,calc’d.409.1357。
The preparation of embodiment 4, fluorescent probe BOD-C
Step a)：Under an inert atmosphere, 0.5g 3- chloro- iso-indoles -1- aldehyde and 0.07mL 2,4- dimethyl pyrrole are added
Enter in anhydrous 1, the 2- dichloroethanes of 10mL, add 0.50mL phosphorus oxychloride, after stirring 30 minutes under ice bath, add 3.9mL
Anhydrous triethylamine and 3.9mL boron trifluoride diethyl etherate, are stirred at room temperature 36 hours.After question response is complete, solvent is spin-dried for, Jing column chromatographies are pure
Change obtains intermediate 3- chlorobenzenes BODIPY0.42g (yield is 49%), red solid.
1H NMR(400MHz,CDCl3) δ 7.69 (d, J=8.2Hz, 1H), 7.65 (d, J=8.2Hz, 1H), 7.43 (t, J
=7.6Hz, 1H), 7.30-7.23 (m, 2H), 7.19 (s, 1H), 5.95 (s, 1H), 2.48 (s, 3H), 2.20 (s, 3H).
Step b)：Under an inert atmosphere, by 0.5g 3- chlorobenzenes BODIPY, 0.20g is anhydrous to methylbenzene phenyl-sulfhydrate and 0.5g
Sodium hydroxide is dissolved in anhydrous 1, the 2- dichloroethanes of 10mL, is stirred at room temperature 30 minutes.After reaction completely, solvent, Jing post layers are spin-dried for
Analysis purification obtains 3- (4- methylphenyl-sulfanyls) benzo BODIPY 0.33g (yield is 48%), red solid.
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=7.9Hz, 1H), 7.44 (d, J=7.6Hz, 2H), 7.27 (t, J
=7.3Hz, 1H), 7.18 (s, 1H), 7.10 (d, J=7.5Hz, 2H), 6.91 (t, J=7.5Hz, 1H), 6.74 (d, J=
8.2Hz,1H),5.93(s,1H),2.48(s,3H),2.30(s,3H),2.20(s,3H)；13C NMR(100MHz,CDCl3)δ
151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,
124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c)：Under an inert atmosphere, 0.15g 3- (4- methylphenyl-sulfanyls) benzo BODIPY is dissolved in into 10mL 1,2- bis-
In ethyl chloride, 0.15g metachloroperbenzoic acids are added in system in three batches, reacted 2 hours under 25 degree.After question response is complete,
Reactant liquor is spin-dried for, Jing column chromatographic isolation and purifications obtain end product BOD-C 68mg (yield 44%), red solid.
1H NMR(400MHz,CDCl3) δ 8.19 (d, J=8.4Hz, 1H), 7.80 (d, J=8.2Hz, 2H), 7.68 (d, J
=8.2Hz, 1H), 7.38 (s, 1H), 7.31 (t, J=7.6Hz, 1H), 7.20 (d, J=6.0Hz, 3H), 7.15 (t, J=
7.7Hz,1H),6.08(s,1H),2.55(s,3H),2.27(s,3H),2.25(s,3H)；HRMS(ESI-TOF):m/z
409.1360[M+H]+,calc’d.409.1357。
The preparation of embodiment 5, fluorescent probe BOD-C
Step a)：Under an inert atmosphere, 0.5g 3- chloro- iso-indoles -1- aldehyde and 0.46mL pyrroles are added to into 10mL anhydrous
In dichloromethane, add 0.29mL phosphorus oxychloride, under ice bath stir 30 minutes after add 3.9mL anhydrous triethylamines and
3.9mL boron trifluoride diethyl etherate, is stirred at room temperature 12 hours.After question response is complete, solvent is spin-dried for, Jing column chromatography purification obtains intermediate
3- chlorobenzenes BODIPY 0.60g (yield is 70%), red solid.
1H NMR(400MHz,CDCl3) δ 7.69 (d, J=8.2Hz, 1H), 7.65 (d, J=8.2Hz, 1H), 7.43 (t, J
=7.6Hz, 1H), 7.30-7.23 (m, 2H), 7.19 (s, 1H), 5.95 (s, 1H), 2.48 (s, 3H), 2.20 (s, 3H).
Step b)：Under an inert atmosphere, by 0.5g 3- chlorobenzenes BODIPY, 0.50g is anhydrous to methylbenzene phenyl-sulfhydrate and 1.0g
Sodium bicarbonate is dissolved in 10mL dry DMFs, is stirred at room temperature 20 minutes.After reaction completely, solvent is spin-dried for, Jing column chromatography purification is obtained
3- (4- methylphenyl-sulfanyls) benzo BODIPY 0.55g (yield is 81%), red solid.
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=7.9Hz, 1H), 7.44 (d, J=7.6Hz, 2H), 7.27 (t, J
=7.3Hz, 1H), 7.18 (s, 1H), 7.10 (d, J=7.5Hz, 2H), 6.91 (t, J=7.5Hz, 1H), 6.74 (d, J=
8.2Hz,1H),5.93(s,1H),2.48(s,3H),2.30(s,3H),2.20(s,3H)；13C NMR(100MHz,CDCl3)δ
151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,
124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c)：Under an inert atmosphere, 0.15g 3- (4- methylphenyl-sulfanyls) benzo BODIPY is dissolved in 10mL DMF,
0.08g metachloroperbenzoic acids are added in system in three batches, is reacted 12 hours under 10 degree.After question response is complete, reaction is spin-dried for
Liquid, Jing column chromatographic isolation and purifications obtain end product BOD-C54mg (yield 35%), red solid.
1H NMR(400MHz,CDCl3) δ 8.19 (d, J=8.4Hz, 1H), 7.80 (d, J=8.2Hz, 2H), 7.68 (d, J
=8.2Hz, 1H), 7.38 (s, 1H), 7.31 (t, J=7.6Hz, 1H), 7.20 (d, J=6.0Hz, 3H), 7.15 (t, J=
7.7Hz,1H),6.08(s,1H),2.55(s,3H),2.27(s,3H),2.25(s,3H)；HRMS(ESI-TOF):m/z
409.1360[M+H]+,calc’d.409.1357。
The spectral quality of compound shown in embodiment 6, formula (I)
4.1mg BOD-C are weighed, 10mL DMSO are dissolved in, mother solution (1mM) is made into, that is, is obtained BOD-C test kits.By 100 μ L
This mother solution be added drop-wise in the phosphate buffer of variable concentrations cysteine, and arrived with corresponding phosphate buffer constant volume
10mL.Measure its fluorescence emission spectrum.Fluorescence emission spectrum is excited with 530nm when determining, and the strength ratio of emission peak is
I584/I552Or I552/I584；The slit width for exciting and launching is respectively 1.5/1.5.
Fig. 2 is color response figure of the BOD-C test kits to aqueous cystein solution.Known by Fig. 2, when addition cysteine water
After solution, the color for being observed visually solution is changed into yellow from redness, while the fluorescence of solution is also changed into bright from orange-yellow fluorescence
Green fluorescence.Prove that test kit of the present invention has intuitively developing response to cysteine.
Fig. 3 is fluorescence response figure of the BOD-C test kits to different aqueous cystein solutions.Known by Fig. 3, with cysteine
The increase of concentration, wavelength are that the fluorescence intensity of the emission peak at 584nm is gradually reduced, and wavelength is the emission peak at 552nm
Fluorescence intensity gradually increases, it was demonstrated that test kit of the present invention has sensitive rate responsive to cysteine.
Ratio Is of the Fig. 4 for fluorescent emission intensity of the BOD-C test kits under wavelength 552nm and 584nm552/I584With half Guang
Propylhomoserin concentration relationship curve.Known by Fig. 4, with the increase of cysteine in water solution concentration, fluorescent emission ratio I552/I584By
It is cumulative big.It is the fluorescence intensity ratio I of emission peak in the range of 0～1mM in semicystinol concentration552/I584With in aqueous solution half
The concentration of cystine is in good linear relationship (R2=0.996).Prove test kit of the present invention to carry out accurately cysteine
Measurement.
Fig. 5 is fluorescence response figure of the BOD-C test kits to common coexisting ion or biological micromolecule.Known by Fig. 5, it is common common
Depositing cation, anion, the addition of biological micromolecule can not make the fluorescent emission ratio I of solution552/I584Change.Card
Bright test kit of the present invention has outstanding selectivity to cysteine.
The measure of embodiment 7, intracellular cysteine content
1) at 37 degree and 5% (v/v) CO2Under the conditions of, with containing 10% (v/v) FBS (hyclone), 100U/mL disk Buddhist nuns
XiLin, the DMEM culture medium culturing HeLa cells of the streptomycin of 100 μ g/mL.Cell is cleaned with PBS using front.
2) PBS (pH 7.4) is added in HeLa cells, add (5 μM) incubation 30min of BOD-C, wash three times with PBS
Afterwards, confocal fluorescent imaging is carried out, wherein excitation wavelength is 510nm, and collection wave band is 530-650nm.Then, to above-mentioned HeLa
The phosphate buffered saline(PBS) of Cys (10 μM) is added in cell, it is after continuing incubation 10min, enterprising in laser confocal microscope
Row imaging, wherein excitation wavelength are 510nm, and collection wave band is 530-650nm.
Known by Fig. 6, the cell for being loaded with BOD-C was presented orange-yellow fluorescence before cysteine is not added, and showed BOD-C with very
Pass through cell membrane well.And after cysteine is added, cell is presented green-fluorescent emission, show BOD-C with the cell
There is specificly-response with cysteine.Prove test kit of the present invention in cell to detect cysteine.
Finally it should be noted that above-described embodiment is only enumerated with BOD-C compounds as fluorometric reagent, remaining fluorometric reagent by
Close in structure and properties, its concentration, experiment excite waveband selection to differ and one list, but which is not intended to limit the present invention.
Any those skilled in the art, without departing from the spirit and scope of the present invention, should so that various modification can be adapted and
Change.
Claims (7)
1. it is a kind of detection cysteine fluorescent probe, it is characterised in that：The structural formula of the probe is compound shown in formula (I),
In formula (I), R1Any one of for hydrogen, or methyl；R2Appointing in for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine
What is a kind of.
2. it is according to claim 1 it is a kind of detection cysteine fluorescent probe, it is characterised in that：The structural formula of the probe
Such as formula (II),
3. a kind of preparation method of the fluorescent probe of detection cysteine, comprises the steps：
Step one：Under phosphorus oxychloride catalysis, the chloro- iso-indoles -1- aldehyde of 3- shown in formula (III) is with azoles organic molten
Reaction in agent obtains 3- chlorobenzenes the BODIPY replaced shown in formula (IV)；
In formula (IV), R1Any one of for hydrogen, or methyl；
Step 2：Under an inert atmosphere, in the presence of a base, compound shown in formula (IV) in organic solvent with formula (V) shownization
Compound reaction obtains the 3- (4-R replaced shown in formula (VI)2- thiophenyl)-benzo BODIPY；
In formula (V), R2Any one of for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine；In formula (VI), R1For hydrogen, or
Any one of methyl；R2Any one of for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine；
Step 3：Under an inert atmosphere, the 3- (4-R for replacing shown in formula (VI)2- thiophenyl)-benzo BODIPY and m-chloro peroxide benzene
Formic acid reacts compound shown in the formula of obtaining final product (I) in organic solvent.
4. it is according to claim 3 it is a kind of detection cysteine fluorescent probe preparation method, it is characterised in that：
Organic solvent described in step one is dichloromethane, acetonitrile, 1,2- dichloroethanes or tetrahydrofuran；The pyroles chemical combination
Thing is pyrroles, 2- methylpyrroles or 2,4- dimethyl pyrroles；Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) and the pyroles
The mol ratio of compound is 1～0.1:1；Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) is 0.5～5 with the mol ratio of phosphorus oxychloride:
1；The reaction temperature is 0～80 degree；Response time is 1～48 hour；
Alkali described in step 2 is 1～5 with the mol ratio of compound shown in formula (V):1；The alkali be organic base in triethylamine,
Pyridine or diisopropyl ethyl amine；Or for the potassium carbonate in inorganic base, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or
Potassium bicarbonate；The mol ratio of replacement phenylmercaptan. and compound shown in formula (IV) shown in formula (V) is 1～20:1；The reaction temperature
For 0～40 degree, the response time is 0.1～2 hour；The reaction dissolvent of step 2 be organic solvent in dichloromethane, acetonitrile, 1,
2- dichloroethanes, tetrahydrofuran or DMF；
Metachloroperbenzoic acid described in step 3 and the 3- (4-R replaced shown in formula (VI)2- phenylmercaptan. base)-benzo BODIPY
Mol ratio is 1～5:1；The organic solvent is chloroform, dichloromethane, acetonitrile, DMF, DMSO or 1,2- dichloroethanes；It is described anti-
Temperature is answered for 0～50 degree, the response time is 1～24 hour.
5. the preparation method of the fluorescent probe of a kind of detection cysteine according to claim 3 or 4, it is characterised in that：
Organic solvent described in step one is dichloromethane；The azoles are 2,4- dimethyl pyrroles；Formula (III) institute
Show that the chloro- iso-indoles -1- aldehyde of 3- is 0.5 with the mol ratio of the azoles：1；Chloro- iso-indoles-the 1- of 3- shown in formula (III)
Aldehyde is 1 with the mol ratio of phosphorus oxychloride：1；The reaction temperature is 25 degree；Response time is 24 hours；
Alkali described in step 2 is 2.5 with the mol ratio of compound shown in formula (V)：1；Replace phenylmercaptan. and formula shown in formula (V)
(IV) mol ratio of compound shown in is 10：1；Reaction temperature is 25 degree；Response time is 1 hour；Described organic solvent is
Dichloromethane；
Metachloroperbenzoic acid described in step 3 and the 3- (4-R replaced shown in formula (VI)2- phenylmercaptan. base)-benzo BODIPY
Mol ratio is 1.5：1；The organic solvent is dichloromethane；The reaction temperature is 25 degree；Response time is 4 hours.
6. it is a kind of detection cysteine fluorescent probe using method, it is characterised in that the method is comprised the following steps：
Step (1)：Compound shown in the formula (I) of same concentrations, configuration are added in the buffer solution of variable concentrations cysteine
The standard solution containing compound shown in formula (I) of at least 3 kinds different cysteine contents；
Shown buffer solution is being phosphate buffered solution, Tris-HCl buffer solution, HEPES buffer solution, boric acid-sodium borate
Any one of buffer solution；
The pH value of shown standard solution is 5～12；
In shown standard solution, the concentration of compound shown in formula (I) is 1nM～10 μM；
In shown standard solution, the content of cysteine is 0.1nM～1mM；
Step (2)：The fluorescence emission spectrum of the standard solution is determined respectively, and excitation wavelength is 530nm, with semicystinol concentration
For abscissa, with I584/I552Or I552/I584For vertical coordinate, standard curve is set up；
I584Represent the standard solution in the fluorescence emission peak intensity level that wavelength is at 584nm；
I552Represent the standard solution in the fluorescence emission peak intensity level that wavelength is at 552nm；
Step (3)：Compound shown in formula (I) is added in testing sample, its concentration is controlled with formula (I) institute in the standard solution
Show that the concentration of compound is equal；Determine its fluorescence emission spectrum under exciting light of the excitation wavelength for 530nm, i.e., it is bent according to standard
Line computation draws the cysteine content of testing sample；
In above-mentioned steps (2) or step (3), fluorescence intensity is detected on luminoscope.
7. it is according to claim 6 it is a kind of detection cysteine fluorescent probe using method, it is characterised in that：Step
(1) in, buffer solution is phosphate buffered solution；The pH value 7.2 of standard solution.
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| US4774339A (en) * | 1987-08-10 | 1988-09-27 | Molecular Probes, Inc. | Chemically reactive dipyrrometheneboron difluoride dyes |
| CN102633694A (en) * | 2012-03-30 | 2012-08-15 | 浙江理工大学 | Fluorescent probe for detecting mercapto compounds as well as preparation method and using method of fluorescent probe |
| CN102827197A (en) * | 2012-09-19 | 2012-12-19 | 中国科学院理化技术研究所 | Fluorescent chemical sensor for detecting thiol-containing compound as well as preparation method and application thereof |
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| US4774339A (en) * | 1987-08-10 | 1988-09-27 | Molecular Probes, Inc. | Chemically reactive dipyrrometheneboron difluoride dyes |
| CN102633694A (en) * | 2012-03-30 | 2012-08-15 | 浙江理工大学 | Fluorescent probe for detecting mercapto compounds as well as preparation method and using method of fluorescent probe |
| CN102827197A (en) * | 2012-09-19 | 2012-12-19 | 中国科学院理化技术研究所 | Fluorescent chemical sensor for detecting thiol-containing compound as well as preparation method and application thereof |
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