CN104610960A - Fluorescence probe for detecting cysteine as well as preparation method and application method thereof - Google Patents
Fluorescence probe for detecting cysteine as well as preparation method and application method thereof Download PDFInfo
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- CN104610960A CN104610960A CN201510083491.5A CN201510083491A CN104610960A CN 104610960 A CN104610960 A CN 104610960A CN 201510083491 A CN201510083491 A CN 201510083491A CN 104610960 A CN104610960 A CN 104610960A
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 235000018417 cysteine Nutrition 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000000523 sample Substances 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 11
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 59
- 239000000243 solution Substances 0.000 claims description 36
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 35
- 150000001875 compounds Chemical class 0.000 claims description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- 238000012360 testing method Methods 0.000 claims description 24
- 239000007850 fluorescent dye Substances 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 22
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 22
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 20
- 239000003960 organic solvent Substances 0.000 claims description 18
- KQAOUPVFTHOBIX-UHFFFAOYSA-N 1-chloro-2h-isoindole Chemical compound C1=CC=CC2=C(Cl)NC=C21 KQAOUPVFTHOBIX-UHFFFAOYSA-N 0.000 claims description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims description 12
- 239000001257 hydrogen Substances 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
- 230000035484 reaction time Effects 0.000 claims description 12
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical compound OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 11
- LULAYUGMBFYYEX-UHFFFAOYSA-N metachloroperbenzoic acid Natural products OC(=O)C1=CC=CC(Cl)=C1 LULAYUGMBFYYEX-UHFFFAOYSA-N 0.000 claims description 11
- 239000003513 alkali Substances 0.000 claims description 10
- 150000003851 azoles Chemical class 0.000 claims description 10
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 10
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 7
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims description 7
- 239000011737 fluorine Substances 0.000 claims description 7
- 229910052731 fluorine Inorganic materials 0.000 claims description 7
- 150000003233 pyrroles Chemical class 0.000 claims description 7
- MFFMQGGZCLEMCI-UHFFFAOYSA-N 2,4-dimethyl-1h-pyrrole Chemical compound CC1=CNC(C)=C1 MFFMQGGZCLEMCI-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- PAPNRQCYSFBWDI-UHFFFAOYSA-N DMP Natural products CC1=CC=C(C)N1 PAPNRQCYSFBWDI-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 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
- 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
- -1 2-thiophenyl Chemical group 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
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- JYISZVSPRRFQBR-UHFFFAOYSA-N benzene;benzenethiol Chemical class C1=CC=CC=C1.SC1=CC=CC=C1 JYISZVSPRRFQBR-UHFFFAOYSA-N 0.000 claims description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 4
- 239000011707 mineral Substances 0.000 claims description 4
- 235000010755 mineral Nutrition 0.000 claims description 4
- 150000007530 organic bases Chemical class 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- WZMPOCLULGAHJR-UHFFFAOYSA-N thiophen-2-ol Chemical compound OC1=CC=CS1 WZMPOCLULGAHJR-UHFFFAOYSA-N 0.000 claims description 4
- TVCXVUHHCUYLGX-UHFFFAOYSA-N 2-Methylpyrrole Chemical compound CC1=CC=CN1 TVCXVUHHCUYLGX-UHFFFAOYSA-N 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 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
- 239000007810 chemical reaction solvent Substances 0.000 claims description 2
- 235000015320 potassium carbonate Nutrition 0.000 claims description 2
- 235000011118 potassium hydroxide Nutrition 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 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
- 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 description 2
- 230000004044 response Effects 0.000 abstract description 23
- 238000001514 detection method Methods 0.000 abstract description 8
- 239000000975 dye Substances 0.000 abstract description 2
- JJHHIJFTHRNPIK-UHFFFAOYSA-N Diphenyl sulfoxide Chemical group C=1C=CC=CC=1S(=O)C1=CC=CC=C1 JJHHIJFTHRNPIK-UHFFFAOYSA-N 0.000 abstract 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 abstract 1
- 239000007787 solid Substances 0.000 description 16
- 238000005160 1H NMR spectroscopy Methods 0.000 description 15
- 238000003756 stirring Methods 0.000 description 15
- 125000005605 benzo group Chemical group 0.000 description 13
- 239000002904 solvent Substances 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 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
- 238000004440 column chromatography Methods 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 6
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
- 238000007445 Chromatographic isolation Methods 0.000 description 5
- 238000011097 chromatography purification Methods 0.000 description 5
- 239000012467 final product Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 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
- UKFBSHNQPVYKIX-UHFFFAOYSA-N 7$l^{4}-thiabicyclo[4.1.0]hepta-1,3,5-triene 7-oxide Chemical compound C1=CC=C2S(=O)C2=C1 UKFBSHNQPVYKIX-UHFFFAOYSA-N 0.000 description 3
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 3
- 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 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
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000007843 reactive sulfur species Substances 0.000 description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 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
- 206010067125 Liver injury Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000019846 buffering salt Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 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
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 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
- 229940049954 penicillin Drugs 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- JBJWASZNUJCEKT-UHFFFAOYSA-M sodium;hydroxide;hydrate Chemical compound O.[OH-].[Na+] JBJWASZNUJCEKT-UHFFFAOYSA-M 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
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Abstract
The invention discloses a fluorescence probe for detecting cysteine as well as a preparation method and an application method thereof. According to the invention, benz-BODIPY with an emission wavelength in a near infrared is taken as a fluorophore, and a phenyl sulfoxide part is introduced to an easily modified 3-position. In the presence of cysteine, the phenyl sulfoxide part is substituted by the cysteine, and then a cysteine-substituted benz-BODIPY is obtained. A ratio meter type cysteine probe for BODIPY dyes and a special detection kit thereof disclosed by the invention have a good response to a cysteine solution, can implement the detection on cysteine in cells, and have the advantages of easy operation, low cost, sensitive response, convenience for promotion and application, and the like.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of fluorine boron two pyrroles methine-benzene sulfoxide derivant as the use of halfcystine fluorescence probe material and preparation method thereof and using method.
Background technology
Halfcystine (Cysteine, Cys) intracellular reactive sulfur species (Intracellular reactive sulfur species is belonged to gsh (GSH) and homocysteine (Hcy), RSS), take part in the multi-signal conductive processes such as cellular redox reaction, play an important role in the physiology and pathologic process of life entity.And a series of physiological problems such as liver injury, cardiovascular disorder, nervous system degenerative disease will be caused when intracellular cysteine is in abnormal levels (see N.J.Pace and E.Weerapana, Diverse Functional Roles of Reactive Cysteines, ACS Chem.Biol., 2013,8:283-296).Therefore, effectively detection or the halfcystine monitored in biological sample or environmental sample have become the study hotspot of association area in recent years.
Fluorescence detection due to its outstanding detection sensitivity and selectivity, and can realize the extensive concern real-time, the on-line checkingi of biological sample being subject to investigator.Fluorine boron two pyrroles methine class (BODIPY) fluorescence molecule has the particular advantages such as good light stability, narrow absorption and emission wavelength, high molar extinction coefficient and quantum yield because of it and becomes one of most important fluorescent parent of the method, is widely used in the fluoroscopic examination of multiple testing molecule.
That at present to have developed designs for the small-molecule fluorescent probe detecting halfcystine mainly reacts based on the specific chemical between sulfydryl and 2,4-dinitrobenzene sulfonamido or 2,4-dinitrobenzene semi-annular jade pendant acyl ester group.When under the condition that there is halfcystine, in probe molecule 2,4-dinitrobenzene sulfonamido or 2,4-dinitrobenzene semi-annular jade pendant acyl ester group can be replaced by the sulfydryl in halfcystine, former 2,4-dinitrobenzene sulfonamido or 2,4-dinitrobenzene semi-annular jade pendant acyl ester group are left away and cause the photoluminescent property of probe molecule to change, thus realize setting the specificity of halfcystine.
But, the halfcystine probe great majority reported are subject to the same amino acid containing sulfydryl in organism, as: the interference of homocysteine (Hcy) and gsh (GSH) is (see J.Bouffard, Y.Kim, T.M.Swager etc., A Highly Selective Fluorescent Probe for ThiolBioimaging, Org.Lett., 2008,10:37-40).In addition, these halfcystine probe great majority reported are that fluorescence " co " or " on-off " type are (see Y.Kim, M.Choi, S.Seo etc., A Selective Fluorescent Probe for Cysteine and Its Imaging in LiveCells, RSC Adv., 2014,4:64183-64186), be vulnerable to testing environment, the impact of the condition such as temperature, concentration and probe concentration during as detected, is difficult to realize the specific detection to halfcystine in the organism of complexity.Therefore need a kind of novelty, there is good biological stability and the halfcystine fluorescent probe that " ratiometer type " detect can be realized.
Summary of the invention
In order to overcome above-mentioned defect of the prior art, the present invention aim to provide a kind of from fluorine boron two pyrroles's methine and benzene sulfoxide for fluorescent probe detecting halfcystine and preparation method thereof and using method.
Core of the present invention is that the benzo BODIPY utilizing emission wavelength to be in near-infrared region is fluorophore, and introduces benzene sulfoxide moiety in the 3-position being easy to modify.When under the condition that there is halfcystine, benzene sulfoxide moiety is replaced by halfcystine leaves away, and then obtains the benzo BODIPY (sulfydryl now on halfcystine is connected to the 3-position of benzo BODIPY) of halfcystine replacement.Because the fluorescence intensity of reacting former and later two compounds is more or less the same but fluorescence emission wavelengths is different, therefore, by such scheme, obtains desirable " ratiometer type " fluorescence response, substantially increase the sensitivity of detection.
First, in order to achieve the above object, the invention provides one such as formula the fluorine boron two pyrroles methine-benzene sulfoxide derivant shown in (I).
In formula (I), R
1for hydrogen, or any one in methyl; R
2for hydrogen, or methyl, or ethyl, or sec.-propyl, or any one in fluorine.
Shown in formula (I), compound 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-isoindole of 3--1-aldehyde and azoles shown in formula (III) are obtained by reacting the 3-chlorobenzene BODIPY that replace shown in formula (IV) in organic solvent.
In formula (IV), R
1for hydrogen, or any one in methyl.
(2) under an inert atmosphere, in the presence of a base, compound shown in formula (IV) is obtained by reacting the 3-(4-R replaced shown in formula (VI) in organic solvent with compound formula (V) Suo Shi
2-thiophenyl)-benzo BODIPY.
In formula (V), R
2for hydrogen, or methyl, or ethyl, or sec.-propyl, or any one in fluorine; In formula (VI), R
1for hydrogen, or any one in methyl; R
2for hydrogen, or methyl, or ethyl, or sec.-propyl, or any one in fluorine.
(3) 3-(4-R under an inert atmosphere, under an inert atmosphere, replaced shown in formula (VI)
2-thiophenyl)-benzo BODIPY and metachloroperbenzoic acid react in organic solvent and obtain compound shown in formula (I).
Above-mentioned preparation method, organic solvent described in step (1) is methylene dichloride, acetonitrile, 1,2-ethylene dichloride or tetrahydrofuran (THF);
Described azoles is pyrroles, 2-methylpyrrole or 2,4-dimethyl pyrrole;
Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and described azoles is 1 ~ 0.1:1;
Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and phosphorus oxychloride is 0.5 ~ 5:1;
Described temperature of reaction is 0 ~ 80 degree; Reaction times is 1 ~ 48 hour;
As preferentially: described in step (1), organic solvent is methylene dichloride; Described azoles is for being 2,4-dimethyl pyrrole; Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and described azoles is 0.5:1; Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and phosphorus oxychloride is 1:1; Described temperature of reaction is 25 degree; Reaction times is for being 24 hours.
Above-mentioned preparation method, shown in alkali described in step (2) and formula (V), the mol ratio of compound is 1 ~ 5:1;
Described alkali is organic bases or mineral alkali;
Described organic bases is triethylamine, pyridine or diisopropyl ethyl amine; Described mineral alkali is salt of wormwood, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or saleratus;
Shown in formula (V), shown in substituted benzene thiophenol and formula (IV), the mol ratio of compound is 1 ~ 20:1;
Described temperature of reaction is 0 ~ 40 degree, and the reaction times is 0.1 ~ 2 hour;
The reaction solvent of step 2 is organic molten; Described organic solvent is methylene dichloride, acetonitrile, 1,2-ethylene dichloride, tetrahydrofuran (THF) or DMF;
As preferably: shown in alkali described in step (2) and formula (V), the mol ratio of compound is 2.5:1; Shown in formula (V), shown in substituted benzene thiophenol and formula (IV), the mol ratio of compound is 10:1; Temperature of reaction is 25 degree; Reaction times is 1 hour; Described organic solvent is methylene dichloride.
Above-mentioned preparation method, the 3-(4-R replaced shown in metachloroperbenzoic acid described in step (3) and formula (VI)
2-thiophenol base) mol ratio of-benzo BODIPY is 1 ~ 5:1;
Described organic solvent is chloroform, methylene dichloride, acetonitrile, DMF, DMSO or 1,2-ethylene dichloride;
Described temperature of reaction is 0 ~ 50 degree, and the reaction times is 1 ~ 24 hour;
As preferably: the 3-(4-R replaced shown in metachloroperbenzoic acid described in step (3) and formula (VI)
2-thiophenol base) mol ratio of-benzo BODIPY is 1.5:1; Described organic solvent is methylene dichloride; Described temperature of reaction is 25 degree; Reaction times is 4 hours.
Invention further provides a kind of test kit detecting halfcystine, comprise compound and solvent shown in formula (I).
Shown in formula (I), the concentration of compound is 0.001mM ~ 100mM, is specially 1mM.
Described solvent is any one in water, ethanol, dimethyl sulfoxide (DMSO).
Shown in formula (I), compound or mentioned reagent box are applied to the detection of cysteine in water solution.
The content of described cysteine in water solution detects especially by following steps:
(1) in the buffering salt of different concns halfcystine, add the shown compound of formula (I) of same concentrations, configure the standardized solution containing compound shown in formula (I) of at least 3 kinds of different cysteine contents.
Shown in buffered soln to be any one in phosphate buffer soln, Tris-HCl buffered soln, HEPES buffered soln, boric acid-sodium borate buffered soln, specifically to be phosphate buffer soln;
Shown in the pH value of standardized solution be 5 ~ 12, specifically being 7.2;
Shown in shown standardized solution Chinese style (I), the concentration of compound is 1nM ~ 10 μM;
In shown standardized solution, the content of halfcystine is 0.1nM ~ 1mM;
(2) measure the fluorescence emission spectrum of described standardized solution respectively, excitation wavelength is 530nm, is X-coordinate, with I with semicystinol concentration
584/ I
552or I
552/ I
584for ordinate zou, Criterion curve.
I
584represent that described standardized solution is the fluorescence emission peak intensity level at 584nm place at wavelength;
I
552represent that described standardized solution is the fluorescence emission peak intensity level at 552nm place at wavelength;
(3) in testing sample, add compound shown in formula (I), control its concentration equal with the concentration of compound described standardized solution Chinese style (I) Suo Shi; Measuring it is fluorescence emission spectrum under the exciting light of 530nm in excitation wavelength, namely calculates the cysteine content of testing sample according to typical curve.
Above-mentioned steps (2) or the middle fluorescence intensity of step (3) detect on luminoscope.
The present invention has following features:
1) fluorescent probe provided by the invention is red solid, has good structure and optical stability.
2) fluorescent probe provided by the invention, its solution is to the concentration sensitive of halfcystine, and along with the increase of semicystinol concentration, the fluorescence observing its aqueous solution under ultraviolet lamp becomes bright green from orange-yellow.
3) fluorescent probe provided by the invention, its emission wavelength is 552nm and 584nm, and be dual wavelength response, when greatly can eliminate detection, testing conditions difference is on the impact of result, improves the sensitivity detected.
4) fluorescent probe provided by the invention is linear to semicystinol concentration, for the accurate measurement of halfcystine.
" ratiometer type " halfcystine probe and the test kit thereof of BODIPY class dyestuff provided by the invention have good response to cysteine solution, the detection to intracellular cysteine can be realized, have easy and simple to handle, with low cost, respond sensitive, be easy to the advantages such as promotion and application.
Accompanying drawing explanation
Fig. 1 is the synthetic route of fluorescent probe BOD-C prepared by embodiment 1.
Fig. 2 is that the BOD-C test kit of embodiment 6 preparation is to the color response figure of aqueous cystein solution.
Fig. 3 is that the BOD-C test kit of embodiment 6 preparation is to the fluorescence response figure of different aqueous cystein solution.
Fig. 4 is the ratio I of the fluorescent emission intensity of BOD-C test kit under wavelength 552nm and 584nm prepared by embodiment 6
552/ I
584with semicystinol concentration relation curve.
Fig. 5 is that the BOD-C test kit prepared of embodiment 6 is to the fluorescence response figure of common coexisting ion or biological micromolecule.
Fig. 6 is that the BOD-C test kit of embodiment 6 preparation is to the fluorescence imaging figure of intracellular cysteine; Wherein, (a) be do not add BOD-C before cell fluorescence image; B () is for adding the cell fluorescence image after BOD-C; C () is for adding cell fluorescence image after BOD-C and halfcystine.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, obtain all from commercial channels.
As shown in Figure 1, the preparation of embodiment 1, fluorescent probe BOD-C
Step is a): under an inert atmosphere, by the chloro-isoindole of 0.5g 3--1-aldehyde and 0.23mL 2,4-dimethyl pyrrole joins in 10mL anhydrous methylene chloride, add 0.25mL phosphorus oxychloride again, stir under ice bath after 30 minutes and add 3.9mL anhydrous triethylamine and 3.9mL boron trifluoride diethyl etherate again, stirring at room temperature 24 hours.After question response is complete, is spin-dried for solvent, obtains intermediate 3-chlorobenzene and BODIPY0.74g (productive rate is 87%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.69(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-chlorobenzene and BODIPY, 0.25g are dissolved in 10mL anhydrous methylene chloride to methylbenzene phenyl-sulfhydrate and 1.0mL anhydrous triethylamine, stirring at room temperature 10 minutes.After reacting completely, be spin-dried for solvent, obtain 3-(4-methylphenyl-sulfanyl) benzo BODIPY 0.59g (productive rate is 87%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.65(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,CDCl
3)δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c): under an inert atmosphere, 0.15g 3-(4-methylphenyl-sulfanyl) benzo BODIPY is dissolved in 10mL methylene dichloride, in system, adds 0.10g metachloroperbenzoic acid in three batches, reaction 4 hours under 0 degree.After question response is complete, is spin-dried for reaction solution, obtains final product BOD-C0.08g (productive rate 52%) through column chromatographic isolation and purification, red solid.
1H NMR(400MHz,CDCl
3)δ8.19(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 is a): under an inert atmosphere, the chloro-isoindole of 0.5g 3--1-aldehyde and 0.54mL 2-methylpyrrole are joined in 10mL anhydrous acetonitrile, add 1.25mL phosphorus oxychloride again, stir under ice bath after 30 minutes and add 3.9mL anhydrous triethylamine and 3.9mL boron trifluoride diethyl etherate again, stir 48 hours under 0 degree.After question response is complete, is spin-dried for solvent, obtains intermediate 3-chlorobenzene and BODIPY 0.65g (productive rate is 76%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.69(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-chlorobenzene and BODIPY, 1.25g were dissolved in 10mL anhydrous acetonitrile to methylbenzene phenyl-sulfhydrate and 1.3mL anhydrous pyridine, 40 degree of stirrings 6 minutes.After reacting completely, be spin-dried for solvent, obtain 3-(4-methylphenyl-sulfanyl) benzo BODIPY 0.50g (productive rate is 74%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.65(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,CDCl
3)δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c): under an inert atmosphere, 0.15g 3-(4-methylphenyl-sulfanyl) benzo BODIPY is dissolved in 10mL chloroform, in system, adds 0.10g metachloroperbenzoic acid in three batches, reaction 1 hour under 50 degree.After question response is complete, is spin-dried for reaction solution, obtains final product BOD-C35mg (productive rate 23%) through column chromatographic isolation and purification, red solid.
1H NMR(400MHz,CDCl
3)δ8.19(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 is a): under an inert atmosphere, the chloro-isoindole of 0.5g 3--1-aldehyde and 2.3mL pyrroles are joined in 10mL anhydrous tetrahydro furan, add 0.13mL phosphorus oxychloride again, stir under ice bath after 30 minutes and add 3.9mL anhydrous triethylamine and 3.9mL boron trifluoride diethyl etherate again, 80 degree of stirrings 1 hour.After question response is complete, is spin-dried for solvent, obtains intermediate 3-chlorobenzene and BODIPY 0.36g (productive rate is 42%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.69(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-chlorobenzene and BODIPY, 0.13g were dissolved in 10mL anhydrous tetrahydro furan to methylbenzene phenyl-sulfhydrate and 2.8g Anhydrous potassium carbonate, 0 degree of stirring 60 minutes.After reacting completely, be spin-dried for solvent, obtain 3-(4-methylphenyl-sulfanyl) benzo BODIPY 0.42g (productive rate is 61%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.65(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,CDCl
3)δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c): under an inert atmosphere, 0.15g 3-(4-methylphenyl-sulfanyl) benzo BODIPY is dissolved in 10mL acetonitrile, in system, adds 0.35g metachloroperbenzoic acid in three batches, reaction 24 hours under 0 degree.After question response is complete, is spin-dried for reaction solution, obtains final product BOD-C 55mg (productive rate 36%) through column chromatographic isolation and purification, red solid.
1H NMR(400MHz,CDCl
3)δ8.19(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 is a): under an inert atmosphere, by the chloro-isoindole of 0.5g 3--1-aldehyde and 0.07mL 2,4-dimethyl pyrrole joins 10mL anhydrous 1, in 2-ethylene dichloride, add 0.50mL phosphorus oxychloride again, stir under ice bath after 30 minutes and add 3.9mL anhydrous triethylamine and 3.9mL boron trifluoride diethyl etherate again, stirring at room temperature 36 hours.After question response is complete, is spin-dried for solvent, obtains intermediate 3-chlorobenzene and BODIPY0.42g (productive rate is 49%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.69(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-chlorobenzene and BODIPY, 0.20g are dissolved in anhydrous 1, the 2-ethylene dichloride of 10mL to methylbenzene phenyl-sulfhydrate and 0.5g without water sodium hydroxide, stirring at room temperature 30 minutes.After reacting completely, be spin-dried for solvent, obtain 3-(4-methylphenyl-sulfanyl) benzo BODIPY 0.33g (productive rate is 48%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.65(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,CDCl
3)δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c): under an inert atmosphere, 0.15g 3-(4-methylphenyl-sulfanyl) benzo BODIPY is dissolved in 10mL 1,2-ethylene dichloride, in system, adds 0.15g metachloroperbenzoic acid in three batches, reaction 2 hours under 25 degree.After question response is complete, is spin-dried for reaction solution, obtains final product BOD-C 68mg (productive rate 44%) through column chromatographic isolation and purification, red solid.
1H NMR(400MHz,CDCl
3)δ8.19(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 is a): under an inert atmosphere, the chloro-isoindole of 0.5g 3--1-aldehyde and 0.46mL pyrroles are joined in 10mL anhydrous methylene chloride, add 0.29mL phosphorus oxychloride again, stir under ice bath after 30 minutes and add 3.9mL anhydrous triethylamine and 3.9mL boron trifluoride diethyl etherate again, stirring at room temperature 12 hours.After question response is complete, is spin-dried for solvent, obtains intermediate 3-chlorobenzene and BODIPY 0.60g (productive rate is 70%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.69(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-chlorobenzene and BODIPY, 0.50g are dissolved in 10mL dry DMF to methylbenzene phenyl-sulfhydrate and 1.0g anhydrous sodium bicarbonate, stirring at room temperature 20 minutes.After reacting completely, be spin-dried for solvent, obtain 3-(4-methylphenyl-sulfanyl) benzo BODIPY 0.55g (productive rate is 81%) through column chromatography purification, red solid.
1H NMR(400MHz,CDCl
3)δ7.65(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,CDCl
3)δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,126.22,124.40,122.57,118.01,116.65,113.93,20.28,13.48,10.25。
Step c): under an inert atmosphere, 0.15g 3-(4-methylphenyl-sulfanyl) benzo BODIPY is dissolved in 10mL DMF, in system, adds 0.08g metachloroperbenzoic acid in three batches, reaction 12 hours under 10 degree.After question response is complete, is spin-dried for reaction solution, obtains final product BOD-C54mg (productive rate 35%) through column chromatographic isolation and purification, red solid.
1H NMR(400MHz,CDCl
3)δ8.19(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)
Take 4.1mg BOD-C, be dissolved in 10mL DMSO, be made into mother liquor (1mM), namely obtain BOD-C test kit.This mother liquor of 100 μ L is added drop-wise in the phosphate buffered saline buffer of different concns halfcystine, and with corresponding phosphate buffered saline buffer constant volume to 10mL.Measure its fluorescence emission spectrum.Fluorescence emission spectrum excites with 530nm when measuring, and the strength ratio of emission peak is I
584/ I
552or I
552/ I
584; The slit width excited and launch is respectively 1.5/1.5.
Fig. 2 is the color response figure of BOD-C test kit to aqueous cystein solution.Known by Fig. 2, after adding aqueous cystein solution, the color being observed visually solution becomes yellow from redness, and the fluorescence of solution also becomes bright green fluorescence from orange-yellow fluorescence simultaneously.Prove that test kit of the present invention has developing response intuitively to halfcystine.
Fig. 3 is the fluorescence response figure of BOD-C test kit to different aqueous cystein solution.Known by Fig. 3, along with the increase of semicystinol concentration, wavelength is that the fluorescence intensity of the emission peak at 584nm place reduces gradually, and the fluorescence intensity that wavelength is the emission peak at 552nm place increases gradually, proves that test kit of the present invention has sensitive rate responsive to halfcystine.
Fig. 4 is the ratio I of the fluorescent emission intensity of BOD-C test kit under wavelength 552nm and 584nm
552/ I
584with semicystinol concentration relation curve.Known by Fig. 4, along with the increase of cysteine in water solution concentration, fluorescent emission ratio I
552/ I
584increase gradually.In the scope that semicystinol concentration is 0 ~ 1mM, the fluorescence intensity ratio I of emission peak
552/ I
584with the linear relationship (R that the concentration of cysteine in water solution is good
2=0.996).Prove that test kit of the present invention is to carry out Measurement accuracy to halfcystine.
Fig. 5 is BOD-C test kit to the fluorescence response figure of common coexisting ion or biological micromolecule.Known by Fig. 5, the common positively charged ion that coexists, negatively charged ion, biological micromolecule add the fluorescent emission ratio I that can not make solution
552/ I
584change.Prove that test kit of the present invention has outstanding selectivity to halfcystine.
The mensuration of embodiment 7, intracellular cysteine content
1) at 37 degree and 5% (v/v) CO
2under condition, with the DMEM culture medium culturing HeLa cell of the Streptomycin sulphate containing 10% (v/v) FBS (foetal calf serum), 100U/mL penicillin, 100 μ g/mL.Cell uses PBS buffer solution for cleaning before using.
2) in HeLa cell, add PBS (pH 7.4), then add BOD-C (5 μMs) and hatch 30min, after washing three times with PBS, carry out confocal fluorescent imaging, wherein excitation wavelength is 510nm, and collection wave band is 530-650nm.Then, in above-mentioned HeLa cell, add the phosphate buffered saline(PBS) of Cys (10 μMs) again, after continuing to hatch 10min, laser confocal microscope carries out imaging, wherein excitation wavelength is 510nm, and collection wave band is 530-650nm.
Known by Fig. 6, the cell being loaded with BOD-C presented orange-yellow fluorescence before not adding halfcystine, showed that BOD-C is with permeate through cell membranes well.And after add halfcystine, cell presents green-fluorescent emission, show BOD-C with in cell with halfcystine generation specificly-response.Prove that test kit of the present invention to detect halfcystine in cell.
Finally it should be noted that above-described embodiment is only enumerated with BOD-C compound for fluorescent reagent, all the other fluorescent reagents due to structure and properties close, its concentration, experiment excite band selection not list one by one, but it 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 to make various changes and modifications.
Claims (7)
1. detect a fluorescent probe for halfcystine, it is characterized in that: the structural formula of this probe is compound shown in formula (I),
In formula (I), R
1for hydrogen, or any one in methyl; R
2for hydrogen, or methyl, or ethyl, or sec.-propyl, or any one in fluorine.
2. a kind of fluorescent probe detecting halfcystine according to claim 1, is characterized in that: the structural formula of this probe such as formula (II),
3. detect a preparation method for the fluorescent probe of halfcystine, comprise the steps:
Step one: under phosphorus oxychloride catalysis, the chloro-isoindole of 3--1-aldehyde and azoles shown in formula (III) are obtained by reacting the 3-chlorobenzene BODIPY that replace shown in formula (IV) in organic solvent;
In formula (IV), R
1for hydrogen, or any one in methyl;
Step 2: under an inert atmosphere, in the presence of a base, shown in formula (IV), compound is obtained by reacting the 3-(4-R replaced shown in formula (VI) in organic solvent with compound formula (V) Suo Shi
2-thiophenyl)-benzo BODIPY;
In formula (V), R
2for hydrogen, or methyl, or ethyl, or sec.-propyl, or any one in fluorine; In formula (VI), R
1for hydrogen, or any one in methyl; R
2for hydrogen, or methyl, or ethyl, or sec.-propyl, or any one in fluorine;
Step 3: under an inert atmosphere, under an inert atmosphere, the 3-(4-R replaced shown in formula (VI)
2-thiophenyl)-benzo BODIPY and metachloroperbenzoic acid react in organic solvent and obtain compound shown in formula (I).
4. a kind of preparation method detecting the fluorescent probe of halfcystine according to claim 3, is characterized in that:
Organic solvent described in step one is methylene dichloride, acetonitrile, 1,2-ethylene dichloride or tetrahydrofuran (THF); Described azoles is pyrroles, 2-methylpyrrole or 2,4-dimethyl pyrrole; Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and described azoles is 1 ~ 0.1:1; Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and phosphorus oxychloride is 0.5 ~ 5:1; Described temperature of reaction is 0 ~ 80 degree; Reaction times is 1 ~ 48 hour;
Shown in alkali described in step 2 and formula (V), the mol ratio of compound is 1 ~ 5:1; Described alkali is organic bases or mineral alkali; Described organic bases is triethylamine, pyridine or diisopropyl ethyl amine; Described mineral alkali is salt of wormwood, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or saleratus; Shown in formula (V), shown in substituted benzene thiophenol and formula (IV), the mol ratio of compound is 1 ~ 20:1; Described temperature of reaction is 0 ~ 40 degree, and the reaction times is 0.1 ~ 2 hour; The reaction solvent of step 2 is organic molten; Described organic solvent is methylene dichloride, acetonitrile, 1,2-ethylene dichloride, tetrahydrofuran (THF) or DMF;
3-(the 4-R replaced shown in metachloroperbenzoic acid described in step 3 and formula (VI)
2-thiophenol base) mol ratio of-benzo BODIPY is 1 ~ 5:1; Described organic solvent is chloroform, methylene dichloride, acetonitrile, DMF, DMSO or 1,2-ethylene dichloride; Described temperature of reaction is 0 ~ 50 degree, and the reaction times is 1 ~ 24 hour.
5. a kind of preparation method detecting the fluorescent probe of halfcystine according to claim 3 or 4, is characterized in that:
Organic solvent described in step one is methylene dichloride; Described azoles is 2,4-dimethyl pyrrole; Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and described azoles is 0.5:1; Shown in formula (III), the mol ratio of the chloro-isoindole of 3--1-aldehyde and phosphorus oxychloride is 1:1; Described temperature of reaction is 25 degree; Reaction times is for being 24 hours;
Shown in alkali described in step 2 and formula (V), the mol ratio of compound is 2.5:1; Shown in formula (V), shown in substituted benzene thiophenol and formula (IV), the mol ratio of compound is 10:1; Temperature of reaction is 25 degree; Reaction times is 1 hour; Described organic solvent is methylene dichloride;
3-(the 4-R replaced shown in metachloroperbenzoic acid described in step 3 and formula (VI)
2-thiophenol base) mol ratio of-benzo BODIPY is 1.5:1; Described organic solvent is methylene dichloride; Described temperature of reaction is 25 degree; Reaction times is 4 hours.
6. detect a using method for the fluorescent probe of halfcystine, it is characterized in that, the method comprises the following steps:
Step (1): the shown compound of formula (I) adding same concentrations in the buffered soln of different concns halfcystine, configures the standardized solution containing compound shown in formula (I) of at least 3 kinds of different cysteine contents;
Shown buffered soln is to be any one in phosphate buffer soln, Tris-HCl buffered soln, HEPES buffered soln, boric acid-sodium borate buffered soln;
The pH value of shown standardized solution is 5 ~ 12;
Shown in shown standardized solution Chinese style (I), the concentration of compound is 1nM ~ 10 μM;
In shown standardized solution, the content of halfcystine is 0.1nM ~ 1mM;
Step (2): the fluorescence emission spectrum measuring described standardized solution respectively, excitation wavelength is 530nm, is X-coordinate, with I with semicystinol concentration
584/ I
552or I
552/ I
584for ordinate zou, Criterion curve;
I
584represent that described standardized solution is the fluorescence emission peak intensity level at 584nm place at wavelength;
I
552represent that described standardized solution is the fluorescence emission peak intensity level at 552nm place at wavelength;
Step (3): add compound shown in formula (I) in testing sample, control its concentration equal with the concentration of compound described standardized solution Chinese style (I) Suo Shi; Measuring it is fluorescence emission spectrum under the exciting light of 530nm in excitation wavelength, namely calculates the cysteine content of testing sample according to typical curve;
Above-mentioned steps (2) or the middle fluorescence intensity of step (3) detect on luminoscope.
7. a kind of using method detecting the fluorescent probe of halfcystine according to claim 6, is characterized in that: in step (1), buffered soln is phosphate buffer soln; The pH value 7.2 of standardized solution.
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| CN107033131A (en) * | 2017-04-26 | 2017-08-11 | 许昌学院 | It is a kind of to be used to detect fluorescence probe of cysteine and its preparation method and application |
| WO2018200498A1 (en) * | 2017-04-24 | 2018-11-01 | Georgetown University | Compositions and methods for analyzing cysteine |
| US11860084B2 (en) | 2018-09-11 | 2024-01-02 | Georgetown University | Quantitative auxiliary-free chirality sensing with a metal probe |
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| CN106753339A (en) * | 2016-12-06 | 2017-05-31 | 北京师范大学 | It is a kind of can high selectivity recognize benzenethiol fluorescence probe, preparation method and application |
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| US11852633B2 (en) | 2017-04-24 | 2023-12-26 | Georgetown University | Compositions and methods for analyzing cysteine |
| CN107033131A (en) * | 2017-04-26 | 2017-08-11 | 许昌学院 | It is a kind of to be used to detect fluorescence probe of cysteine and its preparation method and application |
| US11860084B2 (en) | 2018-09-11 | 2024-01-02 | Georgetown University | Quantitative auxiliary-free chirality sensing with a metal probe |
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