CN103214875B - Preparation method of fluorescence dye using fluorescein as matrix and application thereof - Google Patents
Preparation method of fluorescence dye using fluorescein as matrix and application thereof Download PDFInfo
- Publication number
- CN103214875B CN103214875B CN201310067820.8A CN201310067820A CN103214875B CN 103214875 B CN103214875 B CN 103214875B CN 201310067820 A CN201310067820 A CN 201310067820A CN 103214875 B CN103214875 B CN 103214875B
- Authority
- CN
- China
- Prior art keywords
- compound
- reaction
- fluorescence dye
- fluorescence
- molar ratio
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 46
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000011159 matrix material Substances 0.000 title abstract 2
- 150000001875 compounds Chemical class 0.000 claims abstract description 79
- 238000004043 dyeing Methods 0.000 claims abstract description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 90
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 75
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 72
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 71
- 238000006243 chemical reaction Methods 0.000 claims description 64
- 239000000203 mixture Substances 0.000 claims description 57
- 239000007810 chemical reaction solvent Substances 0.000 claims description 48
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 39
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 26
- RFFLAFLAYFXFSW-UHFFFAOYSA-N 1,2-dichlorobenzene Chemical compound ClC1=CC=CC=C1Cl RFFLAFLAYFXFSW-UHFFFAOYSA-N 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- -1 acryl Chemical group 0.000 claims description 20
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 15
- 229910052739 hydrogen Inorganic materials 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000012472 biological sample Substances 0.000 claims description 10
- 229910052801 chlorine Inorganic materials 0.000 claims description 10
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 10
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 229910052731 fluorine Inorganic materials 0.000 claims description 8
- 229910052794 bromium Inorganic materials 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 5
- 229910052740 iodine Inorganic materials 0.000 claims description 5
- 150000003997 cyclic ketones Chemical class 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000010025 steaming Methods 0.000 claims description 4
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims 1
- 239000000975 dye Substances 0.000 abstract description 31
- 238000001228 spectrum Methods 0.000 abstract description 6
- 210000000170 cell membrane Anatomy 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000035699 permeability Effects 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 4
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 abstract 1
- 235000018417 cysteine Nutrition 0.000 abstract 1
- 230000008774 maternal effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 54
- 230000035484 reaction time Effects 0.000 description 48
- 239000000243 solution Substances 0.000 description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 20
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 20
- 239000000523 sample Substances 0.000 description 16
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 15
- 238000002073 fluorescence micrograph Methods 0.000 description 12
- 238000010586 diagram Methods 0.000 description 11
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 10
- 239000000460 chlorine Substances 0.000 description 8
- 238000003384 imaging method Methods 0.000 description 7
- NPSSWQJHYLDCNV-UHFFFAOYSA-N prop-2-enoic acid;hydrochloride Chemical compound Cl.OC(=O)C=C NPSSWQJHYLDCNV-UHFFFAOYSA-N 0.000 description 7
- 239000011541 reaction mixture Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 206010006187 Breast cancer Diseases 0.000 description 6
- 208000026310 Breast neoplasm Diseases 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 239000006143 cell culture medium Substances 0.000 description 5
- 238000009833 condensation Methods 0.000 description 5
- 230000005494 condensation Effects 0.000 description 5
- 238000013016 damping Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 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 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 229940126062 Compound A Drugs 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 229940125782 compound 2 Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 4
- IFTVAQUNDKGWDD-UHFFFAOYSA-M mitoTracker Green FM Chemical compound [Cl-].O1C2=CC=CC=C2N(C)C1=CC=CC(=[N+](C1=CC(Cl)=C(Cl)C=C11)C=2C=CC(CCl)=CC=2)N1C1=CC=C(CCl)C=C1 IFTVAQUNDKGWDD-UHFFFAOYSA-M 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000010254 subcutaneous injection Methods 0.000 description 4
- 239000007929 subcutaneous injection Substances 0.000 description 4
- 229910020366 ClO 4 Inorganic materials 0.000 description 3
- 101100075747 Drosophila melanogaster Lztr1 gene Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 238000006482 condensation reaction Methods 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 150000002367 halogens Chemical class 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- MLIREBYILWEBDM-UHFFFAOYSA-N cyanoacetic acid Chemical compound OC(=O)CC#N MLIREBYILWEBDM-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- CUONGYYJJVDODC-UHFFFAOYSA-N malononitrile Chemical compound N#CCC#N CUONGYYJJVDODC-UHFFFAOYSA-N 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910052711 selenium Inorganic materials 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 101100520660 Drosophila melanogaster Poc1 gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100520662 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PBA1 gene Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940117389 dichlorobenzene Drugs 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a fluorescence dye using fluorescein as matrix, and a preparation method and application thereof. The fluorescence dye using fluorescein as maternal has the structure in general formula I. The fluorescence dye is used for biological dyeing, and simultaneously one of the dyes is applied to external and internal detections of cysteine, and meanwhile is applied to in vivo detection and other fields. The compound has a certain level of water-solubility, and simultaneously has good cell membrane permeability. The compound of the present invention simultaneously has novel spectrum characteristics, and the fluorescein derivative dyes with good properties are applied to the aspect of the fluorescence imaging.
Description
Technical field
The present invention relates to a class and take the novel fluorescence dyestuff that fluorescein is parent, its preparation method and application, and utilize such fluorescent probe compounds detection of biological sulfhydryl compound in vitro and in vivo, and by application in cell imaging and experiment made on the living.
Background technology
Fluorescence dye is used widely in every field of science and technology as functional pigmented, and especially the research at aspects such as life science, clinical treatment diagnosis, immunoassay detections gets most of the attention in the whole world.In numerous fluorescence dyes, fluorescein is the dyestuff that a class is widely used, and this class dyestuff has large molar extinction coefficient, good light stability, the high advantages such as fluorescence quantum yield.
Fluorescence imaging monitoring bio macromole is one of the most powerful technology in Living system, compare with absorption in visible region and the fluorescent optical sensor of transmitting, in the application of dyestuff near infrared (NIR) scope in Living system, be favourable bio-imaging reagent, because nir dye has minimum light injury, the infiltration of deep tissue, and the autofluorescence of minimum background.Therefore, the chromophore of red-emitting and near infrared region obtained close concern in recent years.
Recently, the cyanine dyes of the NIR fluorophore probe being widely used, is mainly Cy5 and Cy7.Yet as far as we know, cyanine dyes has fatal shortcoming-poor especially light fastness.Therefore, a lot of groups are exhausted their institute can, to develop new near infrared fluorophor, they are mainly based on Rhodamine B, BODIPY dyestuff, had at present the spectrum of a lot of these two classes fluorophores to have extended near infrared region, prior, this two classes fluorophore has particularly preferred light stability.For example, the seminar of Lin Weiying has built Changsha near infrared functional dye.It is reversible near infrared ROS fluorescent probe that the research group of Nagano has developed serial 2-Me TeR.They have designed and synthesized the assorted rhodamine fluorescent probe MMSiR of a kind of novel near infrared Si and have detected hypochlorous acid.Meanwhile, they have also developed the fluorescence dye that a series of new near-infrared wavelengths excite, SiR-NIRs, and this class probe is the assorted rhodamine parent of the Si based on after modifying.Meanwhile, other each groups have also been found more new near infrared BODIPY dyestuff, still, till now, the bibliographical information that seldom has near-infrared fluorescent element derivative to build.
Fluorescein(e) dye has good photochemical properties, as high molar extinction coefficient, and larger fluorescence quantum yield, preferably light stability.Recently, the 2' that fluorescein derivative and chlorine replace, the position of 7' is by extensive exploitation.It should be noted that the hydroxy position of fluorescein, all the time in a kind of state of activation, can modify at an easy rate.The rotatable sensor of fluorescence that the fluorescent probe of hydroxyl protection is built as active platform widely.Many diversified target detect things, comprise hydrogen peroxide, ultra-oxygen anion free radical, the report of fluorescein probe in the process of unique hydroxyl protection on the basis of halfcystine.But the absorption of the probe that these had been reported and emission wavelength are still respectively about 480nm and 520nm left and right.It is reported, short wavelength excites and has easily brought huge biological sample infringement, and therefore, the research and development of the fluorescein analogue of the absorption spectrum near infrared range and transmitting are very desirable development.At this, we have introduced near infrared functional fluorescent dyes of new strategy design, and this dyestuff is based on 2', 7'-dichlorofluorescein (DCF) parent, meanwhile, the mechanism based on D-π-A.It is that further useful application is in the development strategy of biological study that the design of our expection can extend various near infrared Functional dyes.
Summary of the invention
The present invention is based on above-mentioned background technology, develop a kind of new fluorescence dye, this fluorescence dye has the following advantages: good light stability, emission wavelength is at a class novel fluorescence dyestuff that is parent based on fluorescein analogue in nearly " redness " region, dyestuff of the present invention has certain permeability of cell membrane aspect biologic applications simultaneously, and long wavelength's feature makes the spectral range of spectral range and biological sample have larger difference.
In a first aspect of the present invention, the present invention is directed to the deficiency of the fluorescein(e) dye of existing existence, improve on its basis, provide that a class formation is simple, long wavelength and there is the new compound of good permeability of cell membrane, it has following general structure I:
In general formula I:
M is 0,1,2 or 3;
M is selected from H, K
+, Na
+,-R
5,-OR
10with-(CH
2)
n-O-R
10; R
10for C
1-18alkyl, the integer that n is 1-18;
R
0be selected from H, F, Cl, Br and I;
R ' is selected from H, ethanoyl, acryl;
R is selected from H, R
1, R
2, R
3, R
4, R
5, R
6, R
7, R
8and R
9;
X is C (CH
3)
2, O, S or Se;
Y
-for negative ion.
The fluorescence dye with said structure general formula I can be used for biological stain, in embodiment, enumerated, in vitro and in vivo by one of them application in halfcystine detects, also be applied to the experiment in the fields such as live body detection simultaneously, demonstrate this compounds and there is the water-soluble of certain level, there is certain good permeability of cell membrane simultaneously.This compounds of the present invention also has more novel spectral response curve simultaneously, can be applicable to the aspects such as fluorescence imaging.
Another aspect of the present invention object is to improve the conjugate of described fluorescence dye.
The object of one side is to improve the composition for biological sample dyeing again, comprises above-mentioned fluorescence dye or its conjugate.
On the other hand: a kind of method of preparing compound of the present invention is provided, said method comprising the steps of:
(1) fluorescein parent compound II ' is dissolved in the aqueous solution of 50% sodium hydroxide, in temperature of reaction, be to react 10～24h under 10～165 ℃ of conditions, obtain thick thing, then reaction mixture is joined in frozen water, with concentrated hydrochloric acid, be neutralized to neutrality, standing then suction filtration obtains solid II;
Wherein, M is selected from H, K
+, Na
+,-R
10,-OR
10with-(CH
2)
n-O-R
10; R
10for C
1-18alkyl, the integer that n is 1-18;
R
0be selected from H, F, Cl, Br and I;
2) the new compound cyclic ketones III ' steaming is added drop-wise in the vitriol oil of 0 ℃, then by the solid II of step (1): cyclic ketones III ' in molar ratio 1:1～30 joins in reaction system in batches, reaction mixture system heats after 10h～24h under the condition of 10～180 ℃ of temperature of reaction, be cooled to normal temperature, pour in frozen water and produce the rear filtration of precipitation, obtain solid III;
Wherein, m is 0,1,2 or 3;
(3) reaction solvent N, N '-dimethyl formamide and phosphorus oxychloride compound are according to molar ratio 1:1～30, in temperature of reaction, be to react after 20min～2h under 0 ℃ of condition, then the compound of formula II is dissolved in to reaction solvent N, in N '-solvent dimethylformamide, according to compound III and phosphorus oxychloride feed ratio 1:1～30, add, under 10-180 ℃ of condition, react 5-60 hour, preparation compound IV;
(4) compound IV is reacted 10-50h by molar ratio 1:1～50, the compound of preparation formula I with the compound of formula Rx under 10-180 ℃ of condition in reaction solvent;
The compound of described Rx is selected from i, ii, iii, iv, v, vi, vii and viii;
The described reaction solvent of step (4) is for being methylene dichloride, chloroform, ethanol, methyl alcohol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene, the vitriol oil or its mixture.
In above technical scheme, one preferred embodiment in, formula I compound and acrylate chloride are pressed molar ratio 1:1-30, simultaneously and Et
3n also dissolves in reaction solvent according to 1:1-1:30 molar ratio, and under 10～150 ℃ of conditions, mix and blend is 5～50 hours, and then mixed system stirs a night at ambient temperature, the compound of preparation formula I '; Wherein, reaction solvent is methylene dichloride, chloroform, ethanol, methyl alcohol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene, the vitriol oil or its mixture.
The present invention improves the deficiency on existing fluorescein spectrum, designs and synthesizes out long wavelength's fluorescence dye.Such long wavelength's fluorescence dye has the feature of good light stability, and this compounds has the water-soluble of certain level, has certain permeability of cell membrane simultaneously.
Accompanying drawing explanation
Accompanying drawing 8 width of the present invention:
Fig. 1 is general structure I of take the fluorescent probe that fluorescein is parent of the present invention.
Fig. 2 a is compd A
2white the Photomicrograph to viable cell MCF-7 (human breast cancer cell) dyeing, Fig. 2 b is the fluorescence micrograph of 2 pairs of viable cell MCF-7 dyeing of compd A, picture is collected red wave band 640-680nm, Fig. 2 c is the stacking diagram of Fig. 2 a and Fig. 2 b, instrument is confocal laser scanning microscope, CLSM, model: Olympus FV1000-IX81.Exciting light passage: 635nm.
Fig. 3 a is compd A
3to white Photomicrograph of viable cell MCF-7 (human breast cancer cell) dyeing, Fig. 3 b is compd A
3to the fluorescence micrograph of viable cell MCF-7 dyeing, picture is collected red wave band 540-640nm, and Fig. 3 c is the stacking diagram of Fig. 3 a and Fig. 3 b, and instrument is confocal laser scanning microscope, CLSM, model: Olympus FV1000-IX81.Exciting light passage: 488nm.
Fig. 4 a is compd A
3to white Photomicrograph of viable cell MCF-7 (human breast cancer cell) dyeing, Fig. 4 b is the fluorescence micrograph of compound MitoTracker Green FM to viable cell MCF-7 dyeing, and picture is collected red wave band 500-540nm; Fig. 4 c is compd A
3to the fluorescence micrograph of viable cell MCF-7 dyeing, picture is collected red wave band 540-640nm, and Fig. 4 d is Fig. 4 a, the stacking diagram of 4b and 4c, and instrument is confocal laser scanning microscope, CLSM, model: Olympus FV1000-IX81.Exciting light passage: 488nm.
Fig. 5 a is compd A
3to white Photomicrograph of viable cell MCF-7 (human breast cancer cell) dyeing, Fig. 5 b is compd A
3fluorescence micrograph to viable cell MCF-7 dyeing, Fig. 5 c is the stacking diagram of Fig. 5 a and 5b, picture is collected red wave band 540-640nm, Fig. 5 g is the enlarged view of g in Fig. 5 b, Fig. 5 h is the enlarged view of h in Fig. 5 b, instrument is confocal laser scanning microscope, CLSM, model: Olympus FV1000-IX81.Exciting light passage: 488nm.
Fig. 6 characterizes fluorescent probe compounds A of the present invention in embodiment 2
4, the A that the concentration of take is 1mM
1in the HEPES damping fluid that the 3mLpH value that-DMSO solution 3 μ L join is 7.40, then at 25 ℃, add gradually halfcystine (Cys) to test its Absorption and fluorescence spectrum change in adding test system.Fig. 6 a is for adding probe A
4after add again the absorption spectrum variation diagram of Cys, X-coordinate is wavelength (nm), ordinate zou is relative intensity of fluorescence, wherein illustration represents with the naked eye to observe directly the colour-change of solution, has redness to become purple.Fig. 6 b is for adding probe A
4after add again the fluorescence spectrum variation diagram of Cys, X-coordinate is wavelength (nm), ordinate zou is relative intensity of fluorescence, wherein list of illustrations is shown in the change in fluorescence figure directly observing with the naked eye under luminescent lamp, solution is by directly becoming red fluorescence without fluorescence.Instrument is ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.
Fig. 7 a is compd A
4to white Photomicrograph of viable cell MCF-7 (human breast cancer cell) dyeing, Fig. 7 b is compd A
4to the fluorescence micrograph of viable cell MCF-7 dyeing, picture is collected red wave band 640-680nm, and instrument is confocal laser scanning microscope, CLSM, model: Olympus FV1000-IX81.Exciting light passage: 635nm.
Fig. 8 is compd A
4photo to vital staining, wherein in figure, A represents at the micromolar compd A of this region subcutaneous injection 0
4, B represents at the micromolar compd A of this region subcutaneous injection 10
4, C represents at the micromolar compd A of this region subcutaneous injection 20
4, D represents at the micromolar compd A of this region subcutaneous injection 50
4, instrument is small animal living body imager, model: NightOWL II LB983.Exciting light: 630nm.
Embodiment
Unless otherwise indicated, term used herein has following implication.
Term used herein " alkyl " comprises straight chained alkyl and branched-chain alkyl, for example, and " C
1-6alkyl " comprise C
1-4alkyl, C
1-3alkyl, methyl, ethyl, n-propyl, sec.-propyl and the tertiary butyl.Other group that similarly rule is also applicable to use in this specification sheets.
Term used herein " halogen " comprises fluorine, chlorine, bromine and iodine.
Use Y herein
-represent negative ion, it can be any suitable negative ion, includes but not limited to inorganic negative ion or organic negative ion, for example preferred Y
-for halide-ions, ClO
4 -, PF
6 -, BF
4 -, CH
3cOO
-or OTs
-; More preferably Y again
-be selected from halide-ions, ClO
4 -, PF
6 -, BF
4 -, CH
3cOO
-; More preferably Y again
-be selected from halide-ions, ClO
4 -; Y most preferably
-be selected from halogen bromide anion.
Described m is 0,1,2 or 3 herein; M preferably 1.
R is selected from H, R
1, R
2, R
3, R
4, R
5, R
6, R
7, R
8and R
9, R
1, R
2, R
3, R
4, R
5, R
6, R
7, R
8and R
9be respectively aldehyde radical, C
7h
6nO base, C
9h
6nS base, C
11h
7n
2o base, C
10h
9nXY base, C
9h
8o base, C
4hN
2base, C
6h
6nO
2base, C
4h
2nO
2base, wherein X is C (CH
3)
2, O, S or Se; Y
-for negative ion.
In compound of Formula I of the present invention, preferably X is C (CH
3)
2, O or S; More preferably X is C (CH
3)
2or S; Most preferably X is S.
In compound of Formula I of the present invention, R
0be selected from H or halogen; Preferred R
0be selected from H or F, Cl, Br; More preferably R
0be selected from H or F, Cl; Preferred R again
0be selected from H or Cl; R most preferably
0be selected from Cl.
M is selected from H, K
+, Na
+,-R
10,-OR
10with-(CH
2)
n-O-R
10; Preferably M is selected from H ,-R
10,-OR
10with-(CH
2)
n-O-R
10; More preferably M is selected from H or C again
1-18alkyl; Most preferably M is H.Wherein said R
10for C
1-18alkyl; Preferred R
10for C
1-6alkyl; Described n is integer, the integer that preferably n is 1-18.
The preparation method of the novel long wavelength's that the fluorescein of take described in the present invention provides is on the other hand parent fluorescence dye, that is: first prepare respectively intermediate list aldehyde, then will in intermediate, for example, under the effect of organic bases (piperidines), react and obtain described compound with i, ii, iii, iv, v, vi, vii and viii, concrete synthetic schemes be as described below.
(1) fluorescein parent compound II ' is dissolved in the aqueous solution of 50% sodium hydroxide, in temperature of reaction, be to react 10～24h under 10～165 ℃ of conditions, obtain thick thing, then reaction mixture is joined in frozen water, with concentrated hydrochloric acid, be neutralized to neutrality, standing then suction filtration obtains solid II;
Temperature of reaction is 50-160 ℃, and the reaction times is 4-20 hour;
One preferred embodiment in, temperature of reaction is 70-140 ℃, the reaction times is 3～15 hours;
In a preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～10 hours;
In most preferred embodiments, temperature of reaction is 90～100 ℃, and the reaction times is 1～5 hour.
(2) the new compound cyclic ketones III ' steaming is added drop-wise in the vitriol oil of 0 ℃, then by the solid II of step (1): cyclic ketones III ' in molar ratio 1:1～30 joins in reaction system in batches, reaction mixture system heats after 10h～24h under the condition of 10～180 ℃ of temperature of reaction, be cooled to normal temperature, pour in frozen water and produce the rear filtration of precipitation, obtain solid III;
Wherein, m is 0,1,2 or 3;
Temperature of reaction is 50-160 ℃, and the reaction times is 4-20 hour, and the molar ratio of formula II compound and compound cyclic ketones III ' is 1:1～20;
One preferred embodiment in, temperature of reaction is 70-140 ℃, the reaction times is 3～15 hours, the molar ratio of II compound and compound cyclic ketones III ' is 1:3～15;
In a preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～10 hours, and the molar ratio of II compound and compound cyclic ketones III ' is 1:3～10;
In most preferred embodiments, temperature of reaction is 90～100 ℃, and the reaction times is 1～5 hour, and the molar ratio of II compound and compound cyclic ketones III ' is 1:3～5.
(3) reaction solvent N, N '-dimethyl formamide and phosphorus oxychloride compound are according to molar ratio 1:1～30, in temperature of reaction, be to react after 20min～2h under 0 ℃ of condition, then the compound of formula II is dissolved in to reaction solvent N, in N '-solvent dimethylformamide, according to compound III and phosphorus oxychloride feed ratio 1:1～30, add, under 10-180 ℃ of condition, react 5-60 hour, preparation compound IV;
Temperature of reaction is 50-160 ℃, and the reaction times is 4-48 hour, formula III compound and POCl
3the molar ratio of compound is 1:1～20, and the molar ratio of formula III compound and DMF compound is 1:1～20;
One preferred embodiment in, temperature of reaction is 70-140 ℃, the reaction times is 3～30 hours formula IIIs and POCl
3reaction mol ratio be 1:1～15, the molar ratio of formula III compound and DMF compound is 1:3～15;
In a preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, formula III and POCl
3reaction mol ratio be 1:2～10, the molar ratio of formula III compound and DMF compound is 1:3～10;
In most preferred embodiments, temperature of reaction is 90～100 ℃, and the reaction times is 1～10 hour, formula III and POCl
3reaction mol ratio be 1:3～10, the molar ratio of formula III compound and DMF compound is 1:3～5;
3) by step 2) in the single aldehyde IV and the formula i that obtain, ii, iii, the compound condensation reaction of iv, v, vi, vii and viii obtains general formula I:
Compound IV is reacted 10-50h by molar ratio 1:1～50, the compound of preparation formula I with the compound of formula Rx under 10-180 ℃ of condition in reaction solvent;
4) by step 2) in single aldehyde IV of obtaining react and obtain formula V with the compound of formula i:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, methyl alcohol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, and the molar ratio of formula IV compound and Ortho-Aminophenol compound is 1:1～30;
Preferred embodiment, temperature of reaction is 60～160 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from ethanol, methyl alcohol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, and IV is 1:1～20 with the mol ratio of reacting of i;
In further preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from ethanol, methyl alcohol, acetonitrile, ethyl acetate or its mixture, and IV is 1:2～10 with the mol ratio of reacting of i;
In most preferred embodiment, temperature of reaction is 80～90 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from methyl alcohol, and IV is 1:3～5 with the mol ratio of reacting of i;
5) by step 2) in single aldehyde IV of obtaining react and obtain formula VI with the compound condensation of formula ii:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, and the molar ratio of formula IV compound and ii compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 2～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, ethyl acetate or its mixture, and IV is 1:1～15 with the mol ratio of reacting of ii;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 3～24 hours, and reaction solvent is selected from methylene dichloride, ethanol or its mixture, and IV is 1:2～10 with the mol ratio of reacting of ii;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 2～12 hours, and reaction solvent is selected from ethanol, and IV is 1:3～5 with the mol ratio of reacting of ii;
6) by step 2) in the single aldehyde IV and the compound III condensation reaction that obtain obtain formula VII:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, and the molar ratio of formula IV compound and iii compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, ethyl acetate or its mixture, and IV is 1:2～15 with the mol ratio of reacting of iii;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from methylene dichloride, ethanol or its mixture, and IV is 1:3～10 with the mol ratio of reacting of iii;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from ethanol, and IV is 1:4～5 with the mol ratio of reacting of iii;
7) by step 2) in the single aldehyde intermediate compound IV and the compound IV condensation reaction that obtain obtain formula VIII:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, and the molar ratio of formula IV compound and iv compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, ethyl acetate or its mixture, and IV is 1:1～15 with the mol ratio of reacting of iv;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from methylene dichloride, ethanol or its mixture, and IV is 1:2～10 with the mol ratio of reacting of iv;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from ethanol, and IV is 1:3～5 with the mol ratio of reacting of iv;
8) by step 2) in single aldehyde IV of obtaining react and obtain formula IX with the compound condensation of formula v:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene, aqueous sodium hydroxide solution or its mixture, and the molar ratio of formula IV compound and formula v compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, ethyl acetate, aqueous sodium hydroxide solution or its mixture, and IV is 1:1～15 with the mol ratio of reacting of v;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from methylene dichloride, ethanol, aqueous sodium hydroxide solution or its mixture, and IV is 1:2～10 with the mol ratio of reacting of v;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from aqueous sodium hydroxide solution, and IV is 1:3～5 with the mol ratio of reacting of v;
9) by step 2) in single aldehyde intermediate compound IV of obtaining react and obtain formula X with the compound condensation of formula vi:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, neighbour～dichlorobenzene or its mixture, and the molar ratio of formula IV compound and formula vi compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, acetonitrile, ethyl acetate or its mixture, and IV is 1:1～15 with the mol ratio of reacting of vi;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from ethanol, acetonitrile or its mixture, and IV is 1:2～10 with the mol ratio of reacting of vi;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from acetonitrile solution, and IV is 1:3～5 with the mol ratio of reacting of vi;
10) by step 2) in single aldehyde IV of obtaining react and obtain formula XI with the compound condensation of formula vii:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene, aqueous sodium hydroxide solution or its mixture, and the molar ratio of formula IV compound and formula vii compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, acetonitrile, ethyl acetate or its mixture, and IV is 1:1～15 with the mol ratio of reacting of vii;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from methylene dichloride, ethanol, acetonitrile or its mixture, and IV is 1:2～10 with the mol ratio of reacting of vii;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from acetonitrile solution, and IV is 1:3～5 with the mol ratio of reacting of vii;
11) by step 2) in single aldehyde IV of obtaining react and obtain formula XII with the compound condensation of formula viii:
Temperature of reaction is 50～180 ℃, reaction times is 4～48 hours, reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, and the molar ratio of formula IV compound and formula viii compound is 1:1～20;
Preferred embodiment, temperature of reaction is 70～140 ℃, and the reaction times is 3～36 hours, and reaction solvent is selected from methylene dichloride, ethanol, acetonitrile, ethyl acetate or its mixture, and IV is 1:1～15 with the mol ratio of reacting of viii;
In further preferred embodiment, temperature of reaction is 80～120 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from methylene dichloride, ethanol, acetonitrile or its mixture, and IV is 1:2～10 with the mol ratio of reacting of viii;
In most preferred embodiment, temperature of reaction is 90～100 ℃, and the reaction times is 1～12 hour, and reaction solvent is selected from acetonitrile solution, and IV is 1:3～5 with the mol ratio of reacting of viii;
12) by step 4)-11) in the I compound that obtains and acrylate chloride by molar ratio 1:1-30, also according to 1:1-1:30 molar ratio, in reaction solvent, dissolve with Et3N simultaneously, under 10～150 ℃ of conditions, mix and blend is 5～50 hours, then mixed system stirs a night at ambient temperature, the compound of preparation formula I ';
Temperature of reaction is 5-100 ℃, reaction times is 4-48 hour, reaction solvent is for being selected from: DMF, methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene or its mixture, the molar ratio of I compound and acrylate chloride is 1:1-1:20, formula I and Et
3n is 1:1～20 according to molar ratio;
One preferred embodiment in, temperature of reaction is 4-80 ℃, and the reaction times is 3～36 hours, and reaction solvent is for being selected from: methylene dichloride, chloroform, ethanol, acetonitrile, ethyl acetate or its mixture, the molar ratio of I compound and acrylate chloride is 1:1～15, formula I and Et
3n is 1:3～15 according to molar ratio;
In a preferred embodiment, temperature of reaction is 3～60 ℃, and the reaction times is 2～24 hours, and reaction solvent is selected from: methylene dichloride, chloroform or its mixture, the molar ratio of I compound and acrylate chloride is 1:2～10, formula I and Et
3n is 1:3～10 according to molar ratio;
In most preferred embodiments, temperature of reaction is 2～40 ℃, and the reaction times is 1～10 hour, and reaction solvent is selected from methylene dichloride, and the molar ratio of I compound and acrylate chloride is 1:3～10, formula I and Et
3n is 1:3～5 according to molar ratio;
Above-mentioned in description of take the fluorescent probe preparation method that various substituent fluoresceins are parent of the present invention, each substituting group (R
1, R
2, R
3, R
4, R
5, R
6, R
7, R
8, R
9and R
10) definition and preferably, all with in the present invention to the definition in the description of compound and preferably identical.
The present invention is adopted to the synthetic novel class fluorescein fluorescent probe compounds of aforesaid method, adopts nmr spectrum or mass spectrum to confirm its structure, and be aided with carbon spectrum, high resolution mass spectrum is tested and assist its structure of confirmation.
Of the present inventionly take the fluorescent probe that fluorescein is parent and possess following characteristics:
The fluorescent emission wavelength of described compound part molecule is to be greater than 600nm place to have emission peak, can be used for fluorescent probe and cell imaging and living imaging, avoids the fluorescence background of biology self to disturb;
Described compound has excellent light stability, has low bio-light bleaching property while being applied to biological sample imaging;
Described new compound product side effect is little, and raw material is easy to get, simple in structure, is easy to preparation, and easily industrialization, by 2 step reactions.
These feature and advantage of the present invention and other feature and advantage will become apparent with reference to the following drawings with after the specific embodiment of the present invention.
Given this, the fluorescent probe compounds of class fluorescein derivative of the present invention can be used for the parent detecting as biological sulfhydryl compound.In described composition, should comprise one of class fluorescein derivative fluorescent probe compounds provided by the present invention of significant quantity.In addition, can also comprise needed other component of biological sample dyeing, such as solvent, pH adjusting agent etc.These components are all that one's own profession is known in the art.
The compounds of this invention can be for the dyeing of biological sample, and described compound is fluorescence conjugate." conjugate " used herein refers to the compound that dyestuff that fluorescein analogue of the present invention is parent forms by two keys and other minute sub-connection.The molecule with cell or cellular constituent specific binding be can be with the molecule that fluorescence dye of the present invention is puted together, antibody, antigen, acceptor, part, enzyme, substrate, coenzyme etc. included but not limited to.Conventionally, test sample and fluorescence conjugate constant-temperature incubation for some time, make this fluorescence conjugate and some cell or the cellular constituent specific binding tested in sample, the combination of this fluorescence conjugate and cell or cellular constituent also can be called as dyeing.This staining procedure can carry out repeatedly successively, or carries out multiple dyeing by multiple conjugates simultaneously.After having dyeed, sample is analyzed in fluorescence-activated cell sorter, the fluorescence dye of the present invention in excitation light source excites conjugate wherein, and determinator is measured the utilizing emitted light being produced by the fluorescence dye exciting.
The present invention also provides the method for the fluorescent probe compounds labeled cell sample of the class fluorescein derivative that uses the invention described above, and the method comprises the step that described compound is contacted with biological sample.Term used herein " contact " can be included in solution and contact.
The present invention's composition mentioned above can exist with aqueous solution form, or other suitable form that can be formulated as solution with water before use exists.
Embodiment 1
Prepare fluorescent dye compound A1
(1) midbody compound 1 is synthetic
DCF (6.30g, 15.75mmol) is dissolved in and contains 60mL, in the round-bottomed flask of 50% aqueous sodium hydroxide solution.Stir this mixture, be heated to 165 ℃, continue to stir 1h, mixture becomes thick.Then mixture joins in 400mL frozen water, with 100mL concentrated hydrochloric acid, is neutralized to acid system, presents muddy state, after standing 2 hours, then with Büchner funnel, filters, and washes precipitation 3 times with water.After draining, under infrared(ray)lamp, dry, obtain red midbody compound 1 solid (8.10g).
(2) midbody compound 2 is synthetic
The new pimelinketone (6.6mL, 63.7mmol) steaming is added drop-wise to the vitriol oil (7.0mL), is then cooled to 0 ℃.Then, midbody compound 1 (32mmol) joins in system in batches, and reaction mixture system heats 1.5h under the condition of 90 ℃, cool down, pour into (300g) in frozen water, produce sedimentation and filtration and fall, with cold wash (100mL), obtain midbody compound 2.Thereby midbody compound 2 is not further purified as next step raw material.
(3) dye composition A
1synthetic
By POC1
3(10.0mL, 0.107mol) is added dropwise to the DMF (25mL) of stirring by separating funnel, under 0 ℃ of condition over 35 minutes.Compound 2 (4.735g, 0.0134mol) is dissolved in DMF (1mL), then the material having dissolved is added drop-wise in the mixture of reaction slowly, then under 90 ℃ of conditions, react 3h.Then mixture is poured in frozen water, the throw out of generation is filtered, with cold wash (100mL).Then obtain orange solids A
1(1.95g), orange solids need not be carried out purifying and directly be carried out next step reaction.
Embodiment 2
Prepare fluorescent dye compound A
2:
(1) dye composition A
1synthetic
According to the method for embodiment 1, prepare dye composition A
1.Obtain orange solids A
1(1.95g), thus be not further purified as next step raw material.
(2) dye composition A
2synthetic
Under nitrogen atmosphere, by A
1(100mg, 0.41mmol) and propane dinitrile (vi) (40mg, 0.61mmol) be dissolved in acetonitrile (in 45mL, this mixture 4h that refluxes under 85 ℃ of conditions of nitrogen.Reaction mixture cools to room temperature.Then be to evaporate solvent under vacuum condition, by after column chromatography purifying at silica gel (eluent methylene dichloride: ethyl acetate=40/3, v/v) obtains an intense violet color solid phase prod A
2(0.0793g, 0.185mmol, output 45%), R
f=0.40, (5% CH
3oH/ methylene dichloride).
1H?NMR(400MHz,MeOD)δ8.03(d,J=6.9Hz,1H),8.00(d,J=11.4Hz,1H),7.67–7.42(m,2H),7.10(dd,J=7.3,1.4Hz,1H),6.80(s,1H),6.67(s,1H),2.79(t,J=6.1Hz,2H),2.44–2.15(m,2H),1.80–1.58(m,2H).
13C?NMR(101MHz,DMSO)δ164.31,156.80,152.71,151.73,142.39,138.45,134.02,132.91,132.61,130.90,125.27,123.45,122.69,121.10,119.29,113.42,107.26,61.01,33.84,30.75,28.74,25.54,24.40,21.63.MS(ESI+Tof)m/z?Found429.0M～1,calculated430.072for?C
24H
15ClN
2O
4 ～。
Embodiment 3
Prepare fluorescent dye compound A
3:
(1) dye composition A
1synthetic
According to the method for embodiment 1, prepare dye composition A
1.Obtain orange solids A
1(1.95g), thus be not further purified as next step raw material.
(2) dye composition A
3synthetic
Under nitrogen atmosphere, by A
1(766mg, 2.0mmol) and 2-cyanoacetic acid (i.e. vii) (813mg, 7.2mmol) be dissolved in ethanol (45mL), and then add in 0.2mL piperidines (3.2mmol) and this reaction system of 0.4mL acetum, this mixture 10h that refluxes under 85 ℃ of conditions of nitrogen.Reaction mixture cools to room temperature.Then be to evaporate solvent under vacuum condition, by after column chromatography purifying, at silica gel, (methylene chloride/methanol for eluent=200/3 v/v) obtains dark red solid A
3(productive rate is 20% for 150mg, 0.33mmol), Rf=0.30, (5% CH
3oH/ methylene dichloride).
1H?NMR(400MHz,MeOD)δ8.68(s,1H),8.68(s,1H),8.19(d,J=7.8Hz,1H),7.74(t,J=7.0Hz,1H),7.64(t,J=7.2Hz,1H),7.24(d,J=7.4Hz,1H),6.86(s,1H),6.50(s,1H),4.31(q,J=7.1Hz,4H),2.94–2.82(m,4H),2.48–2.12(m,4H),1.72(s,6H),1.37(t,J=7.1Hz,6H)。
Embodiment 4
Prepare probe compound A
4
(1) dye composition A
2synthetic
According to the method for embodiment 2, prepare dye composition A
2.Obtain intense violet color solid phase prod A
2(0.0793g, 0.185mmol, output 45%), R
f=0.40, (5% CH
3oH/ methylene dichloride).
(3) dye composition A
4synthetic
Dye composition A
2(18.6mg, 0.043mmol) is dissolved in the anhydrous CH of 20mL
2cl
2in, acrylate chloride (4eq, 14 μ L) and Et
3n (4eq, 24 μ L) is added dropwise in this system at 0 ℃.At this temperature, stir 90min, mixed system stirs a night at ambient temperature.Solvent is fallen in underpressure distillation, (the eluent methylene dichloride: ethyl acetate=200/5, v/v) obtains the finished product 5.0mg (0.01035mmol, productive rate is 24%) as elutriant of column chromatography for product.Rf=0.30, (5% CH
3oH/ methylene dichloride),
1h NMR (400MHz, MeOD) δ 8.23 (d, J=1.0Hz, 1H), 8.21 (s, 1H), 7.76 (td, J=7.5,1.3Hz, 1H), 7.66 (td, J=7.7,1.3Hz, 1H), 7.42 (s, 1H), 7.28 (dd, J=7.6,1.0Hz, 1H), 6.68 (s, 1H), 6.64 (dd, J=17.3,1.2Hz, 1H), 6.40 (dd, J=17.3,10.5Hz, 1H), 6.16 (dd, J=10.5,1.2Hz, 1H), 2.83 (t, J=6.1Hz, 2H), 2.39 – 2.12 (m, 2H), 1.74 (d, J=3.1Hz, 2H).
13c NMR (101MHz, MeOD) δ 168.29,167.14,157.59,150.91,150.80,148.17,141.44,135.09,133.28,131.31,130.64,130.03,129.53,126.12,125.83,122.49,122.10,116.97,115.49,112.19,111.32,71.33,56.84,26.88,24.45,19.91,19.53,17.63.MS (ESI+Tof) m/z Found483.0M～1, calculated484.04for C
27h
17clN
2o
5 ~.
Embodiment 5
Under confocal laser scanning microscope, CLSM, observe compd A
2dyeing to viable cell MCF-7: by compd A
2(40 μ L, A
2dMSO mother liquid concentration be 1mM) be added in the nutrient solution of MCF-7 cell of 2mL, at 37 ℃, 5%CO
2cell culture incubator in hatch 120min.Then, PBS shakes rinsing 5min * 3, then adds cell culture medium, confocal laser scanning microscope, CLSM (Olympus) observation of cell form.Choose representative area, 488nm passage excites, and observes, in triplicate with oily mirror (100 *).Fig. 2 a is compd A
2to white Photomicrograph of viable cell MCF-7 dyeing, Fig. 2 b is compd A
2to the red channel fluorescence micrograph of viable cell MCF-7 dyeing, Fig. 2 c is the stacking diagram of Fig. 2 a and Fig. 2 b, as figure can be observed compd A
2to MCF-7 cell dyeing, instrument is confocal laser scanning microscope, CLSM, model: Olympus.Exciting light passage: 488nm.
Embodiment 6
Under confocal laser scanning microscope, CLSM, observe compd A
3dyeing to viable cell MCF-7: by compd A
3(40 μ L, A
3dMSO mother liquid concentration be 1mM) be added in the nutrient solution of MCF-7 cell of 2mL, at 37 ℃, 5%CO
2cell culture incubator in hatch 120min.Then, PBS shakes rinsing 5min * 3, then adds cell culture medium, confocal laser scanning microscope, CLSM (Olympus) observation of cell form.Choose representative area, 488nm passage excites, and observes, in triplicate with oily mirror (100 *).Fig. 3 a is compd A
3to white Photomicrograph of viable cell MCF-7 dyeing, Fig. 3 b is compd A
3to the red channel fluorescence micrograph of viable cell MCF～7 dyeing, Fig. 3 c is the stacking diagram of Fig. 3 a and Fig. 3 b, as figure can be observed compd A
3to MCF-7 cell dyeing, instrument is confocal laser scanning microscope, CLSM, model: Olympus.Exciting light passage: 488nm.
Embodiment 7
Under confocal laser scanning microscope, CLSM, observe compd A
3with the redye experiment of Mitotracker Green FM to viable cell MCF-7: by compd A
3(40 μ L, A
3dMSO mother liquid concentration be 1mM) be added in the nutrient solution of MCF-7 cell of 2mL, at 37 ℃, 5%CO
2cell culture incubator in hatch 120min.Then, then add Mitotracker Green FM, concentration is 200nmol, in cell culture incubator, hatch 30min, then with PBS, shake rinsing 5min * 3, then add cell culture medium, confocal laser scanning microscope, CLSM (Olympus) observation of cell form.Choose representative area, 488nm passage excites, and observes, in triplicate with oily mirror (100 *).Fig. 4 a is compd A
3to white Photomicrograph of viable cell MCF-7 dyeing, Fig. 4 b is the green channel fluorescence micrograph of compound Mitotracker Green FM to viable cell MCF-7 dyeing, and Fig. 4 c is compd A
3to the red channel fluorescence micrograph of viable cell MCF-7 dyeing, Fig. 4 d is the stacking diagram of Fig. 3 a, Fig. 3 b and Fig. 3 c, as figure can be observed compd A
3to MCF～7 cell dyeing, instrument is confocal laser scanning microscope, CLSM, model: Olympus.Exciting light passage: 488nm.
Embodiment 8
Under confocal laser scanning microscope, CLSM, observe compd A
3dyeing to viable cell MCF-7: by compd A
3(40 μ L, A
3dMSO mother liquid concentration be 1mM) be added in the nutrient solution of MCF-7 cell of 2mL, at 37 ℃, 5%CO
2cell culture incubator in hatch 120min.Then, PBS shakes rinsing 5min * 3, then adds cell culture medium, the form of the different damages of confocal laser scanning microscope, CLSM (Olympus) observation of cell.Choose representative area, 488nm passage excites, and observes, in triplicate with oily mirror (100 *).Fig. 5 a is compd A
3to white Photomicrograph of viable cell MCF-7 (human breast cancer cell) dyeing, Fig. 5 b is compd A
3to the fluorescence micrograph of viable cell MCF-7 dyeing, Fig. 5 c is the stacking diagram of Fig. 5 a and 5b, and picture is collected red wave band 540-640nm; Fig. 5 g is the enlarged view of g in Fig. 5 b, and Fig. 5 h is the enlarged view of h in Fig. 5 b, and instrument is confocal laser scanning microscope, CLSM, model: Olympus FV1000-IX81.Exciting light passage: 488nm.
Embodiment 9
Probe compound A
4specific detection experiment to halfcystine
Compd A
4be combined the mensuration of Absorption and fluorescence emmission spectrum with halfcystine: the compd A that configuration concentration is 1mM
4dMSO (dimethyl sulfoxide (DMSO)) solution, get 3 μ L, then add HEPES damping fluid/DMSO of pH7.36,20mM to be diluted to 3mL, be placed in cuvette, measure its absorption spectrum and fluorescence intensity change.Configure the aqueous solution of certain density halfcystine, demarcating its concentration is 10mg/mL.Separately get respectively the compd A that concentration is 1mM
4dMSO (dimethyl sulfoxide (DMSO)) solution 3 μ L in cuvette, adding wherein respectively concentration is the bovine serum albumen solution of 10mg/mL again, HEPES damping fluid/the DMSO that finally adds pH7.36,20mM is diluted to 3mL, measures absorption spectrum and fluorescence intensity under its different concns.Relative intensity of fluorescence increases with linear relationship ratio, and Fig. 6 a is for adding probe A
4after add again the absorption spectrum variation diagram of halfcystine, Fig. 6 a illustration is for adding probe A
4after add again the naked eyes of halfcystine to observe figure, Fig. 6 b is for adding probe A
4after add again the fluorescence spectrum figure of halfcystine, Fig. 6 b illustration is for adding probe A
4after add again the naked eyes of halfcystine to observe fluorogram, model: Hp8453; Spectrophotofluorometer, model: FP～6500.
Embodiment 10
Under confocal laser scanning microscope, CLSM, observe compd A
4dyeing to viable cell MCF～7: by compd A
4(20 μ L, A
4dMSO mother liquid concentration be 1mM) be added in the nutrient solution of MCF～7 cell of 2mL, at 37 ℃, 5%CO
2cell culture incubator in hatch 120min.Then, PBS shakes rinsing 5min * 3, then adds cell culture medium, confocal laser scanning microscope, CLSM (Olympus) observation of cell form.Choose representative area, 488nm passage excites, and observes, in triplicate with oily mirror (100 *).Fig. 7 a is compd A
4to white Photomicrograph of viable cell MCF～7 dyeing, Fig. 7 b is compd A
4red channel fluorescence micrograph to the dyeing of viable cell MCF～7, as figure can be observed compd A
4to MCF～7 cell dyeing, can in living cell body, detect halfcystine, instrument is confocal laser scanning microscope, CLSM, model: Olympus.Exciting light passage: 488nm.
Embodiment 11
Under small animal living body Image-forming instrument, observe compd A
4to vital staining: add the compd A of being furnished with different concns
4(0 μ M, 10 μ M, 20 μ M, 50 μ M) add in the HEPES damping fluid of 0.1mL, then the damping fluid of this different concns are injected into nude mice belly epithelium different zones and hatch 10min.Then, use the fluorescence of NightOWL II LB983 living imaging systematic observation mouse web portion different zones.Fig. 8 is compd A
4photo to mouse living imaging.As figure can be observed compd A
4can detect halfcystine in vivo to vital staining, instrument is small animal living body imager, model: NightOWL II LB983.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.It as fluorescence dye, is a kind of purposes of new compound of the present invention; can not assert that compound of the present invention is only for fluorescence dye; for general technical staff of the technical field of the invention; under the consideration as the same function mechanism of fluorescence dye based on the compounds of this invention; can also make some simple inferences; draw other application purpose of compound of the present invention, all should be considered as belonging to protection scope of the present invention.
Claims (6)
1. a class be take the fluorescence dye that fluorescein analogue is parent, has the structure of general formula I:
Wherein:
M is 1;
M is H;
R
0be selected from H, F, Cl, Br and I;
R ' is selected from H, acryl;
R is selected from R
7, R
8
2. fluorescence dye claimed in claim 1, is characterized in that, described R
0be selected from H, F, Cl.
3. the conjugate of fluorescence dye claimed in claim 1.
4. the preparation method of fluorescence dye claimed in claim 1, comprises the steps:
(1) fluorescein parent compound II ' is dissolved in the aqueous solution of sodium hydroxide of 50wt%, under 10～165 ℃ of conditions, reacts 10～24h, the thick thing obtaining is added in frozen water, with concentrated hydrochloric acid, be neutralized to neutrality, standing rear suction filtration obtains solid II;
Wherein, M is H;
R
0be selected from H, F, Cl, Br and I;
(2) the new cyclic ketones III ' steaming is added drop-wise in the vitriol oil of 0 ℃, then solid II step (1) being obtained: cyclic ketones III ' is after 1:1～30 join in reaction system in batches in molar ratio, after 10～180 ℃ of heating 10h～24h, be cooled to normal temperature, pour in frozen water and produce the rear filtration of precipitation, obtain solid III;
Wherein, m is 1;
(3) N, N '-dimethyl formamide and phosphorus oxychloride in molar ratio 1:1～30 are mixed, and react after 20min～2h, then add compound III according to compound III and phosphorus oxychloride molar ratio 1:1～30 under 0 ℃ of condition, under 10-180 ℃ of condition, react 5-60 hour, preparation compound IV;
(4) compound IV and compound R x press molar ratio 1:1～50, under 10-180 ℃ of condition, react 10-50h in reaction solvent, preparation Compound I; Wherein, described compound R x is selected from vi, vii;
The described reaction solvent of step (4) is selected from methylene dichloride, chloroform, ethanol, methyl alcohol, acetonitrile, ethyl acetate, toluene, dimethylbenzene, o-dichlorobenzene, the vitriol oil or its mixture.
5. for a composition for biological sample dyeing, comprise fluorescence dye claimed in claim 1 or fluorescence dye conjugate claimed in claim 3.
6. a dyeing process for biological sample, comprises the step that fluorescence dye claimed in claim 1, fluorescence dye conjugate claimed in claim 3 or composition claimed in claim 5 are contacted with biological sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310067820.8A CN103214875B (en) | 2013-03-04 | 2013-03-04 | Preparation method of fluorescence dye using fluorescein as matrix and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310067820.8A CN103214875B (en) | 2013-03-04 | 2013-03-04 | Preparation method of fluorescence dye using fluorescein as matrix and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103214875A CN103214875A (en) | 2013-07-24 |
| CN103214875B true CN103214875B (en) | 2014-08-27 |
Family
ID=48813061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310067820.8A Active CN103214875B (en) | 2013-03-04 | 2013-03-04 | Preparation method of fluorescence dye using fluorescein as matrix and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103214875B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104710815B (en) * | 2013-12-17 | 2017-05-10 | 中国科学院大连化学物理研究所 | Novel rhodafluor fluorescent dye with characteristics of large stokes shift and near-infrared fluorescence emitting, and synthesis method thereof |
| CN104710816B (en) * | 2013-12-17 | 2017-05-10 | 中国科学院大连化学物理研究所 | Large Stokes shift and near infrared fluorescence emitting new rhodamine fluorescent dye and synthetic method thereof |
| CN106632215B (en) * | 2016-11-17 | 2019-04-12 | 陕西师范大学 | A kind of fluorescence protein coloring agent and its preparation method and application |
| CN107954994A (en) * | 2017-12-14 | 2018-04-24 | 大连理工大学 | Long-life phosphors element derivative with the targeting of weary oxygen, its synthesis and biologic applications |
| CN108853495A (en) * | 2018-06-01 | 2018-11-23 | 大连理工大学 | Composite nanoparticle, preparation method and application of the one kind based on fluorescein derivative dye |
| CN109320490A (en) * | 2018-10-26 | 2019-02-12 | 济南大学 | A kind of fluorescence probe of near-infrared specific detection cysteine |
| CN109988141B (en) * | 2018-11-30 | 2021-07-16 | 大连理工大学 | Application of fluorescein derivative compound with up-conversion luminescence property based on thermal activation delayed fluorescence |
| CN114605376A (en) * | 2022-04-21 | 2022-06-10 | 济南大学 | Difunctional fluorescent probe for detecting cysteine and viscosity and preparation thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1273584A1 (en) * | 2001-07-03 | 2003-01-08 | Dyomics GmbH | 2- or 4-Chromenyliden based merocyanines and use thereof |
| JP2004138407A (en) * | 2002-10-15 | 2004-05-13 | Arkray Inc | Test piece for creatinine measurement |
| CN101115507A (en) * | 2004-12-13 | 2008-01-30 | 通用电气医疗集团股份有限公司 | Fluorescent contrast agents |
| CN101451018A (en) * | 2007-12-03 | 2009-06-10 | 西北大学 | Yellow fluorochrome and synthetic method and use thereof |
| CN101613337A (en) * | 2008-06-23 | 2009-12-30 | 天津市化学试剂研究所 | The preparation method of salicyl fluorone |
-
2013
- 2013-03-04 CN CN201310067820.8A patent/CN103214875B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1273584A1 (en) * | 2001-07-03 | 2003-01-08 | Dyomics GmbH | 2- or 4-Chromenyliden based merocyanines and use thereof |
| JP2004138407A (en) * | 2002-10-15 | 2004-05-13 | Arkray Inc | Test piece for creatinine measurement |
| CN101115507A (en) * | 2004-12-13 | 2008-01-30 | 通用电气医疗集团股份有限公司 | Fluorescent contrast agents |
| CN101451018A (en) * | 2007-12-03 | 2009-06-10 | 西北大学 | Yellow fluorochrome and synthetic method and use thereof |
| CN101613337A (en) * | 2008-06-23 | 2009-12-30 | 天津市化学试剂研究所 | The preparation method of salicyl fluorone |
Non-Patent Citations (2)
| Title |
|---|
| A Unique Class of Near-Infrared Functional Fluorescent Dyes with Carboxylic-Acid-Modulated Fluorescence ON/OFF Switching: Rational Design, Synthesis, Optical Properties, Theoretical Calculations, and Applications for Fluorescence Imaging in Living Animals;Yuan, Lin等;《Journal of the American Chemical Society》;20111215;第134卷(第2期);1200-1211 * |
| Yuan, Lin等.A Unique Class of Near-Infrared Functional Fluorescent Dyes with Carboxylic-Acid-Modulated Fluorescence ON/OFF Switching: Rational Design, Synthesis, Optical Properties, Theoretical Calculations, and Applications for Fluorescence Imaging in Living Animals.《Journal of the American Chemical Society》.2011,第134卷(第2期),1200-1211. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103214875A (en) | 2013-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103214875B (en) | Preparation method of fluorescence dye using fluorescein as matrix and application thereof | |
| CN103087545B (en) | Fluorochrome taking fluorescein as matrix, as well as preparation method and application thereof | |
| CN107089937B (en) | The fluorescence probe and its preparation method and application of Mitochondrially targeted measurement viscosity | |
| CN106566532A (en) | Amphiphilic illuminant with aggregation induced emission characteristics and applications thereof | |
| CN108033907A (en) | A kind of Heptamethine cyanines active fluoro probe and preparation method and application | |
| CN103382313B (en) | Naphthalimide fluorochrome and its preparation and application | |
| CN102146284B (en) | Ratiometric fluorescent probe and application thereof | |
| CN104945322B (en) | Detect the compound and preparation method thereof of tumor hypoxia | |
| CN108864056B (en) | Near infrared fluorescent compound and its preparation method and application with AIE performance | |
| KR20170101360A (en) | Compound based cyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same | |
| Zhang et al. | Diketopyrrolopyrrole-based ratiometric fluorescent probe for the sensitive and selective detection of cysteine over homocysteine and glutathione in living cells | |
| CN103820104B (en) | Near infrared fluorescent probe, its method for making and the application of one class taking Nile blue as parent | |
| Wei et al. | Engineering a lipid droplet targeting fluorescent probe with a large Stokes shift through ester substituent rotation for in vivo tumor imaging | |
| CN102702769B (en) | Green fluorescence cyanine dye and preparation method as well as application thereof | |
| CN110423487A (en) | A kind of Rhodol derivative dye and its application | |
| Jin et al. | Ratiometric upconversion luminescence nanoprobes from construction to sensing, imaging, and phototherapeutics | |
| CN105111773B (en) | One class amino cyanine fluorochrome and its preparation method and application | |
| CN111793371B (en) | 3, 5-asymmetrically modified BODIPY near-infrared fluorescent dye and preparation method thereof | |
| Zhang et al. | A series of novel NIR fluorescent dyes: Synthesis, theoretical calculations and fluorescence imaging applications in living cells | |
| CN109456250A (en) | Hot activation delayed fluorescence (TADF) nano-probe and preparation method thereof and the application in bio-imaging | |
| CN106634964B (en) | Oxazine compound is preparing the application near infrared fluorescent probe | |
| CN107814796A (en) | A kind of environment sensitive dyestuff based on benzofuraxan and its preparation method and application | |
| CN102617554B (en) | Two-photon fluorescent probe taking naphthaline as matrix as well as preparation method and application thereof | |
| CN105085340B (en) | Diphenylethene group dual cyano benzene two-photon fluorescence probe for detecting and displaying intracellular Hg ion | |
| CN111303111A (en) | Novel cyanine near-infrared two-region dye, preparation method and fluorescent imaging application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |