CN106905389A - A kind of beta galactosidase fluorescence probe with Intracellular retention ability - Google Patents
A kind of beta galactosidase fluorescence probe with Intracellular retention ability Download PDFInfo
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- CN106905389A CN106905389A CN201710059283.0A CN201710059283A CN106905389A CN 106905389 A CN106905389 A CN 106905389A CN 201710059283 A CN201710059283 A CN 201710059283A CN 106905389 A CN106905389 A CN 106905389A
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- beta galactosidase
- fluorescence probe
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- 239000000523 sample Substances 0.000 title claims abstract description 96
- 108010005774 beta-Galactosidase Proteins 0.000 title claims abstract description 51
- 102000005936 beta-Galactosidase Human genes 0.000 title claims abstract description 50
- 230000031852 maintenance of location in cell Effects 0.000 title description 7
- 150000001875 compounds Chemical class 0.000 claims abstract description 56
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 15
- 238000006467 substitution reaction Methods 0.000 claims abstract description 8
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 6
- 125000005466 alkylenyl group Chemical group 0.000 claims abstract description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 5
- 239000001257 hydrogen Substances 0.000 claims abstract description 5
- 125000004093 cyano group Chemical group *C#N 0.000 claims abstract description 3
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims 3
- 238000012360 testing method Methods 0.000 abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 63
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 52
- 210000004027 cell Anatomy 0.000 description 47
- 239000000243 solution Substances 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 238000001816 cooling Methods 0.000 description 18
- 238000010992 reflux Methods 0.000 description 18
- 239000002904 solvent Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 description 14
- 238000010898 silica gel chromatography Methods 0.000 description 11
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 10
- 238000003384 imaging method Methods 0.000 description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000000799 fluorescence microscopy Methods 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 239000000741 silica gel Substances 0.000 description 7
- 229910002027 silica gel Inorganic materials 0.000 description 7
- 229960001866 silicon dioxide Drugs 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 6
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 description 6
- 229910000024 caesium carbonate Inorganic materials 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 6
- 239000012074 organic phase Substances 0.000 description 6
- 150000003053 piperidines Chemical class 0.000 description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical class [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 206010033128 Ovarian cancer Diseases 0.000 description 5
- 206010061535 Ovarian neoplasm Diseases 0.000 description 5
- 230000031709 bromination Effects 0.000 description 5
- 238000005893 bromination reaction Methods 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000012046 mixed solvent Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000002027 dichloromethane extract Substances 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- -1 beta galactose glycosides Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000032696 parturition Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000007541 cellular toxicity Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 231100000048 toxicity data Toxicity 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 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
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000007421 fluorometric assay Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229960003082 galactose Drugs 0.000 description 1
- 150000008195 galaktosides Chemical class 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Abstract
The present invention relates to a kind of beta galactosidase fluorescence probe.The beta galactosidase fluorescence probe be compound shown in Formulas I or its in pharmaceutically acceptable salt.Test shows that the beta galactosidase fluorescence probe that the present invention is provided has stronger retentivity after being activated in cell, and the cell of beta galactosidase expression high can be carried out to be imaged within (12 hours) for a long time.Wherein, R1It is C1~C4Alkyl, sulfonic group substitution C1~C4Alkyl or phenyl；R2、R3And R4It is respectively and independently selected from：Hydrogen or C1~C3One kind in alkyl, or R2With R3Be combined as phenyl, R4It is H；R5The group shown in cyano group or Formula II.
Description
Technical field
The present invention relates to a kind of fluorescence probe, specifically, it is related to a kind of beta galactosidase fluorescence probe.
Background technology
Beta galactosidase (β-galactosidase) is a kind of by beta-galactosidase gene (lacZ gene) coding
Hydrolase, it can be by a molecule lactose hydrolysis an into molecule glucose and a molecule galactolipin, and it is widely distributed in various dynamic
Thing, in plant, and microorganism.The property of lactose is hydrolyzed using it, beta galactosidase is initially mainly used in food industry
The lactose content of dairy products is reduced, it is significant to improving lactose intolerance.In bioengineering field, beta galactose glycosides
Enzyme gene is a kind of widely used reporter gene, by detecting the expression of beta galactosidase come research purpose gene
Transfection efficiency (such as, blue hickie screening) is evaluated in transcription control.Additionally, research shows that beta galactosidase is thin in aging
Its activity can be significantly raised in born of the same parents and primary ovarian cancer cell, can be as a kind of important biomarker.Therefore, detect
Betagalactosidase activity has very important significance to biomedical and medical diagnosis on disease.
In recent years, the detection to betagalactosidase activity has also attracted substantial amounts of research.Use colorimetric method earliest
Detection beta galactosidase (US.5491069A), the chloro- 3- indoles-β-D- galactopyranosides (X-gal) of the bromo- 4- of 5- and β-half
The product of blueness can be generated after lactoside enzyme reaction, by blue product come qualitative determination beta galactosidase, the method lacks
It is relatively low sensitivity to fall into, and is difficult to quantitative determination, and response speed is slow.Compared to colorimetric method, Fluorometric assay has spirit
Sensitivity is high, can quantitative determination, fast response time, and it is easy to operate the advantages of.2005, professor Nagano reported one
Beta galactosidase Fluorescence Increasing type probe (J.Am.Chem.Soc., 2005,127,4888-4894), the probe is in β-gala
Very strong fluorescence signal can be produced under glucosides enzyme catalysis, beta galactosidase can be detected according to fluorescence intensity, and in fact
The detection of beta galactosidase in cell is showed.But, the dye molecule after activation is easy to be discharged cell, causes fluorescence to be believed
Number fidelity is very poor.In order to retention time is short in solving the problems, such as cell, the seminar reports one by molecular structure alteration
New fluorescence probe (J.Am.Chem.Soc., 2011,133,12960-12963) is planted, the probe is anti-with beta galactosidase
Should after show Fluorescence Increasing, while generate product net charge be zero, probe be used for cell imaging when efficiently avoid
Dye molecule spreads through cell membrane, enhances the holdup time in cell.Only drawback is that, this strategy is to dyestuff
The improvement of reserve capability is limited (about 30 minutes) in cell.
Therefore, in beta galactosidase problem is detected using fluorescence probe, reservation of the enhancing dye molecule in cell
Time, particularly the long-time imaging to beta galactosidase is still a huge challenge.
The content of the invention
The present inventor extensively and in-depth study, designs and has synthesized a kind of with Intracellular retention ability
Beta galactosidase fluorescence probe.Test shows, after the beta galactosidase fluorescence probe that the present invention is provided is activated in cell
With stronger retentivity, and the cell of beta galactosidase expression high can be carried out to be imaged within (12 hours) for a long time.
It is an advantage of the invention to provide a kind of beta galactosidase fluorescence spy with Intracellular retention ability
Pin.
Beta galactosidase fluorescence probe of the present invention be compound shown in formula I or its in pharmaceutically acceptable salt：
In formula I, R1It is C1~C4Alkyl, sulfonic group (SO3H) the C of substitution1~C4Alkyl or phenyl；
R2、R3And R4It is respectively and independently selected from：Hydrogen (H) or C1~C3One kind in alkyl, or R2With R3Combination (R2+R3) be
Phenyl, R4It is H；R5It is cyano group (- CN) or group shown in formula II (curve mark is substitution position, similarly hereinafter)；
It is another object of the present invention to provide the method for compound shown in a kind of formula I, methods described includes as follows
Step：
(1) compound reacts with parahydroxyben-zaldehyde through Knoevenagel as shown in formula III, obtains compound shown in formula IV
The step of；
(2) by the compound of formula IV and 2,3,4,6- tetra--O- acetyl group-α-D- galactopyranose bromidesInstead
Should, the step of obtain compound shown in formula V；With,
(3) in the basic conditions, the compound as shown in formula V sloughs blocking group (acetyl group), obtains target (shown in formula I
Compound) the step of.
Wherein, the synthesis of compound can be found in document shown in formula III：Horwitz, L., J.Am.Chem.Soc., 1955,
77,1687 and G.G.Badcock, F.M.Dean, A.Robertson and W.B.Whalley, J.Chem.Soc., 1950,
903；R1~R5Definition it is identical with described previously.
Brief description of the drawings
Fig. 1 probe Is -1 (10 μM, detailed in Example 1) are continuously increased water content in dimethyl sulfoxide (DMSO) and water mixed solvent
Abosrption spectrogram；
Wherein, abscissa is wavelength (nm), and ordinate is absorbance.
Fig. 2 probe Is -1 (10 μM) are continuously increased the fluorescence emission of water content in dimethyl sulfoxide (DMSO) and water mixed solvent
Spectrogram；
Wherein, abscissa is wavelength (nm), and ordinate is fluorescence intensity (a.u.), and excitation wavelength is 434nm.
Fig. 3 probe Is -1 (10 μM) abosrption spectrogram front and rear with beta galactosidase (6U) reaction；
Wherein, abscissa is wavelength (nm), and ordinate is absorbance.
The fluorescence emission spectrogram of compound that Fig. 4 probe Is -1 (10 μM) are changed over time after being reacted with beta galactosidase (6U)；
Wherein, abscissa is wavelength (nm), and ordinate is fluorescence intensity (a.u.).
Fig. 5 probe Is -1 (10 μM) are to different enzymes, amino acid and biomolecule selectivity block diagram；
Wherein, abscissa is different enzymes, amino acid and biomolecule, and ordinate is relative intensity of fluorescence (I/I0)。
The cell toxicity data figure of Fig. 6 probe Is -1；
Wherein, abscissa is concentration (μM), and ordinate is versus cell survival rate (Relative cell
viability)。
Fig. 7 probe Is -1 (10 μM) and Human embryo kidney cell (293T cells) are incubated fluorescence imaging figure after 30min.
Fig. 8 probe Is -1 (10 μM) are incubated fluorescence imaging figure after 30min to ovarian cancer cell (SKOV-3 cells).
Fig. 9 probe Is -1 (10 μM) are incubated the fluorescence imaging figure of different time with ovarian cancer cell (SKOV-3 cells).
Figure 10 probe Is -5 (10 μM) are incubated the fluorescence imaging figure of different time with ovarian cancer cell (SKOV-3 cells).
Specific embodiment
In a preferred technical scheme of the invention, R1It is C1~C4Alkyl, sulfonic group (SO3H) the C of substitution1~C4
Alkyl or phenyl.
In presently preferred technical scheme, R2、R3And R4It is respectively and independently selected from：Hydrogen (H) and C1~C3Alkyl
In one kind, or R2With R3Combination (R2+R3) it is phenyl, R4It is H.
In a further preferred technical solution of the present invention, R2、R3And R4It is respectively and independently selected from：H and C1~C3In alkyl
It is a kind of；R1It is C1~C4Alkyl or phenyl；
Further preferred technical scheme is：R2、R3And R4It is respectively and independently selected from：One kind in H or methyl；R1It is butyl
Or phenyl.
In a further preferred technical solution of the present invention, R2With R3Combination (R2+R3) it is phenyl, R4It is H, R1It is C1~
C4Alkyl or sulfonic group substitution C1~C4Alkyl；
Further preferred technical scheme is：R2With R3Combination (R2+R3) it is phenyl, R4It is H, R1It is ethyl or SO3H takes
The propyl group in generation.
The method of compound, specifically includes following steps shown in the formula I that the present invention is provided：
(1) parahydroxyben-zaldehyde of compound and 1.5~2.0 equivalents shown in the formula III of 1 equivalent is dissolved in acetonitrile, piperidines
Used as catalyst, reflux state is kept at least 1 hour, stops backflow, and cooling, reaction solution is separated through silica gel column chromatography, obtains formula
Compound shown in IV；
(2) by compound shown in the formula IV of 1 equivalent and 2,3,4,6- tetra--O- acetyl group-α-D- pyrroles of 1.2~1.5 equivalents
Galactolipin bromide of muttering is dissolved in ethanol, and using cesium carbonate as acid binding agent, reflux state is kept at least 12 hours, stops backflow,
Cooling, filtering, filtrate is spin-dried for, then washed twice with unsaturated carbonate potassium solution, with dichloromethane extract three times (3 ×
30mL), organic phase anhydrous sodium sulfate drying after extraction, is spin-dried for methylene chloride, and silica gel column chromatography is separated, and obtains the institute of formula V
Show compound；With,
(3) compound shown in formula V and MeONa/MeOH solution are placed in reactor, at least 24 is kept in reflux state
Hour, stopping backflow, the reaction solution of cooling is separated through silica gel column chromatography, obtains object (compound shown in formula I).
The present invention has following features：
(1) fluorescence probe of the invention has excellent water solubility, can be applied to living things system；
(2) fluorescence probe of the invention has response speed (35min) faster；
(3) fluorescence probe of the invention has very low cytotoxicity；
(4) fluorescence probe of the invention has cell permeable；
(5) using the change of molecular conformation during AIE, fluorescence probe of the invention has Intracellular retention ability, and
And can be used in long-time cell imaging (up to 12 hours), there is potential application value in field of biological detection.
Below by drawings and Examples, the present invention is further elaborated, its purpose be only that be best understood from it is of the invention
Content.Therefore, the cited case is not limited the scope of the invention.
Embodiment 1
The synthesis of compound shown in Formulas I -1 (being abbreviated as " probe I -1 ")：
Compound (300mg, 1.27mmol), parahydroxyben-zaldehyde shown in formula III -1 are added in 100mL single port bottles
(233mg, 1.91mmol), piperidines (1.0mL) and acetonitrile (20mL), reflux state keep 1 hour, stop backflow, cooling it is anti-
Liquid is answered to obtain compound shown in formula IV -1 through silicagel column column chromatography for separation (solvent DCM: MeOH=80: 1, v/v)；
By compound (180mg, 0.53mmol) shown in formula IV -1,2,3,4,6- tetra--O- acetyl group-α-D- pyranose brominations
Thing (321mg, 0.78mmol), cesium carbonate (863mg, 2.65mmol) and ethanol (40mL) are placed in reactor, in reflux state
Kept for 12 hours, stop backflow, be spin-dried for for filtrate, then washed twice with unsaturated carbonate potassium solution by cooling, filtering, uses dichloro
Methane extracts three times (3 × 30mL), and organic phase anhydrous sodium sulfate drying after extraction is spin-dried for solvent (dichloromethane), silicagel column
Chromatography (DCM: MeOH=100: 1, v/v), obtains compound shown in formula V -1；
The MeONa/MeOH solution of compound (211mg, 0.32mmol) and 30mL shown in formula V -1 is placed in reactor,
Reflux state is kept for 24 hours, stops backflow, the reaction solution of cooling separate through silica gel column chromatography (solvent DCM: MeOH=40:
1, v/v) 128mg faint yellow solids (title compound, i.e. probe I -1) are obtained, gross production rate is 20%.
1HNMR (400MHz, DMSO-d6, ppm):δ=1.41 (t, J=6.4Hz, 3H ,-CH2CH3), 3.44-3.63 (m,
4H ,-OH), 3.72 (s, 2H ,-CH2OH), 4.57 (t, J=6.8Hz, 2H ,-CH2CH3), 4.68 (s, 2H, galactose-H),
4.93 (d, J=8.4Hz, 2H, galactose-H), 5.22 (d, J=4.4Hz, 1H, galactose-H), 7.03 (s, 1H,
Pyrrole-H), 7.11 (d, J=8.4Hz, 2H, phenyl-H), 7.42 (s, 2H, alkene-H), 7.62 (t, J=8.0Hz,
1H, phenyl-H), 7.79 (d, J=8.4Hz, 2H, phenyl-H), 7.93 (t, J=8.0Hz, 1H, phenyl-H), 8.10
(d, J=8.8Hz, 1H, phenyl-H), 8.94 (d, J=8.4Hz, 1H, phenyl-H)
Mass spectrometry(ESI positive ion mode for[M+H]+):M/z calcd.for are (theoretical
Value, similarly hereinafter)：C28H28N3O6：502.1978；Found (test value, similarly hereinafter)：502.1976.
Embodiment 2
The synthesis of compound shown in Formulas I -2 (being abbreviated as " probe I -2 ")：
Compound (400mg, 1.14mmol), parahydroxyben-zaldehyde shown in formula III -2 are added in 100mL single port bottles
(209mg, 1.71mmol), piperidines (1.0mL) and acetonitrile (20mL), reflux state keep 1 hour, stop backflow, cooling it is anti-
Liquid is answered to obtain compound shown in formula IV -2 through silicagel column column chromatography for separation (solvent DCM: MeOH=10: 1, v/v)；
By compound (319mg, 0.70mmol) shown in formula IV -2,2,3,4,6- tetra--O- acetyl group-α-D- pyranose brominations
Thing (412mg, 1.0mmol), cesium carbonate (1140mg, 3.50mmol) and ethanol (30mL) are placed in reactor, in reflux state
Kept for 12 hours, stop backflow, be spin-dried for for filtrate, then washed twice with unsaturated carbonate potassium solution by cooling, filtering, uses dichloro
Methane extracts three times (3 × 30mL), and organic phase anhydrous sodium sulfate drying after extraction is spin-dried for solvent (dichloromethane), silicagel column
Chromatography (DCM: MeOH=15: 1, v/v) obtains compound shown in formula V -2；
The MeONa/MeOH solution of compound (251mg, 0.32mmol) and 30mL shown in formula V -2 is placed in reactor,
Reflux state is kept for 24 hours, stops backflow, the reaction solution of cooling separate through silica gel column chromatography (solvent DCM: MeOH=5: 1,
V/v 112mg faint yellow solids (title compound, i.e. probe I -2)) are obtained, gross production rate is 16%.
1H NMR (400MHz, DMSO-d6, ppm):δ=2.18 (m, 2H ,-CH2CH2CH2SO3-), 2.63-2.68 (m,
2H ,-CH2SO3-), 3.49-3.70 (m, 4H), 4.56 (t, J=6.0Hz, 1H), 4.68 (t, J=5.6Hz, 1H), 4.75 (t, J
=8.0Hz, 2H ,-NCH2CH2-), 4.92 (d, J=5.6Hz, 2H), 4.96 (d, J=5.6Hz, 2H), 5.22 (d, J=5.2Hz,
1H), 6.72 (d, J=8.0Hz, 2H, phenyl-H), 7.13 (s, 1H, pyrrole-H), 7.38 (d, J=15.6Hz, 1H,
Alkene-H), 7.49 (d, J=16.0Hz, 1H, alkene-H), 7.56 (t, J=8.0Hz, 1H, phenyl-H), 7.79 (d, J
=8.0Hz, 2H, phenyl-H), 7.90 (t, J=8.0Hz, 1H, phenyl-H), 8.20 (d, J=8.0Hz, 1H, phenyl-
H), 8.92 (d, J=8.0Hz, 1H, phenyl-H)
Mass spectrometry(ESI negative ion mode for[M-Na]-):m/z calcd.for
C29H28N3O9S:594.1546；found:594.1542.
Embodiment 3
The synthesis of compound shown in Formulas I -3 (being abbreviated as " probe I -3 ")：
Compound (300mg, 1.32mmol), parahydroxyben-zaldehyde shown in formula III -3 are added in 100mL single port bottles
(242mg, 1.98mmol), piperidines (1.0mL) and acetonitrile (20mL), reflux state keep 1 hour, stop backflow, cooling it is anti-
Liquid is answered to obtain compound shown in formula IV -3 through silicagel column column chromatography for separation (solvent DCM: MeOH=80: 1, v/v)；
By compound (199mg, 0.60mmol) shown in formula IV -3,2,3,4,6- tetra--O- acetyl group-α-D- pyranose brominations
Thing (370mg, 0.90mmol), cesium carbonate (977mg, 3.0mmol) and ethanol (30mL) are placed in reactor, are protected in reflux state
Hold 12 hours, stop backflow, be spin-dried for for filtrate, then washed twice with unsaturated carbonate potassium solution by cooling, filtering, uses dichloromethane
Alkane extracts three times (3 × 30mL), and organic phase anhydrous sodium sulfate drying after extraction is spin-dried for solvent (dichloromethane), silica gel column layer
Analysis separates (DCM: MeOH=100: 1, v/v) and obtains compound shown in formula V -3；
The MeONa/MeOH solution of compound (159mg, 0.24mmol) and 30mL shown in formula V -3 is placed in reactor,
Reflux state is kept for 24 hours, stops backflow, the reaction solution of cooling separate through silica gel column chromatography (solvent DCM: MeOH=20:
1, v/v) 153mg faint yellow solids (title compound, i.e. probe I -3) are obtained, gross production rate is 23%.
1H NMR (400MHz, DMSO-d6, ppm):δ=1.01 (t, J=8.0Hz, 3H ,-CH2CH2CH3), 1.42-1.47
(m, 2H ,-CH2CH2CH3), 1.65-1.71 (m, 2H ,-CH2CH2CH3), 2.41 (s, 3H ,-CH3), 3.46-3.65 (m, 4H),
4.04 (t, J=7.8Hz, 2H ,-NCH2CH2-), 4.50-4.52 (m, 2H), 4.66 (d, J=5.6Hz, 1H), 4.90-4.95 (m,
3H), 5.20 (d, J=5.2Hz, 1H), 6.62 (d, J=7.6Hz, 2H), 6.78-6.82 (m, 3H, phenyl-H＆alkene-
H), 6.98 (s, 1H, pyrrole-H), 7.16 (d, J=15.6Hz, 1H, alkene-H), 7.42 (d, J=8.0Hz, 1H,
phenyl-H).
Mass spectrometry(ESI positive ion mode for[M+H]+):m/z calcd.for
C27H32N3O6:494.2291；found:494.2296.
Embodiment 4
The synthesis of compound shown in Formulas I -4 (being abbreviated as " probe I -4 ")：
Compound (350mg, 0.99mmol), parahydroxyben-zaldehyde shown in formula III -4 are added in 100mL single port bottles
(178mg, 1.46mmol), piperidines (1.0mL) and acetonitrile (20mL), reflux state keep 1 hour, stop backflow, cooling it is anti-
Liquid is answered to obtain compound shown in formula IV -4 through silicagel column column chromatography for separation (solvent DCM: MeOH=30: 1, v/v)；
By compound (202mg, 0.44mmol) shown in formula IV -4,2,3,4,6- tetra--O- acetyl group-α-D- pyranose brominations
Thing (218mg, 0.53mmol), cesium carbonate (717mg, 2.2mmol) and ethanol (30mL) are placed in reactor, are protected in reflux state
Hold 12 hours, stop backflow, be spin-dried for for filtrate, then washed twice with unsaturated carbonate potassium solution by cooling, filtering, uses dichloromethane
Alkane extracts three times (3 × 30mL), and organic phase anhydrous sodium sulfate drying after extraction is spin-dried for solvent (dichloromethane), silica gel column layer
Analysis separates (DCM: MeOH=50: 1, v/v) and obtains compound shown in formula V -4；
The MeONa/MeOH solution of compound (229mg, 0.29mmol) and 30mL shown in formula V -4 is placed in reactor,
Reflux state is kept for 24 hours, stops backflow, the reaction solution of cooling separate through silica gel column chromatography (solvent DCM: MeOH=10:
1, v/v) 112mg dark red solids (title compound, i.e. probe I -4) are obtained, gross production rate is 18%.
1HNMR (400MHz, DMSO-d6, ppm)：δ=1.07 (t, J=8.0Hz, 3H ,-CH2CH2CH3), 1.47-1.56
(m, 2H ,-CH2CH2CH3), 1.85-1.96 (m, 2H ,-CH2CH2CH3), 2.60 (s, 3H ,-CH3), 3.50-3.68 (m, 4H),
4.17 (t, J=8.0Hz, 2H ,-NCH2CH2-), 4.53 (d, J=5.6Hz, 1H), 4.68-4.79 (m, 2H), 4.92-4.99 (m,
3H), 5.26 (d, J=5.6Hz, 1H), 6.71-6.77 (m, 4H), 7.02-7.06 (m, 2H, phenyl-H), 7.18 (d, J=
8.0Hz, 2H, phenyl-H), 7.34-7.43 (m, 2H), 7.49-7.56 (m, 3H, phenyl-H)；
Mass spectrometry(ESI positive ion mode for[M+Na]+):m/z calcd.for
C36H36N4O6Na:643.2533；found:643.2530.
Embodiment 5
The synthesis of compound shown in Formulas I -5 (being abbreviated as " probe I -5 ")：
Compound (300mg, 0.80mmol), parahydroxyben-zaldehyde shown in formula III -5 are added in 100mL single port bottles
(146mg, 1.20mmol), piperidines (1.0mL) and acetonitrile (20mL), reflux state keep 1 hour, stop backflow, cooling it is anti-
Liquid is answered to obtain compound shown in formula IV -5 through silicagel column column chromatography for separation (solvent DCM: MeOH=20: 1, v/v)；
By compound (215mg, 0.45mmol) shown in formula IV -5,2,3,4,6- tetra--O- acetyl group-α-D- pyranose brominations
Thing (259mg, 0.63mmol), cesium carbonate (733mg, 2.25mmol) and ethanol (40mL) are placed in reactor, in reflux state
Kept for 12 hours, stop backflow, be spin-dried for for filtrate, then washed twice with unsaturated carbonate potassium solution by cooling, filtering, uses dichloro
Methane extracts three times (3 × 30mL), and organic phase anhydrous sodium sulfate drying after extraction is spin-dried for solvent (dichloromethane), silicagel column
Chromatography (DCM: MeOH=40: 1, v/v) obtains compound shown in formula V -5；
The MeONa/MeOH solution of compound (226mg, 0.28mmol) and 30mL shown in formula V -5 is placed in reactor,
Reflux state is kept for 24 hours, stops backflow, the reaction solution of cooling separate through silica gel column chromatography (solvent DCM: MeOH=10:
1, v/v) 103mg dark red solids (title compound, i.e. probe I -5) are obtained, gross production rate is 20%.
1HNMR (400MHz, DMSO-d6, ppm)：δ=2.58 (s, 3H ,-CH3), 3.47-3.68 (m, 4H), 4.38-4.44
(m, 2H), 4.62 (d, J=5.2Hz, 1H), 4.83 (d, J=5.6Hz, 2H), 4.91 (d, J=5.2Hz, 1H), 5.24 (d, J=
5.6Hz, 1H), 6.58 (s, 1H, pyrrole-H), 6.64 (d, J=7.6Hz, 2H, phenyl-H), 6.96-7.05 (m, 2H),
7.08 (d, J=8.0Hz, 2H, phenyl-H), 7.10 (d, J=8.0Hz, 2H, phenyl-H), 7.18 (d, J=16.0Hz,
1H, alkene-H), 7.24-7.32 (m, 3H, phenyl-H＆alkene-H), 7.42 (d, J=7.6Hz, 2H, phenyl-H),
7.48-7.53 (m, 3H, phenyl-H)；
Mass spectrometry(ESI positive ion mode for[M+H]+):m/z calcd.for
C38H33N4O6:641.2400；found:641.2402.
Embodiment 6
As water content is continuously increased in water/dimethyl sulfoxide (DMSO) (DMSO) mixed solvent, it absorbs and fluorescence probe I -1
Spectrum change
Probe I -1 is dissolved in analytically pure DMSO, 1.0 × 10 are made-2The storing solution of M.Then different water are prepared respectively
The water of content (0-95%)/DMSO mixed solvents 2mL.Take the different water contents that the 2 above-mentioned storing solutions of μ L are added separately to prepare
Water/DMSO mixed solvents in, it is well mixed after be transferred to its absorption of test in optical quartz cuvette (10 × 10mm) respectively
Spectrum and fluorescence spectrum.As shown in figure 1, probe I -1 shows one group of obvious doublet between 300-500nm, and with
Being continuously increased for water content, the position of absworption peak is held essentially constant.With maximum absorption band 434nm, to excite optical tests, its is glimmering
Light spectrum.As shown in Fig. 2 being continuously increased with water content, the fluorescence intensity of probe I -1 is very weak, can be ignored, explanation
In the mixed system of different water contents, because it has good water solubility, not there is aggregation and produce fluorescence in probe I -1.
Also there is same phenomenon probe I -2~5, and this is no longer going to repeat them.
Embodiment 7
The spectral response of -1 pair of beta galactosidase of probe I
By the μ L of storing solution 2 of probe I -1 in 10mL centrifuge tubes, 2mL PBSs (PBS, pH=are subsequently adding
7.4) dilute the storing solution of probe I -1 so that the concentration of probe I -1 is 10 μM, add beta galactosidase (6U) and be well mixed,
Its Absorption and fluorescence spectrum is tested after the different times are incubated under the conditions of 37 DEG C.As seen from Figure 3, when probe I -1 and β -
After galactoside enzyme reaction, absorbance drastically declines in its absorption spectrum.With 434nm as exciting light, with incubation time not
Disconnected to increase, the fluorescence intensity at its 560nm also gradually strengthens (Fig. 4).
Also there is same phenomenon probe I -2~5, and this is no longer going to repeat them.
Embodiment 8
The selectivity of probe I -1
By the μ L of storing solution 2 of probe I -1 in 10mL centrifuge tubes, 2mL PBSs (PBS, pH=are subsequently adding
7.4) storing solution of probe I -1 is diluted so that the concentration of probe I -1 is 10 μM, then the competitive species for being separately added into 100 equivalents
(cellulase, reductase, lysozyme, esterase, cysteine, homocysteine, reduced glutathione, dithiothreitol (DTT),
Hydrogen peroxide and hydrogen sulfide) and beta galactosidase (6U) it is well mixed, test its fluorescence after being incubated 30min under the conditions of 37 DEG C
Spectrum.By Fig. 5 it can be found that other competitive species do not influence substantially on the fluorescence of probe I -1, and beta galactosidase adds
It is remarkably reinforced the fluorescence of probe I -1 after entering.
Also there is identical selectivity probe I -2~5, and this is no longer going to repeat them.
Embodiment 9
The cytotoxicity test of probe I -1
The cytotoxicity of probe I -1 is tested using the MTT experiment of standard.Specific experiment step is as follows：Cell is spread first
To in 96 orifice plates, cell density is 1 × 104Cells/well, and the overnight incubation in cell culture incubator.The probe I -1 of various concentrations
(1-10 μM, 100 μ L/ holes) is added separately in different holes, and the DMSO (0.5%, 100 μ L/ holes) of culture medium dilution is used as cloudy
Property control group.Cell cultivates 24h in cell culture incubator, and 10 μ LMTT solution (5mg/mL) are then added in every hole.It is incubated 4
After hour, the liquid in 96 orifice plates is removed, and add DMSO (100 μ L/ holes), be finally measured with ELIASA.By thin in Fig. 6
Shown in cellular toxicity data, when the concentration of probe I -1 increases to 10 μM, cell survival rate is substantially unaffected, and shows probe I -1
With very low cytotoxicity.
Equally, tested through identical method, the cytotoxicity of probe I -2~5 is also very low, and this is no longer going to repeat them.
Embodiment 10
- 1 pair of endogenous beta galactosidase of probe I carries out cell imaging
By probe I -1 be applied in ovarian cancer cell (SKOV-3 cells, height expression beta galactosidase) to endogenous β -
Galactosidase carries out fluorescence imaging, while choosing Human embryo kidney cell (293T cells) work for not expressing beta galactosidase
It is negative control group.Specific experiment step is as follows：
The storing solution of probe I -1 is added in the nutrient solution for giving birth to SKOV-3 cells or 293T cells in cell culture incubator
Middle incubation 0.5h (ultimate density of probe I -1 is 10 μM), then cell imaging is carried out with Laser Scanning Confocal Microscope, wherein excitation source is
404nm laser, detector receiving wave range is set to 500-650nm.The 293T cell imaging situations that observation probe I -1 was incubated,
It was found that not observing fluorescence signal (Fig. 7).The SKOV-3 cells that probe I -1 was incubated are observed under same experimental conditions, can
It was observed that obvious green florescent signal (Fig. 8), illustrate probe I -1 endogenous beta galactosidase can be carried out cell into
Picture.
Also there is same phenomenon probe I -2~5, and this is no longer going to repeat them.
Embodiment 11
- 1 pair of SKOV-3 cell of probe I carries out long-time fluorescence imaging
The storing solution of probe I -1 is added in the nutrient solution for giving birth to SKOV-3 cells and is incubated respectively in cell culture incubator
0.5h, 6h and 12h (ultimate density of probe I -1 is 10 μM), then carry out cell imaging, wherein excitation source with Laser Scanning Confocal Microscope
It is 404nm laser, detector receiving wave range is set to 500-650nm.By Fig. 9 it can be found that when probe I -1 and SKOV-3 cells
After being incubated 12h, remain able to observe green fluorescence in SKOV-3 cells, illustrate that probe I -1 has stronger Cellular retention energy
Power, can for a long time carry out cell imaging.
Embodiment 12
- 5 pairs of SKOV-3 cells of probe I carry out long-time fluorescence imaging
To take during probe I -5 is added to the nutrient solution for giving birth to SKOV-3 cells and be incubated 0.5h respectively in cell culture incubator,
6h and 12h (ultimate density of probe I -5 is 10 μM), then cell imaging is carried out with Laser Scanning Confocal Microscope, wherein excitation source is
560nm laser, detector receiving wave range is set to 650-700nm.By Figure 10 it can be found that when probe I -5 and SKOV-3 cells
After being incubated 12h, red fluorescence is also able to observe that in SKOV-3 cells, illustrates that probe I -5 also has stronger Cellular retention energy
Power, can be applied to long-time cell imaging.
Also there is same cell retentivity probe I -2~4, and this is no longer going to repeat them.
Claims (9)
1. a kind of beta galactosidase fluorescence probe, it is characterised in that the beta galactosidase fluorescence probe is Formulas I shownization
Compound or its in pharmaceutically acceptable salt：
Wherein, R1It is C1~C4Alkyl, sulfonic group substitution C1~C4Alkyl or phenyl；R2、R3And R4It is respectively and independently selected from：
Hydrogen or C1~C3One kind in alkyl, or R2With R3Be combined as phenyl, R4It is H；R5The group shown in cyano group or Formula II.
2. beta galactosidase fluorescence probe as claimed in claim 1, it is characterised in that wherein, R1It is C1~C4Alkyl,
The C of sulfonic group substitution1~C4Alkyl or phenyl.
3. beta galactosidase fluorescence probe as claimed in claim 1, it is characterised in that wherein, R2、R3And R4Independently
It is selected from：Hydrogen and C1~C3One kind in alkyl.
4. beta galactosidase fluorescence probe as claimed in claim 3, it is characterised in that wherein, R1It is C1~C4Alkyl or
Phenyl.
5. beta galactosidase fluorescence probe as claimed in claim 4, it is characterised in that wherein, R2、R3And R4Independently
It is selected from：One kind in H or methyl；R1It is butyl or phenyl.
6. beta galactosidase fluorescence probe as claimed in claim 5, it is characterised in that described beta galactosidase fluorescence
Probe is one kind in following compounds：
7. beta galactosidase fluorescence probe as claimed in claim 1, it is characterised in that wherein, R2With R3Be combined as benzene
Base, R4It is H, R1It is C1~C4Alkyl or sulfonic group substitution C1~C4Alkyl.
8. beta galactosidase fluorescence probe as claimed in claim 7, it is characterised in that wherein, R1It is that ethyl or sulfonic group take
The propyl group in generation.
9. beta galactosidase fluorescence probe as claimed in claim 8, it is characterised in that described beta galactosidase fluorescence
Probe is one kind in following compounds：
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| CN111778014A (en) * | 2019-04-04 | 2020-10-16 | 复旦大学 | Beta-galactosidase near-infrared fluorescent probe and preparation method and application thereof |
| CN111778014B (en) * | 2019-04-04 | 2024-01-02 | 复旦大学 | Near infrared fluorescent probe of beta-galactosidase, preparation method and application thereof |
| CN110028446A (en) * | 2019-04-10 | 2019-07-19 | 华东理工大学 | A kind of methods and applications of fluorescence probe and its measurement critical micelle concentration based on aggregation-induced emission feature |
| CN111763234A (en) * | 2020-06-12 | 2020-10-13 | 武汉工程大学 | Fluorescent probe for detecting beta-galactosidase and preparation method and application thereof |
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