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WO2008018129A1 - Procédé de détection de protéine et colorant fluorescent utilisé à cet effet - Google Patents

Procédé de détection de protéine et colorant fluorescent utilisé à cet effet Download PDF

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WO2008018129A1
WO2008018129A1 PCT/JP2006/315751 JP2006315751W WO2008018129A1 WO 2008018129 A1 WO2008018129 A1 WO 2008018129A1 JP 2006315751 W JP2006315751 W JP 2006315751W WO 2008018129 A1 WO2008018129 A1 WO 2008018129A1
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protein
fluorescent dye
derivative
fluorescence
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Japanese (ja)
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Shinichiro Isobe
Michinori Waki
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    • 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"

Definitions

  • the present invention relates to a method for detecting a protein and a fluorescent dye used therefor.
  • proteome analysis which comprehensively analyzes the properties and expression dynamics of all proteins contained in one organism or cell using genomic information, is produced by gene expression. It is necessary to detect and identify small amounts of proteins with high sensitivity.
  • diseases such as cancer and infections caused by viruses generate special proteins, these special proteins can be treated as markers of diseases, and can be applied to diagnosis and treatment of diseases.
  • it is necessary to detect and identify these special proteins with high sensitivity.
  • a sample protein is labeled with a fluorescent dye (Non-patent Document 1), separated by electrophoresis, and then mass spectrometer such as MALDI-TOF MS is used. The molecular weight of the fractionated protein is measured, and database search is performed to identify the protein.
  • protein chips using DNA chip technology are used for blotting methods such as Western blotting and further for expression analysis and analysis of protein-protein interactions (for example, Non-patent Document 2).
  • a protein chip simultaneous analysis of expression dynamics and interaction of many types of proteins can be performed easily and quickly.
  • separation of proteins using electrophoresis is carried out by placing proteins on a gel and conducting electricity on the electrodes set at both ends to move on the chromatograph, and separation is performed according to differences in molecular weight and the like. Do. After this, fluorescent labeling is performed by immersing the gel in a fluorescent dye solution (for example, Permitted documents 1 and 2). However, since the fluorescence quenching occurs when the gel is dried, the quantitative determination can not be accurately performed because of the thickness of the swollen gel although the quantitative measurement is performed in the wet state.
  • a fluorescent dye solution for example, Permitted documents 1 and 2
  • Patent Document 1 Japanese Published Patent Application No. 2003-531946
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2004-317297
  • Non Patent Literature 1 Michael Brinkley, Bioconjugate Chem “1992, 3, 2-13
  • Non Patent Literature 2 Paul Cutler, Proteomics, 2003, 3, 3-18
  • the object of the present invention is to solve the above-mentioned problems, and to provide a method for detecting a protein with high sensitivity and easy operation, and a fluorescent dye used therefor.
  • the present inventors have made earnest efforts to solve the above problems, and as a result, they have found that it is possible to detect a completely new protein different from conventional detection methods based on changes in fluorescence intensity, and completed the present invention. It is a thing.
  • the method for detecting a protein of the present invention is a method for detecting a protein labeled with a fluorescent dye, which has a shorter wavelength than the first fluorescence wavelength observed in the free state, and binds to the protein. And detecting the fluorescence by measuring the fluorescence based on the second fluorescence wavelength observed in the According to the present invention, even when the fluorescence intensity in the bound state is weak, the fluorescence wavelength is shorter than the first fluorescence wavelength observed in the free state, and is observed in the protein bound state.
  • the fluorescence of the second fluorescence wavelength may be measured, for example, the second fluorescence wavelength value or the fluorescence intensity thereof may be measured, so that detection with higher sensitivity can be performed compared to the conventional detection method.
  • the second fluorescence wavelength shifts to a short wavelength as well as the amount of binding to the protein increases, so the relationship between the shift value from the first fluorescence wavelength and the amount of protein The protein can be quantified from
  • the second step is performed before the protein is provided to the separation means.
  • the protein can be labeled with a first fluorescent dye that generates a fluorescence wavelength of Furthermore, prior to or simultaneously with the labeling of the protein with the first fluorescent dye, the protein is labeled with the second fluorescent dye in which the first fluorescent wavelength is not shifted to a short wavelength while bound to the protein, and then the separation means is used. It can also be provided.
  • a protein and a first fluorescent dye that generates the second fluorescence wavelength are reacted in a solution, and the solution is spotted on a measurement substrate.
  • the fluorescence image based on the second fluorescence wavelength from the measurement substrate can be measured.
  • the second fluorescent dye which does not shift the first fluorescent wavelength to a short wavelength when bound to the protein is a protein. And in solution.
  • the first fluorescent dye have an anionic group that electrostatically bonds to a protein.
  • the second fluorescent dye have a covalent bond group that covalently bonds to a protein. Examples of the covalent bond include an amide bond, an imide bond, a urethane bond, an ester bond or a guanidine bond.
  • an anionic fluorescent dye in which an anionic group binding to a protein is bound to an organic EL dye directly or via a linking moiety may be used.
  • the above-mentioned anion group is a carboxyl group, sulfonyl group, sulfur Any of acid groups, phosphate groups and combinations thereof can be used.
  • H-0)-(n is an integer from 1 to 10),-CH CH--C ⁇ C--Ar_ and-CO-Ar_NR-
  • At least one functional group selected from the group consisting of
  • organic EL dye a fused polycyclic compound composed of a 5-membered ring compound containing one or more kinds of heteroatoms, selenium atoms or boron atoms and a 6-membered ring compound having a conjugated system is used. That ability S can.
  • an azole derivative represented by any one of the following general formulas (1), (2) or (3) can be used.
  • R R R R independently represents a hydrogen atom, a halogen atom, or an alkyl group
  • aromatic hydrocarbon group which may have a substituent such as a group, a hydrocarbon group, a heterocyclic group or an aromatic group containing a hetero atom in the ring
  • X may have a substituent Good nitrogen atom or sulfur atom or oxygen atom or selenium atom or boron atom
  • R ′ represents an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring or an aromatic hydrocarbon group
  • An — represents a halide ion such as CI—, Br— or I—, CF 2 SO—, BF—, or PF—.
  • R and R one selected from the group consisting of thiophen derivatives, furan derivatives, pyrrol derivatives, imidazol derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives and pyrazine derivatives is used.
  • a phenyl group having a sulfonyl group can be used.
  • an imidazole derivative represented by the following general formula (4), (5), (6), (7) or (8) can also be used.
  • R, R, R, R and R are each independently a hydrogen atom, a halogen atom,
  • Aromatic hydrocarbon group or hydrocarbon group or heterocyclic group which may have a group Represents an aromatic group containing a group or a heteroatom in the ring, and R, R, R, R and R are the same or different
  • R ⁇ R is an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring,
  • An- is a halogen such as Cl-, Br-, or. Indicating fluoride ion, CF2SO-, BF-, PF-.
  • One selected from the group consisting of toluene derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives and pyrazine derivatives can be used.
  • a phenyl group having a sulfonyl group can also be used.
  • the anion fluorescent dye of the present invention does not quench even when the sample is dried, high sensitivity detection is possible even in the dry state. For example, it is possible to react an anion fluorescent dye and a protein in a solution, spot the solution on a substrate of a protein chip, and image it for detection with an image scanner or the like. Further, the anion fluorescent dye used in the present invention can be used also in the recently developed dry assay, and is a reagent of which method of use is not selected. In addition, it is stable to heat and can withstand long-term storage at room temperature, so it is easy to handle.
  • the above-mentioned anion fluorescent dye can be suitably used in the detection method of the present invention. According to the findings of the inventor of the present invention, no shift of the fluorescence wavelength was observed even when using conventional fluorescent dyes having an anionic group such as methyl orange and orange G. On the other hand, when the above-mentioned anion fluorescent dye was used, the fluorescence wavelength was shifted to a shorter wavelength (blue shift), and the fluorescence intensity increased. In addition, the absorption wavelength shifted to a longer wavelength (red shift) and the intensity decreased.
  • Anion fluorescent dye electrostatically bonds to a positively charged group of a protein, for example, an amino group, but when an organic EL dye is used for the color forming part, the energy of the organic EL dye is easily released due to the interaction with the adjacent protein. Possibly due.
  • an organic EL dye is used for the color forming part, the energy of the organic EL dye is easily released due to the interaction with the adjacent protein. Possibly due.
  • neither blue shift of the fluorescence wavelength nor a fluorescence increase was observed even when using a dye whose coloring part is an organic EL dye.
  • the fluorescent dye is considered to react with only the functional group on the surface of the protein to form a covalent bond.
  • a fluorescent dye having a binding site comprising an anionic group Since it has a small molecular weight and low steric hindrance, it bonds not only to the amino group on the surface of the protein but also to the amino group in the deep part by electrostatic bonding, so it is positioned in the deep part (hydrophobic field) of the protein as described above. This is considered to have caused a blue shift and an increase in fluorescence intensity.
  • the detection method of the present invention has the following effects.
  • the fluorescent dye used in the present invention is easier to handle and cheaper than Cy3 and Cy5, since no thermal fading is observed, which is higher in thermal stability than Cy3, Cy5, and Alexa dyes. Protein can be detected at lower cost. Further, since the fluorescent dye of the present invention exhibits the largest quantum yield when dried, when the protein is separated by electrophoresis, the thickness can be made as thin as possible by drying the gel as compared to the conventional method. Accurate quantification can be performed Brief description of the drawings
  • FIG. 1 is a photograph showing an example of the change in color tone of the fluorescent dye in the present invention.
  • FIG. 2 shows changes in UV spectrum tone of the fluorescent dye in Example 1 of the present invention.
  • FIG. 3 is a view showing a change in fluorescence spectrum of the fluorescent dye in Example 1 of the present invention.
  • FIG. 4 is a view showing the change in UV spectrum tone of the fluorescent dye in Example 1 of the present invention.
  • FIG. 5 is a graph showing changes in the fluorescence spectrum of the fluorescent dye in Example 2 of the present invention.
  • FIG. 6 is a graph showing the relationship between the insulin concentration and the fluorescence peak wavelength in Example 2 of the present invention.
  • FIG. 7 is a graph showing the relationship between insulin concentration and fluorescence intensity in Example 2 of the present invention. Ru.
  • FIG. 8 is a view showing the change of the fluorescence spectrum of the fluorescent dye in Example 3 of the present invention.
  • the detection method of the present invention is a method for detecting a protein labeled with a fluorescent dye, which has a shorter wavelength than the first fluorescence wavelength observed in the free state, and is in a state bound to the protein.
  • the protein is detected by measuring the fluorescence based on the second fluorescence wavelength observed in.
  • the fluorescence wavelength of the anionic fluorescent dye used in the present invention gradually shifts from the free fluorescence wavelength to a shorter wavelength with an increase in the amount of binding to the protein, and further does not shift the fluorescence wavelength force S and the fluorescence intensity Also leads to saturation that does not increase. Therefore, the presence or absence of the protein in the sample can be confirmed and quantified by using the fluorescence wavelength value in the intermediate state from the free state to the saturated state and the fluorescence intensity thereof.
  • proteins may be added stepwise to an anionic fluorescent dye, and proteins may be quantified from the relationship between the amount of added protein and the shift value from the fluorescence wavelength in the free state.
  • the sample targeted by the present invention is not particularly limited as long as it contains a protein. It is possible to label collagen, which is a simple protein, and to label it after electrophoresis. In addition, it can be used for detection of sugar chains and peptides having an amino group. In addition, it is also possible to use for the antigen-antibody reaction which is good in handling as in the past in the labeling of antibodies. For example, antibody response can be observed by any method such as an antibody / antigen chip or an evanescent wave fluorescence immunoassay.
  • the detection method of the present invention can be applied to either a solution state sample or a solid state sample.
  • a sample in a solution state for example, a sample solution containing a protein is added to a solution in which an anionic fluorescent dye having a predetermined concentration is dissolved, and the fluorescence vector of the solution is measured using a fluorescence spectrophotometer or the like.
  • the presence or absence of the fluorescence wavelength of 2 and the fluorescence intensity or shift value of the fluorescence wavelength The sample concentration is detected.
  • the measurement wavelength is fixed to the second fluorescence wavelength, and the sample concentration is detected from the fluorescence intensity.
  • a method in which an anion fluorescent dye is dissolved in a sample solution containing proteins can also be used.
  • the following method can be used.
  • the protein is reacted with an anionic fluorescent dye in a solution state, then the solution is spotted and spotted on a measurement substrate, for example, a protein chip, and the chip is imaged using an image scanner or the like to determine the sample concentration.
  • a measurement substrate for example, a protein chip
  • the chip is imaged using an image scanner or the like to determine the sample concentration.
  • the first fluorescence wavelength observed in the free state refers to the fluorescence wavelength observed when an anionic fluorochrome is present alone in a solution or solid.
  • the second fluorescence wavelength observed in the bound state to the protein is a fluorescence wavelength based on the anionic fluorescent dye bound to the protein, and is a shorter wavelength than the first fluorescence wavelength.
  • the shift value of the second fluorescence wavelength from the first fluorescence wavelength depends on the type of anionic fluorescent dye bound to the protein, and is at least 2 nm or more, more preferably 10 nm or more.
  • anion fluorescent dye to be used in the detection method of the present invention
  • a substance in which an anionic group capable of binding to a protein is directly bonded to an organic EL dye, and an anionic group capable of binding to an organic EL dye via a linking part Included are those that are combined.
  • the anion group any one of a carboxyl group, a sulfonyl group, a sulfate base, a phosphate group and a combination thereof can be used which is electrostatically bonded to a positively charged group such as an amino group of a protein. It is preferable to use a group.
  • the fluorescent dye having a covalent bond group used as the second fluorescent dye includes one in which a covalent bond group to be bound to a protein is bound to an organic EL dye directly or via a linkage.
  • the covalent bond may, for example, be an amide bond, an imide bond, a urethane bond, an ethenole bond or a guanidine bond.
  • covalent bond group examples include isothiocyanate group, isocyanato group, epoxy group, sulfonyl halide group, sulfonyl chloride group, acinole group, halogenated alkyl group, glyoxal group, anoredide group, triazine group, carbodiimide group and active esterification And the like can be used.
  • isothiocyanate group, isocyanato group, epoxy group, halogenated alkyl group It is preferable to use any one kind selected from a riadine group, a carppositimide group and an activated esterified carbonyl group.
  • an isocyanate group an epoxy group, a halogen alkyl group, a triazine group, a carppositimide group and an active ester group.
  • they are a triazine group, a carpodiimide group or an activated esterified carbonyl group.
  • these organic EL dyes have a carboxylic acid group, it is also possible to directly modify the amino group and imino group present in the target molecule in the presence of a carbidoimide derivative or triazine derivative.
  • the anionic fluorescent dye can also contain a covalent bonding group. Thereby, stronger binding can be formed between target molecules.
  • the combination of the covalent bond group and the anion group is not particularly limited, and a combination of the above functional group and the above anion group such as a sulfonyl group or a carboxyl group may be mentioned.
  • the linking part used in the fluorescent dye of the present invention is a constituent part linking a chromogenic moiety and an anion group or a covalent bonding group, and is a moiety containing a covalent bond or an atomic chain,-(CH 2) )-(
  • n n is an integer of 1 to 4
  • _NHC _ _, _ C NH NH-, _C _ _ _, _ C _ _ _, _ SO NH _, _ HN _ C ( NH) _ NH _
  • the linking portion may be composed of only one type of functional group selected from the above group, or may be configured to include two or more types of functional groups. In addition, it may be configured to include two or more selected one functional groups.
  • XI and X2 are each independently-(CH 2)-(n is an integer of 1 to 4), -NHCOO-, _C
  • One type of functional group selected from the group consisting of NR— can be used. Preferred combinations are CONH-COO-, -CH- 0-, -CH-NR-, _CCNH- (CH 2)-, CCNH- (C
  • H) at least one functional group selected from the group consisting of -NH-, -0-, -S-, -NR-, -CH CH-, -C ⁇ C-, -Ar- and -C0_Ar-NR-
  • R1 and R2 each independently represent a hydrogen atom, or an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring, or an aromatic hydrocarbon group, and if necessary, a sulfonyl group, Any one selected from the group consisting of a hydroxyl group, a quaternary amino group and a carboxyl group and substituted by one type of charged group can be used.
  • Ar is a aryl group, preferably a phenyl group or a naphthylene group, which may be optionally substituted with a sulfonyl group.
  • p and q are each independently an integer of 0 to 20, preferably an integer of 0 to 10, more preferably an integer of 0 to 5, and p + q l l.
  • this linkage part are: _ (CH2) p-CONH- (CH2) q-,-(CH2) p-COO- (CH2) q-, _ (CH2) p-CH (-R1-S03H )-(CH2) q-, _ (CH2) p-CH (-Rl_N + H3)-(CH2) q-,-(CH 2) p-CH (-Rl-COOH)-(CH2) q-,- (CH2) p_CH (-Rl-OH)-(CH2) q-, _ (CH2) p- (0_CH-) n- (CH2) q-, _ (CH2) p-CONH (-Rl_S03H)-(CH2) q-, _ (CH2) p-CONH (-Rl-S03H) _ (CH 2) q-, _ (CH 2) p-C0 NH (-Rl-N + H3) _ (CH 2) q-, _ (CH2)
  • X4 and X5 are each independently -NHCOO-, -CONH-, -COO_, -SO NH-
  • R 3 is a hydrogen atom, or an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring, or an aromatic hydrocarbon group, and if necessary, a sulfonyl group, a hydroxyl group, a quaternary amine Any one selected from the group consisting of a group and a carboxyl group and substituted by one type of charged group can be used.
  • Ar is a aryl group, preferably a phenylene group or a naphthylene group, which may be optionally substituted with a sulfonyl group.
  • r is an integer of 0 to 20, preferably an integer of 0 to 10, and more preferably an integer of 0 to 5. Specific examples of this spacer include -CONH- (CH2) r-COO-, -CONH-CH (-R3-OH) -COO-, -CONH-CH (-R3-COOH)- COO-,-CONH-CH (R3-S03H) -COO-,-COO- (CH2) r-COO-, etc. can be mentioned.
  • a peptide linker which also forms an amino acid or an amino acid power of 2 to 20 at the junction.
  • amino acids natural or synthetic amino acids can be used.
  • natural amino acids include glycine, analanin, norin, leucine, isoleucine, 4-amino-2-butyric acid, homoserine, serine, threonine, aspartic acid, gnoletamic acid, aspartic acid, gnoletamine, lysine, hydroxylysine, Anoreginin, cystine, cystic acid, 2-amino-3-sulfosulfanipropanoic acid, 2-amino-3-sulfoxypropanoic acid, cystine, methionine, ferricyanin, tyrosine, tryptophan, histidine, proline and 4- Hid Roxyproline etc. are included.
  • Synthetic amino acids include the D-form of the above-mentioned naturally occurring amino acids, and modified amino acids having at least an amino group and a carboxyl group in the molecule.
  • Modified amino acids can be represented by the general formula: H—N (R1) — (R2—C ⁇ ) — OH.
  • R1 and R2 each independently consist of a sulfonyl group, a hydroxyl group, a quaternary amine group, and a carboxyl group with or without an ester, ethanol, thioester, thioether, amide, carbamide or thiocarbamide. Any of the groups selected from the group represents a hydrocarbon group or an aromatic group or a heterocyclic group substituted by four charged groups.
  • each of the hydrocarbon group, the aromatic group or the heterocyclic group may be substituted with at least one of a halogen atom, an anolequinole group, an alkenyl group, an alkynyl group or an alkoxy group.
  • amino acids having a sulfonyl group such as cysteine acid, 2-amino-3-sulfosulfanipropanoic acid, 2-amino-3-sulfopropanoic acid, And any one member selected from the group consisting of tyrosine having a hydroxyl group, threonine, 4-amino-2-hydroxybutanoic acid, homoserine and serine. More preferably, cystic acid, homoserine or serine.
  • peptide linkers -C (-Rl) -CONH-C (-R2)-, -C (-Rl) -CONH-C (-R2) -CONH-C (-R3)-, respectively. It is preferable to use a dipeptide, tripeptide or tetrapeptide represented by _C (-Rl) -CONH_C (-R2) -CONH_C (-R3) -CONH_C (-R4)-.
  • R 1, R 2, R 3 and R 4 each represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alcohol group, an alcohol group, an indolate group, a hydroxyphenyl group, a benzyl group, a guanidine group, a thioether group, an alkyl thiol group, an imidazole Represents a substituent such as a group or an alkylamine group.
  • These peptides may be homo or hetero peptides.
  • Ala_Ser, Glu_Ala, Glu-Ala-Leu, Gly_Pro, Gly-Pro-Asn, lie-Val, Ile-Va ⁇ Met, etc. can be used.
  • peptide linker one having at least one type of charged group selected from the group consisting of sulfonyl group and carboxyl group, if necessary, as a part of the peptide linker.
  • a peptide linker one containing one or more of these amino acids having four or more charged groups can be used. By this, it is impossible to introduce an anion group at the connecting part. Anions can be attached to the fluorescent dye.
  • cysteine acids having sulfonyl groups 2-amino-3-sulfosulfanipropanoic acid, 2-amino-3-sulfoxypropanoic acids, tyrosines having hydroxyl groups, threonine, 4-amino-2- A peptide linker comprising at least one amino acid selected from the group comprising hydroxybutanoic acid, homoserine and serine can be used.
  • the fluorescent dye of the present invention can be synthesized, for example, by using any of triazine group, carpimidimide group and activated esterified carbonyl group, more preferably activated esterified carboxyl group.
  • activated esterified carbonyl group N-hydroxy-succinimide ester or maleimide ester can be used.
  • N-hydroxy-succinimide ester is used.
  • N-hydroxy monosuccinimide as shown in the reaction formula I of the following scheme 1, by using DCC as a condensing agent, by using an amide bond via an N-hydroxy mono succinimidate ester, an EL dye And the target molecule bind.
  • a triazine derivative can also be used for the activated esterified carbonyl group.
  • carbidoimide group carbimidoimide reagents such as ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC) and 1-cyclohexyl-3- (2_morpholinoethyl) carbimide can be used.
  • the EL dye can be linked to the target molecule via an amide bond via a carbidoimide (Scheme III).
  • an EL dye in which a carpodiimide group and a triazine group are introduced in advance in the molecule can be directly bonded to the amino group and imino group in the biological molecule (Scheme IV).
  • R represents an aromatic hydrocarbon group containing an anionic group as a substituent, a hydrocarbon group, a heterocyclic group or an aromatic group containing a hetero atom in the ring.
  • the method of reaction formula V can be used.
  • the fluorescent dyes of the present invention also include those containing a reactive group that forms a covalent bond, in addition to the anionic group. It is preferable to use an activated esterified carbonyl group as a reactive group which forms a covalent bond. It can form stronger bonds with proteins.
  • the functional group other than the functional group directly bonded to the color forming part secures the physical distance between the color forming part and the anion group, and the color forming part and the anion While ensuring the freedom of selection of the molecular skeleton of the sex group, it has the effect of making it easier for the anion group to bond to the deep positive charge group of the protein. This also makes it possible to selectively label only specific proteins.
  • a nitrogen atom or the like as a functional group directly bonded to the color forming portion When the hetero atom of is used, the whole molecule can be made into a more rigid structure, so that the sticking between the colored portions can be suppressed.
  • an oxygen atom or the like it becomes a flexible molecular structure, and it is possible to control the sta- turing strength.
  • the organic EL dye for use in the present invention is held in a solid state between the pair of anode and cathode when the holes injected from the anode recombine with the electrons injected from the cathode. It is not particularly limited as long as it is a dye that can emit light by the energy of.
  • polycyclic aromatic compounds such as tetraphenyl butadiene and perylene, cyclopentadiene derivatives, oxadiazole derivatives, coumarin derivatives, distyrylvirazine derivatives, ataridon derivatives, quinacdoline derivatives, stilbene derivatives, futonothiazine derivatives, pyrazinoviridines Derivative, amino group derivative, imidazole derivative, force rubazole derivative, thiophen derivative and the like can be used.
  • organic EL dye examples include, as polycyclic aromatic compounds, rubrene, anthracene, tetracene, pyrene, perylene, thalicene, decacyclene, coronene, tetraphenyl butadiene, tetraphenynorecyclobutadiene, pentaphenynore Force S to mention cyclobutadiene.
  • cyclopentadiene derivatives include 1,2,3,4-tetraphenylidene 1,3 cyclopentadiene, 1,2,3,4,5 pentaphenynore 1,3 cyclopentadiene.
  • oxadiazole derivative 2 (4 't butyl phenyl) -one 5- (4'-biphenyl) 1, 3, 4- oxadiazole, 2, 5- bis (4- jetylamino fenyl) 1, 3, 4- oxadiazole
  • coumarin derivatives include coumarin 1, coumarin 6, coumarin 7, and coumarin 30.
  • distyrylbirazin derivatives include 2,5 bis (2- (4-biphenyl) ethenyl) pyrazine, 2,5 bis (4-ethysteryl) pyrazine, 2,9_bis_ (4-methoxysteryl ) Pyrazine can be mentioned.
  • Atalidone and its derivatives can be mentioned as the atalidone derivatives.
  • quinacdoline derivatives include quinacdoline and its derivatives.
  • stilbene derivatives include 1,1,4,4-tetraphenyl-2-1,1,3-butadiene and 4,4'-bis (2,2-diphenylvinyl) biphenyl.
  • Asorenol derivatives, imidazole derivatives, force rubazole derivatives and thophen derivatives, those described in the general formula herein can be used.
  • a preferred organic EL dye used in the detection method of the present invention is a compound containing a five-membered ring compound having a conjugated system, and the five-membered ring compound is one or more kinds of heteroatoms, selenium atoms or boron. Those containing atoms can be mentioned. Further, specifically, there can be mentioned a monocyclic compound consisting of a 5-membered ring compound having a conjugated system and a condensed polycyclic compound consisting of a 5-membered ring compound and a 6-membered cyclic compound having a conjugated system. This is because even in the solid state, the quantum yield exhibits a strong fluorescence.
  • azole derivatives or imidazole derivatives are preferred. Furthermore, it is preferable that the azole derivative or imidazole derivative has one or more quaternary ammonium groups. It is because it can improve the water solubility.
  • condensed polycyclic compound described below is used as an anion fluorescent dye by binding to an anion group via the above-mentioned linking part.
  • condensed polycyclic compounds in which an anionic group is directly bonded can be used as an anionic fluorescent dye itself.
  • fused polycyclic compounds can be bound to a covalent bond group directly or via a linkage to be used as a second fluorescent dye.
  • fused polycyclic compounds are all preferably used in the detection method of the present invention: preferably diazole derivative 3, imidazole derivative 2, thiadiazole derivative, force rubazole derivative, thiazole derivative, Preferred are oxazolopyridine derivatives (oxazazolopyridine derivatives).
  • each of R, R, R, R, R and R independently has a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a cyano group or a sulfonyl group. Or an aromatic group containing an aromatic hydrocarbon group, a hydrocarbon group, a heterocyclic group or a hetero atom in the ring.
  • R, R, R, R, R and R may be the same or different.
  • R ′ is an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring,
  • An— is a halide ion such as Cl—, Br— or ⁇ , CF 2 SO— , BF-,
  • R and R each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, an amino group, an aromatic hydrocarbon group which may have a substituent such as a sulfonyl group, or a hydrogen hydrocarbon.
  • the aromatic group which contains a group or a heterocyclic group or a hetero atom in a ring is shown.
  • R and R may be the same or different. The same applies to the following general formulas unless otherwise stated.
  • n is an integer of 1 or more, preferably:! To 5, and the same applies to the following general formulae.
  • R, R, R and R each independently represent a hydrogen atom, a halogen atom or a hydride
  • An aromatic hydrocarbon group which may have a substituent such as an oral xyl group, an amino group, a cyano group or a sulfonyl group, an aromatic hydrocarbon group containing an aromatic hydrocarbon group or a hydrocarbon group or a heterocyclic group or a hetero atom in the ring Show.
  • R, R, R, R, R may be the same or different.
  • R, R is
  • X is a nitrogen atom, a sulfur atom, an oxygen atom, a selenium atom or a boron atom which may have a substituent, and the same applies to the following general formulas unless otherwise specified.
  • R 1 and R 2 each represent a hydrogen atom, a halogen atom, a hydroxyl group,
  • R may be the same or different.
  • R 1 is an orephine group which may have a substituent
  • n is an integer of 1 to 3, preferably 1. The same applies to the following general formulas unless otherwise stated.
  • the above diazole derivative which is not particularly limited as long as it is the diazole derivative, can be suitably used an oxadiazo-open pyridine derivative represented by the following general formula.
  • oxazolopyridine derivative is used, for example, by using ⁇ , ⁇ '-dicyclohexylcarbodimide (DCC) as a condensing agent by the reaction shown in the following scheme 2. It is preferable to use one derived into an active ester containing a ⁇ ⁇ ⁇ ⁇ ⁇ -hydroxy-succinimide ester.
  • DCC dicyclohexylcarbodimide
  • R 2 R 3 and R 4 are each independently a hydrogen atom, linear, branched or cyclic
  • Ar and Ar each represent a substituted or unsubstituted aryl group; May form a nitrogen-containing heterocycle with the nitrogen atom to which it is attached.
  • Y and Y are
  • Ar to Ar each independently represent a substituted or unsubstituted aryl group
  • imidazole may be used as the 5-membered ring compound, and an imidazole derivative represented by the following general formula may be used.
  • the imidazole skeleton is plural at any position on the central benzene ring R 1, R 2, R 3, R 4
  • R 2 is an orephine group which may have a substituent or
  • n is an integer of 1 to 3, preferably 1.
  • a 5-membered ring compound having a conjugated system which is a single ring compound containing one or more kinds of heteroatoms, selenium atoms or boron atoms.
  • an azole derivative represented by the following general formula can be used.
  • R, R and R each independently represent a hydrogen atom, a halogen atom or a hydroxyl group
  • R, R and R may be the same or different.
  • the organic EL dye used for the fluorescent dye of the present invention is not particularly limited as long as it is the fused polycyclic compound and the monocyclic compound described above, and a diazole derivative or an imidazole derivative represented by the following general formula Can be suitably used.
  • diazothiopyridine derivatives or imidazolopyridine derivatives can be suitably used.
  • Particularly preferred fluorescent dyes of the present invention are those containing the above-mentioned diazophoric pyridine derivative or imidazolo pyridine derivative in the color forming part, and can be represented by the following general formula.
  • R and R it is preferable to use an aromatic hydrocarbon group or a hydrocarbon group which may have a substituent.
  • a green fluorescent dye corresponding to Cy3 can be obtained.
  • the aromatic hydrocarbon group is preferably a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group or a tolyl group.
  • R and R described above are selected from the group consisting of optionally substituted thiophen group, furan group, pyrrolic group, imidazole group, imidazole group, oxazole group, thiazole group, pyrazole group and pyridine group. It is also possible to use one of them, more preferably a thiophen group, an imidazole group or a furan group. A red fluorescent dye corresponding to Cy5 can be obtained.
  • the detection method of the present invention can be applied to any detection method as long as it is a detection method for measuring the fluorescence of a solution, solid or semi-solid state protein.
  • a protein in a sample is labeled with an anionic fluorescent dye, the labeled protein is subjected to separation means, the molecular weight of the fraction is measured by a mass spectrometer such as MALDI-TOF MS, and a database search is performed to obtain a protein.
  • a mass spectrometer such as MALDI-TOF MS
  • a database search is performed to obtain a protein.
  • the separation means ion exchange column HPLC, reverse phase partition HPLC, gel filtration HPLC, or electrophoresis can be used.
  • electrophoresis primary and secondary electrophoresis can be used, and after electrophoresis, the gel can be dried and quantified.
  • the following detection method can be achieved by using a covalent fluorescent dye and an anionic fluorescent dye into which an anionic group is introduced.
  • the covalent fluorochrome is one that does not change its fluorescence wavelength even when the protein is labeled accordingly.
  • label with a covalent fluorescent dye Thereafter, electrophoresis is performed to perform separation. Furthermore after migration
  • the fluorescence wavelength changes. Since the anionic fluorescent dye can label an amino residue located deep in a protein, the change in fluorescence wavelength is due to the difference in protein structure. Therefore, it is also possible to predict protein structure from changes in fluorescence wavelength.
  • the fluorescent dye used has the same structure except for the anionic group, the performance such as the quantum yield of the fluorescent dye is the same. Therefore, highly accurate quantification is possible.
  • the present invention can be applied as follows to a detection method using a protein chip.
  • a protein and an anionic fluorescent dye are reacted in a solution, the solution is spotted on a measurement substrate, and a fluorescence image based on a second fluorescence wavelength from the measurement substrate can be measured.
  • the protein can be immobilized on the substrate by leaving it at a predetermined temperature, and incubation can be carried out if necessary.
  • fluorescence quenching does not occur even in the dry state, stable observation is possible even in the dry state.
  • the detection method using the above-mentioned covalent fluorescent dye and the anionic fluorescent dye into which an anionic group is introduced can also be used.
  • the chromophore (3) was synthesized according to scheme 2 below.
  • the active ester (3) was reacted with taurine in DMF to induce sulfonation (4) (scheme 3).
  • an organic EL dye As an organic EL dye, a synthetic pyridine derivative used in Synthesis Example 1 is used as a synthetic organic dye, and a cystenic acid is used as a linking part, and a reactive group is both an activated esterified carbonyl group and a sulfonium group which is an anion group. Introduced. Oxadiazo-open pyridine active ester (3) was reacted with cystine acid to synthesize a carboxylic acid (5) into which a linkage was introduced. Thereafter, the carboxylic acid (5) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxadiazo-opening pyridine active ester (6) having a linkage introduced. An example of the reaction is shown below.
  • the organic EL dye used in Synthesis Example 1 is used as a synthetic organic light-emitting dye, and is used as a coupling moiety Used serine.
  • Oxadiazo-open pyridine active ester (3) was reacted with serine to synthesize a carboxylic acid (7) into which a linkage was introduced.
  • An example of the reaction is shown below.
  • the color tone of Buffer solution containing sulfonyls changed from yellow to yellowish green when BSA was added (Fig. 1).
  • the UV spectrum of sulfonyl 4 is shown in FIG. From this result, sulfonyl
  • the maximum absorption wavelength of body (4) was found to be 397 nm.
  • the fluorescence spectrum was measured using 397 nm as the excitation wavelength. The results are shown in Figure 3.
  • BSA is added to a concentration of 1.6 ⁇ M.
  • the fluorescence spectrum showed a blue shift of 18 nm by addition of BSA, and the fluorescence intensity increased about 5 times.
  • the active ester (3) binds to a protein through an amide bond formed by the nucleophilic substitution reaction between an active ester group and an amino group, but an amino acid located in the deep part of BSA due to steric hindrance such as a succinimide molecule. It is thought that it does not bond to a group, but bonds to only the surface amino group.
  • the sulfonyl compound 4 is bonded not only to the amino group on the surface of BSA but also to the amino group in the deep part by electrostatic bonding, it is located in the deep part (hydrophobic field) of BSA as described above. This is considered to have caused a blue shift and an increase in fluorescence intensity.
  • Example 2 the procedure was performed in the same manner as in Example 1 except that insulin (Inslin) was used as the protein.
  • Sulfonyl compound (4) 26.7 ⁇ was prepared in a fluorescent cell in 2000 ⁇ l, and 0 to 232 ⁇ of insulin was added thereto in six divided doses.
  • the fluorescence spectrum when insulin is added is shown in FIG. As in the case of BSA, a blue shift of 19 nm and an increase in fluorescence intensity were observed.
  • the relationship between the insulin concentration in the cell when added and the peak wavelength FLmax at each concentration is shown in Figure 6, where the insulin concentration and peak wave
  • the relationship with the fluorescence intensity ⁇ Int. (Value obtained by subtracting the fluorescence intensity of only the sulfonyl form) in the length is shown in FIG.
  • the peak wavelength FLmax linearly shifted to a lower wavelength.
  • Example 2 the procedure was carried out in the same manner as in Example 1 except that lysozyme was used as the protein.
  • a 26.7 ⁇ of sulfonyl group (4) was prepared in a fluorescent cell to 2000 ⁇ l, and 0 to 4 6.6 ⁇ of lysozyme was added thereto in four portions.
  • the fluorescence spectrum when lysozyme is added is shown in FIG. Even when lysozyme was added, neither increase in fluorescence intensity nor blue shift of peak wavelength was observed. This indicates that the sulfonate form (4) does not bind to lysozyme. This is considered to mean that the sulfonyl group does not bind to the amino group of lysozyme since the amino group located on the surface of the protein has a different conformation depending on the type of protein. . However, since sulfonyl form (4) binds to BSA and insulin, it is considered possible to use sulfonyl form (4) as a fluorescent reagent capable of selectively labeling proteins.
  • the active ester (6) of Synthesis Example 2 was used as a gel, and the change in color tone when BSA was added was observed in the spectrum by the same method as in Example 1.
  • the fluorescence spectrum was blue-shifted by 18 nm by addition of BSA, and the fluorescence intensity was increased about 5 times.
  • the carboxylic acid (7) of Synthesis Example 3 was used as a gel, and the change in color tone when BSA was added was observed in the spectrum by the same method as in Example 1.
  • the fluorescence specknore is shifted by 19 nm by the addition of BSA and the fluorescence intensity is about 4.5. It has doubled.
  • the detection method of the present invention it is possible to perform simple and highly accurate quantification of protein detection. Furthermore, since the anionic fluorescent dye used in the present invention is not observed to have a higher thermal stability than Cy3 or Cy5 or Alexa dye, it is easy to handle and is cheaper than Cy3 or Cy5. Can detect protein at lower cost

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Abstract

L'invention concerne un procédé de détection de protéine, à sensibilité élevé, à l'aide d'une procédure adéquate et d'un colorant fluorescent. L'invention concerne notamment un procédé de détection dans lequel une protéine est détectée par mesure d'une fluorescence basée sur une seconde longueur d'onde de fluorescence observée dans l'état de liaison à la protéine plus courte qu'une première longueur d'onde observée à l'état libre. Le colorant fluorescent tel que décrit ci-dessus possède une stabilité thermique supérieure à celle des colorants Cy3, Cy5 et Alexa et ne présente aucune élimination de fluorescence. Ainsi, il peut être manipulé plus facilement et de manière moins coûteuse que les colorants Cy3 et Cy5 et, permet la détection d'une protéine à un coût inférieur.
PCT/JP2006/315751 2006-08-09 2006-08-09 Procédé de détection de protéine et colorant fluorescent utilisé à cet effet Ceased WO2008018129A1 (fr)

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US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels

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CN101861562B (zh) * 2006-09-06 2016-05-25 苹果公司 通过应用启发法来确定命令的触摸屏设备、方法和图形用户界面
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US11919887B2 (en) 2019-12-06 2024-03-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US12247021B2 (en) 2019-12-06 2025-03-11 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US12258333B2 (en) 2021-06-04 2025-03-25 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels

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