WO2008018129A1

WO2008018129A1 - Method of detecting protein and fluorescent dye to be used therefor

Info

Publication number: WO2008018129A1
Application number: PCT/JP2006/315751
Authority: WO
Inventors: Shinichiro Isobe; Michinori Waki
Original assignee: Shinichiro Isobe; Michinori Waki
Priority date: 2006-08-09
Filing date: 2006-08-09
Publication date: 2008-02-14
Also published as: JP5244596B2; JPWO2008018129A1

Abstract

It is intended to provide a method of detecting protein at a high sensitivity with the use of a convenient procedure and a fluorescent dye to be used therefor. Namely, a detection method whereby a protein is detected by measuring a fluorescence based on a second fluorescence wavelength observed in the state of binding to the protein which is shorter than a first fluorescence wavelength observed in a free state. The fluorescent dye as described above is superior in heat stability to Cy3, Cy5 and Alexa dyes and shows no fluorescence elimination. Thus, it can be handled more easily and less expensively than Cy3 and Cy5 and, therefore, makes it possible to detect a protein at a lower cost.

Description

Specification

Method for detecting protein and fluorescent dye used therefor

The present invention relates to a method for detecting a protein and a fluorescent dye used therefor.

Background art

[0002] In recent years, the full size of the human genome has been clarified, and post-genome research for gene therapy, gene diagnosis and the like has been actively conducted. In particular, 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. In addition, since 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. However, it is necessary to detect and identify these special proteins with high sensitivity.

[0003] As a high sensitivity analysis method of protein, for example, 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. In addition, 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). For protein chips, use a protein labeled with a fluorescent dye for labeling, a hydrophobic substance, ion exchanger or metal ion, etc., for protein expression analysis, or an antibody, etc., for protein interproteins. Used for analysis of interaction. By using a protein chip, simultaneous analysis of expression dynamics and interaction of many types of proteins can be performed easily and quickly.

[0004] In addition, 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.

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

Disclosure of the invention

Problem that invention tries to solve

However, in the case of measuring the change in fluorescence intensity, it is difficult to detect a protein at high sensitivity if the increase in fluorescence intensity is not reproducible and the fluorescence intensity is weak and the difference from the free state is small. There is a problem of If weak fluorescence intensity is to be measured, the intensity of excitation light has to be increased, and there is also the problem that the light source becomes large and damage to the sample becomes large. Furthermore, when the measurement is performed using a protein chip, there is a problem that the fluorescence of the fluorescent dye bound to the protein is quenched by drying the sample on the chip. In particular, in the case of a trace amount sample such as π, it is a very serious problem because the sample is easily dried. In addition, depending on the fluorescent dye, if it takes time to measure the temperature stability is low, it may be considered that a problem may occur in the quantitativity.

[0006] Therefore, 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.

Means to solve the problem

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.

That is, 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. In addition, it is not necessary to increase the intensity of the excitation light in order to measure the weak fluorescence intensity. In addition, even unskilled inspectors can easily distinguish. In addition, when the protein is added stepwise, 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

In one embodiment of the detection method of the present invention, in the case of a detection method in which the protein in the sample is subjected to separation means and the separated fraction is subjected to mass spectrometry, 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.

Further, as another embodiment of the detection method of the present invention, 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. Furthermore, prior to or simultaneously with the reaction of the protein with the first fluorescent dye in the solution, 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.

In addition, it is preferable that the first fluorescent dye have an anionic group that electrostatically bonds to a protein. In addition, it is preferable that 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.

In the detection method of the present invention, as the first fluorescent dye, 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. it can. Here, the above-mentioned anion group is a carboxyl group, sulfonyl group, sulfur Any of acid groups, phosphate groups and combinations thereof can be used.

In addition, in the above-mentioned connection part, -CH--NHCOO--CONH--CH NH--CH NR--C

2 2 2

00--SO NH--HN-C (= NH) _NH-_0 _ -S--NR- (R is an alkyl group),-(CH-C

twenty two

H-0)-(n is an integer from 1 to 10),-CH = CH--C≡C--Ar_ and-CO-Ar_NR-

2 n

At least one functional group selected from the group consisting of

Further, as the 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.

In addition, as the above-mentioned fused polycyclic compound, an azole derivative represented by any one of the following general formulas (1), (2) or (3) can be used.

[Formula 1]

(3)

Here, in the formula, R R R R independently represents a hydrogen atom, a halogen atom, or an alkyl group

1 2 3 4

Group, alkenyl group, alkynyl group, amino group, alkoxy group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group, heterocyclic group, aromatic group containing a hetero atom in the ring An 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, and X may have a substituent Good nitrogen atom or sulfur atom or oxygen atom or selenium atom or boron atom, and 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—.

[0018] In addition, as 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. Can.

In addition, as R and R described above, a phenyl group having a sulfonyl group can be used.

Further, as the above-mentioned fused polycyclic compound, an imidazole derivative represented by the following general formula (4), (5), (6), (7) or (8) can also be used.

[Formula 2]

(7) (8) Here, in the formula, R, R, R, R and R are each independently a hydrogen atom, a halogen atom,

1 2 3 4 5

Norelyl group, alkenyl group, alkynyl group, amino group, alkoxy group, hydroxyl group, cyano group, sulfonyl group, sulfonyl group, aromatic hydrocarbon group, heterocyclic group, aromatic group containing hetero atom in ring, etc. 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

1 2 3 4 5

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-.

3 3 4 6

In the above R and R, thiophen derivative, furan derivative, pyrrole derivative, imidazo

twenty three

One selected from the group consisting of toluene derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives and pyrazine derivatives can be used.

In addition, as R and R described above, a phenyl group having a sulfonyl group can also be used.

twenty three

Since 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 following reasons can be considered as to the reason why 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. In addition, in the case of not having a binding moiety consisting of an anionic group, 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. When a fluorescent dye is used which does not have a binding site composed of an anionic group, the fluorescent dye is considered to react with only the functional group on the surface of the protein to form a covalent bond. On the other hand, 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.

Effect of the invention

[0027] The detection method of the present invention has the following effects.

That is, by measuring the second fluorescence wavelength value observed in the bound state to the protein, which is shorter than the first fluorescence wavelength observed in the free state, and the fluorescence intensity thereof, compared to the conventional case, More sensitive detection is possible. In addition, since the second fluorescence wavelength shifts to a shorter wavelength as the amount of binding to protein increases, a protein is added stepwise to the fluorescent dye to shift the fluorescence wavelength from the first fluorescence wavelength. Protein can also be quantified from the value. In addition, since fluorescence can be emitted even when the sample is in a dry state, protein detection can be performed easily and accurately. Furthermore, 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. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

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. Alternatively, 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.

[0030] 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.

[0031] The detection method of the present invention can be applied to either a solution state sample or a solid state sample.

In the case of 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. Alternatively, if the second fluorescence wavelength is known in advance, the measurement wavelength is fixed to the second fluorescence wavelength, and the sample concentration is detected from the fluorescence intensity. Can. Alternatively, a method in which an anion fluorescent dye is dissolved in a sample solution containing proteins can also be used.

In the case of a solid state sample, for example, 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. To detect. Alternatively, it is possible to previously fix an anionic fluorescent dye on top and then spot a sample solution containing protein on the measurement substrate.

Here, 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. On the other hand, 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. Here, 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.

In the 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. Here, as 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.

In addition, 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. Examples of the covalent bond group 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. Preferably, 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. More preferably, it is preferable to use any one kind selected from an isocyanate group, an epoxy group, a halogen alkyl group, a triazine group, a carppositimide group and an active ester group. This is because it can form an amide bond with an amino group of a protein and can directly bond to an imino group in the protein. More preferably, they are a triazine group, a carpodiimide group or an activated esterified carbonyl group. When 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.

In addition to the anionic group, 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.

[0037] 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) )-(

2 n n is an integer of 1 to 4), _NHC _ _, _ C NH NH-, _C _ _ _, _ C _ _ _, _ SO NH _, _ HN _ C (= NH) _ NH _

2

, -O_, -S-, -NR- (R is an alkyl group),-(CH-CH-O) _ (n is an integer of 1 to 10), -CH =

2 2 η

It is possible to use one containing one or more functional groups selected from the group consisting of CH 2 —, _C≡C 2-, ArAr— and —CO_Ar—NR—.

That is, 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.

[0038] For example, when composed of only one type of functional group, -CONH-, _COO-, _0_, -NR-, etc. are preferred. Moreover, when comprising with two or more types of functional groups, it can be set as the following aspects.

(1) When consisting of two functional groups

What is represented by the following general formula (I) can be used.

-X1-X2- (I)

Here, XI and X2 are each independently-(CH 2)-(n is an integer of 1 to 4), -NHCOO-, _C

2 n ONH-,-COO-, -SO NH-,-HN-C (= NH) _NH-,-0 _, _S-,-NR-(R is an alkyl group)

2

,-(CH-CH-O)-(n is an integer of 1 to 10),-CH = CH-,-C≡C-,-Ar-and-CO-Ar-

2 2 η

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

2 2 2 n

H-CH-))-.

2 2 η

(2) When consisting of three or more functional groups

(i) It is preferable to use what is represented by the following general formula (Π).

-(CHRl) p-X3- (CHR2) q- (II)

In the formula, X3 is a direct bond or _NHCOO-, -C0NH-, -C00_, -SO NH-, -HN-C (= N

2

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- A group can be used, preferably -C__, -co NH-,-0_, -CH = CH-, -C3 C- or -Ar-, more preferably -C〇_,-CONH -,-0-or-Ar-can be used. 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. In addition, 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.

Specific examples of 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-, _ (CH 2) p C0 NH (-Rl-OH)-(CH2) q-, _ (CH2) p-CONH (-Rl-COOH)-(CH2) q-, _ (CH2) p-C00_R1 (_S03H)-(CH2) q-, _ (CH2) p-COO-Rl (_OH)-(CH2) q-, _ (CH2) p_C00_Rl (-N + H3)-(CH2) q-,-(CH2) p-C00_Rl (-COOH)-( CH2) q-, _ (CH 2) p-Ar_ (CH 2) q-, _ (CH 2) p-(Ar-COO)-(CH 2) q-, _ (CH2) p- (Ar-S03H)-(CH2) q-, _ (CH2) p- (Ar-N + H3) _ (CH2) q-, _ (CH2) p- (Ar- OH)-(CH2) q-, _ (CH2) p- (Ar-COOH)-(CH2) q-, _ (CH2) p-C≡C_ (CH2) q-,-(CH2) p-C = C_ (CH2) q-, _ (CH2) p-NR- (CH2) q-,-(CH2) p-0_ (CH2) q-,-(CH2) pS- (CH2) q-,-(CH2) p-HN-C (= NH) -NH- (CH2) q-, _ (CH2) p-CO_Ar-NR- (CH2) q- and the like can be mentioned. More preferably, they are _ (CH2) p-CONH_ (CH2) q-,-(CH2) p_COO- (CH2) q-.

(ii) It is preferable to use what is represented by the following general formula (III).

-X4- (CHR3) r-X5- (III)

Here, X4 and X5 are each independently -NHCOO-, -CONH-, -COO_, -SO NH-

2

-HN_C (= NH) -NH-, -CH NH-, _CH NR-, _〇-, _S-, _NR-, _CH2 CH-, _C≡C_

twenty two

It is one kind of functional group selected from the group consisting of -Ar-and _CO-Ar_NR-, preferably -CCNH- and -COO-, -C〇0- and -CO〇-, -COO It is a combination of-and-NR-etc. 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. In addition, 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. In addition, it is possible to use a peptide linker which also forms an amino acid or an amino acid power of 2 to 20 at the junction. As amino acids, natural or synthetic amino acids can be used. Here, 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〇) — OH. Here, 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. Further, 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.

[0041] More preferred amino acids for use in the linking part of the present invention are 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.

[0042] As 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)-. Here, 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. As a specific example, Ala_Ser, Glu_Ala, Glu-Ala-Leu, Gly_Pro, Gly-Pro-Asn, lie-Val, Ile-Va 卜 Met, etc. can be used.

In addition, it is possible to use 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. For example, 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. For example, 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. As the activated esterified carbonyl group, N-hydroxy-succinimide ester or maleimide ester can be used. Preferably, N-hydroxy-succinimide ester is used. By using 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. Further, as shown in the reaction formula II of Scheme 1, a triazine derivative can also be used for the activated esterified carbonyl group. In addition, as the carbidoimide group, carbimidoimide reagents such as Ν, Ν'-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). In addition, 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). Here, 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. In order to introduce a sulfonyl group into the active ester, for example, the method of reaction formula V can be used.

[Formula 3]

R *

Bye-N = C = N-R _s H

E

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.

In the linking part containing two or more types of functional groups, 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. In addition, by introducing 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. For example, 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.

Specific examples of the organic EL dye include, as polycyclic aromatic compounds, rubrene, anthracene, tetracene, pyrene, perylene, thalicene, decacyclene, coronene, tetraphenyl butadiene, tetraphenynorecyclobutadiene, pentaphenynore Force S to mention cyclobutadiene. Examples of cyclopentadiene derivatives include 1,2,3,4-tetraphenylidene 1,3 cyclopentadiene, 1,2,3,4,5 pentaphenynore 1,3 cyclopentadiene. As the 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 It can be mentioned. Examples of coumarin derivatives include coumarin 1, coumarin 6, coumarin 7, and coumarin 30. Examples of 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. Examples of quinacdoline derivatives include quinacdoline and its derivatives. Examples of 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. For 5-membered ring compounds, 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.

The condensed polycyclic compound described below is used as an anion fluorescent dye by binding to an anion group via the above-mentioned linking part. In particular, condensed polycyclic compounds in which an anionic group is directly bonded can be used as an anionic fluorescent dye itself. In addition, the following fused polycyclic compounds can be bound to a covalent bond group directly or via a linkage to be used as a second fluorescent dye.

The following 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).

Hereinafter, specific examples of the fused polycyclic compound will be described.

(Monoazole derivative 1)

[Formula 4]

Here, in the formula, 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 Γ, CF 2 SO— , BF-,

Indicates PF—. The same applies to the following general formulas unless otherwise stated.

(Monoazole derivative 2)

[Chem. 5]

Here, in the formula, 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. In addition, n is an integer of 1 or more, preferably:! To 5, and the same applies to the following general formulae.

(Diazole derivative 1)

[Chemical 6]

(Diazole derivative 2) [Formula 7]

Here, in the formula, R, R, R and R each independently represent a hydrogen atom, a halogen atom or a hydride

1 2 3 4

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, R may be the same or different. R, R is

It is preferable to use an aromatic hydrocarbon group which may have 1 2 3 4 6 7 2 3 substituents. Further, 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.

(Diazole derivative 4)

[Chem. 9]

(Diazole derivative 5)

[Chemical 10]

(Diazole derivative 7) [Chemical formula 12]

(Diazole derivative 8) [Chemical formula 13-1]

[Formula 13-2]

Here, in the formula, R 1 and R 2 each represent a hydrogen atom, a halogen atom, a hydroxyl group,

10 11

It is an aromatic hydrocarbon group which may have a substituent such as an aromatic group, a cyano group or a sulfonyl group, or an aromatic group containing a hydrocarbon group, a heterocyclic group or a hetero atom in the ring. R

1

, R may be the same or different. And R 1 is an orephine group which may have a substituent

0 11 12

Or paraffin group, n is an integer of 1 to 3, preferably 1. The same applies to the following general formulas unless otherwise stated.

(Diazole derivative 9)

[Chemical 14-1]

[z-m ^ [goo]

TS .SlC / 900Zdf / X3d 82 6ΖΪ8Ϊ0 / 800Ζ OAV

[0065] 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.

[Formula 15]

[0067] After the synthesis of the carboxylic acid derivative, oxazolopyridine derivative is used, for example, by using Ν, ジ '-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 ヒドロキシ -hydroxy-succinimide ester.

[Chem. 16]

bcneme 1

(Triazole Derivative 1)

[Chemical formula 17]

(Triasole derivative 2)

[Equation 18]

(Triazole derivative 3)

[Chem. 19]

(Triazole derivative 4) [Chemical formula 20]

As a 5-membered ring compound, the following derivatives containing a thiophen group can also be used. (Thiophen derivative 1)

[Chemical formula 21]

(Thiophen derivative 2) [Formula 22]

(Thiophen derivative 3)

In the case of thiophen derivatives, 2,3,4,5-tetraphenyldithiophene derivatives represented by the following general formula can also be used.

[Formula 23]

And R 2, R 3 and R 4 are each independently a hydrogen atom, linear, branched or cyclic

12 13

Represents a substituted or unsubstituted aryl group or a substituted or unsubstituted aryl group; 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. Also, Y and Y are

12 hydrogen atom, halogen atom, linear, branched or cyclic alkyl group, linear, branched or cyclic alkoxy group, substituted or unsubstituted aryl group, substituted or unsubstituted aryl group, substituted or unsubstituted group It represents a substituted amino group.

[0076] (Thiophen derivative 4)

In addition, 2,3,4,5-tetraphenyldithiophene derivatives represented by the following general formula can also be used.

[Formula 24]

Here, in the formula, Ar to Ar each independently represent a substituted or unsubstituted aryl group,

1 6

Furthermore, Ar and Ar, Ar and Ar, and Ar and Ar together with the nitrogen atom to which they are bound

1 2 3 4 5 6

It may form a ring.

Alternatively, imidazole may be used as the 5-membered ring compound, and an imidazole derivative represented by the following general formula may be used.

(Imidazole Derivative 1)

[Formula 25]

(Imidazole Derivative 2)

[Chemical formula 26]

Imidanol derivatives 3

[Formula 27]

(Imidazole derivative 5)

[Chemical 28-1]

[Formula 28-2]

Here, the imidazole skeleton is plural at any position on the central benzene ring R 1, R 2, R 3, R 4

8 9 10 11

Good, even if the units are combined. Also, R 2 is an orephine group which may have a substituent or

12

Paraffin group, n is an integer of 1 to 3, preferably 1.

(Force rubazole derivative)

In addition, force rubazole derivatives represented by the following general formula can also be used. [Chem. 29]

Further, it is also possible to use 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. Although not particularly limited, for example, an azole derivative represented by the following general formula can be used.

[Chemical Formula 30]

Here, in the formula, R, R and R each independently represent a hydrogen atom, a halogen atom or a hydroxyl group

1 4 5

It represents an aromatic hydrocarbon group which may have a substituent such as a cinole group, an amino group, a cyano group or a sulfonyl group, an aromatic group containing a hydrocarbon group or a heterocyclic group or a hetero atom in the ring. R, R and R may be the same or different.

1 4 5

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.

[Chemical Formula 31]

(3)

[Formula 32]

Further, among the diazole derivatives and the imidazole derivatives described above, 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.

[Formula 33]

) p-X3- (CHR ") q-Z

[Chemical Formula 34]

[0093]-(CHR ') p-X3- (CHR ") q- represents the above-mentioned linkage, and Z represents an anion group.

Here, as 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.

Alternatively, 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.

[0095] 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. The same applies to peptides, antibodies, and sugars having an amino group. For example, 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. Can be identified. Here, as the separation means, ion exchange column HPLC, reverse phase partition HPLC, gel filtration HPLC, or electrophoresis can be used. For electrophoresis, primary and secondary electrophoresis can be used, and after electrophoresis, the gel can be dried and quantified.

Further, 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. Here, the covalent fluorochrome is one that does not change its fluorescence wavelength even when the protein is labeled accordingly. First, label with a covalent fluorescent dye. Thereafter, electrophoresis is performed to perform separation. Furthermore after migration When the gel substrate is labeled with an anionic fluorescent dye, 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. At this time, since 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.

Further, 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. After spotting, the protein can be immobilized on the substrate by leaving it at a predetermined temperature, and incubation can be carried out if necessary. In addition, when capturing a protein labeled with this fluorescent dye on a protein chip, since it emits stable fluorescence under any circumstances, it may not be handled as nervous as in the past. In addition, since fluorescence quenching does not occur even in the dry state, stable observation is possible even in the dry state. In addition, 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.

Example

Hereinafter, the present invention will be described in more detail using examples.

Synthesis example 1.

An example of synthesis of an active ester type anionic fluorescent dye is shown.

(1) Synthesis of chromophore (3)

The chromophore (3) was synthesized according to scheme 2 below.

[Formula 35]

Sch me 2 [0100] In a 50 mL three-necked flask, 1.0 g (0.0026 moL) of oxadiazo-opening pyridinecarboxylic acid (1) and 0.30 g (0.0026 moL) of N-hydroxysuccinimide (2) were dissolved in 20 mL of DMF. To this was added 0.54 g (0.0026 moU of N, Ν'-dicyclohexylcarbodiimide dropwise over 30 minutes. After dropwise addition, stirring was carried out at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography ( The resultant product was isolated and purified in a form of black) to obtain 0.76 g of Oxadiazo oral pyridine active ester (3) in a yield of 62%.

(2) Introduction of sulfonyl group

The active ester (3) was reacted with taurine in DMF to induce sulfonation (4) (scheme 3).

[Chemical 36]

Scheme 3

Synthesis Example 2.

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.

[Formula 37]

scheme 4.

The following shows a synthesis procedure of only a portion different from Synthesis Example 1.

(1) Synthesis of carboxylic acid (5)

In a 50 mL three-necked flask, 100 mg (0.21 mmol) of Oxadiazo-open pyridine active ester (3) and 39 mg (0.23 mmol) of cystic acid were dissolved in 20 mL of DMF. Then, it stirred at room temperature for 12 hours. After completion of the reaction, DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform: methanol = 7: 3) to obtain 98 mg (yield 88%) of a carboxylic acid (5).

(2) Synthesis of Active Ester Form (6)

Then, 80 mg (0.15 mmol) of oxadiazo-open pyridinecarboxylic acid (5) and 19 mg (0.17 mmol) of N-hydroxysuccinimide were dissolved in 20 mL of DMF in a 50 mL three-necked flask. To this was added dropwise 35 mg (0.17 mmol) of N, Ν'-dicyclohexylcarbodiimide dissolved in 5 mL of DMF over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. The DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain 73 mg (yield 78%) of an active ester (6).

Synthesis Example 3.

Synthesis example 4.

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.

[Formula 38]

scheme 5.

The following shows the synthesis procedure of only the portion different from Synthesis Example 1.

(1) Synthesis of carboxylic acid (7)

In a 50 mL three-necked flask, 100 mg (0.21 mmol) of Oxadiazo-opened pyridine active ester (3) and 26 mg (0.25 mmol) of serine were dissolved in 20 mL of DMF. Then, it stirred at room temperature for 12 hours. After completion of the reaction, DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography mono (chromatography-methanol = 7: 3) to obtain 79 mg (yield 81%) of carboxylic acid (7).

Example 1

(experimental method)

After dissolving the sulfonyl form (4) of the active ester in pure water and mixing 0.1 M HEPES Buffer (2- [4- (2-Hydroxyethyl) -l-piperazinyl] ethanesulfonic acid) (H 7.3), BSA (Bovine) Serum Albumin was added, and in order to observe changes in color tone in the spectrum, the UV spectrum of the sulfonyl compound was measured, and then the fluorescence spectrum was measured to observe the interaction between the sulfonyl compound (4) and BSA. The UV spectrum was measured by preparing fluorescence reagents of 13 μΜ and 26 / iΜ in 2000 cells. In addition, the fluorescence spectrum was measured by preparing a solution of 300 μg / mL in a cell and adding BSA to a predetermined concentration.

(Result)

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. Next, the fluorescence spectrum was measured using 397 nm as the excitation wavelength. The results are shown in Figure 3. Here, BSA is added to a concentration of 1.6 μM. The fluorescence spectrum showed a blue shift of 18 nm by addition of BSA, and the fluorescence intensity increased about 5 times. This is considered to be due to the fact that the sulfonyl form (4) is electrostatically bonded to the amino group of BSA and the blue shift is observed due to the interaction between the BSA surface and the sulfonyl form. In addition, since the sulfonyl compound (4) is located in the hydrophobic field of BSA, it is considered that the interaction with water is eliminated to some extent and the fluorescence intensity is increased.

Next, 2000 μl of a 26 μl aqueous solution of a sulfonyl compound was prepared, and BSA 0 to 15 Μ was added thereto in eight divided portions. The UV spectrum at that time is shown in FIG. While the addition of BSA shows a light color effect, the peak wavelength is red-shifted (15 nm red shift at 7 nm). From this, the sulfonyl form (4) is considered to be located in the deep part (hydrophobic field) of BSA.

Note that, for comparison, a change in peak wavelength of the force BSA and an increase in fluorescence intensity were not observed using the active ester body (3) instead of the sulfonyl body (4). 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. On the other hand, since 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

(experimental method)

Next, 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 μΜ was prepared in a fluorescent cell in 2000 μl, and 0 to 232 μΜ of insulin was added thereto in six divided doses.

(Result)

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 Δ Int. (Value obtained by subtracting the fluorescence intensity of only the sulfonyl form) in the length is shown in FIG. As the insulin concentration increased, the peak wavelength FLmax linearly shifted to a lower wavelength. In addition, as the insulin concentration increased, the result was obtained that the fluorescence intensity Δ Int. Increased linearly. From this, it is understood that the concentration of insulin can be calculated from the change in fluorescence intensity or from the change in peak wavelength.

Example 3

(experimental method)

Next, the procedure was carried out in the same manner as in Example 1 except that lysozyme was used as the protein. A 26.7 μΜ of sulfonyl group (4) was prepared in a fluorescent cell to 2000 μl, and 0 to 4 6.6 μΜ of lysozyme was added thereto in four portions.

(Result)

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.

Example 4

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.

Example 5

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.

[0120] The fluorescence specknore is shifted by 19 nm by the addition of BSA and the fluorescence intensity is about 4.5. It has doubled.

Comparative Example 1.

The procedure was carried out in the same manner as in Example 1 except that conventionally used methyl orange was used. Even when methyl orange was added, neither increase in fluorescence intensity nor blue shift of peak wavelength was observed.

As described above, according to 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

Claims

The scope of the claims

[1] A method for detecting a protein labeled with a fluorescent dye, the method comprising:

A method for detecting a protein, which comprises detecting a protein by measuring fluorescence based on a second fluorescence wavelength which is shorter than a first fluorescence wavelength observed in a free state and is observed in a state of being bound to a protein.

[2] The above detection method is a detection method in which the proteins in the sample are subjected to separation means, and the separated fractions are subjected to mass analysis,

The method according to claim 1, wherein the protein is labeled with a first fluorescent dye that generates the second fluorescence wavelength before the protein is subjected to separation means.

[3] Prior to, or simultaneously with, labeling 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. The detection method according to claim 2, which is then subjected to separation means.

[4] The above-mentioned protein and the first fluorescent dye that generates the second fluorescence wavelength are reacted in a solution, the solution is spotted on the measurement substrate, and the second fluorescence wavelength from the measurement substrate is used. The detection method according to claim 1, wherein the fluorescence image is measured on the basis.

[5] Prior to or at the same time as the reaction of the above-mentioned protein with the above-mentioned first fluorescent dye in a solution, a second fluorescent dye in which the first fluorescence wavelength is not shifted to a short wavelength while bound to the protein The detection method according to claim 4, wherein the protein is reacted in solution.

[6] The detection method according to any one of claims 2 to 5, wherein the first fluorescent dye has an anionic group that binds to a protein.

[7] The detection method according to claim 4 or 5, wherein the second fluorescent dye has a covalent bond group that binds to a protein.

[8] A fluorescent dye for protein detection comprising an anionic fluorescent dye comprising an organic EL dye to which an anionic group binding to a protein is directly or via a linking moiety.

[9] The fluorescent dye according to claim 8, wherein the anion group is any of a carboxyl group, a sulfonyl group, a sulfate base, a phosphate group and a combination thereof.

[10] The above linked part is -CH-,-NHCOO-,-CONH-,-CH NH-,-CH NR-,-COO-,-S

2 2 2

0 NH-, _HN-C (= NH) -NH-, _0 _, -S-, -NR-(R is an alkyl group), _ (CH -CH _0)-(

2 2 2 η The fluorescent dye according to claim 8, comprising at least one functional group selected from the group consisting of n is an integer of 1 to 10), _CH = CH 2-, ≡C≡C 2-, ArAr- and COCO_Ar_NR-.

[11] The above-mentioned organic EL dye is a condensed polycyclic compound consisting 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. Fluorescent dye.

12. The fluorescent dye according to claim 11, wherein the fused polycyclic compound is an azole derivative represented by any one of the following general formulas (1), (2) or (3).

(3)

(Wherein, R, R, R and R are each independently a hydrogen atom, a halogen atom, an alkyl group,

1 2 3 4

Aromatic hydrocarbon which may have a substituent such as alkenyl group, alkynyl group, amino group, alkoxy group, hydroxyl group, cyano group, amino group, sulfonyl group, aromatic hydrocarbon group, heterocyclic group, etc. Group or a hydrocarbon group or a heterocyclic group, X is a substituent, X is a nitrogen atom or a sulfur atom, or an oxygen atom or a selenium atom, or a boron atom, and R 'is a aromatic ring Aliphatic hydrocarbon group or aromatic hydrocarbon group such as alkyl group or alkenyl group which may contain, An-is a halide ion such as CI-, Br-or I-, CF 2 SO-, BF-, PF- Indicates —. )

[13] The above R and R each independently represent a thiophen derivative, a furan derivative or a pyrrole derivative

twenty three

The fluorescent dye according to claim 12, which is one selected from the group consisting of a conductor, an imidazole derivative, an oxazole derivative, a thiazole derivative, a pyrazole derivative and a pyridine derivative.

[14] The fluorescent color according to claim 12, wherein R and R are each a phenyl group having a sulfonyl group.

twenty three

Raw.

[15] The fluorescent dye according to claim 11, wherein the fused polycyclic compound is an idazole derivative represented by the following general formula (4), (5), (6), (7) or (8).

(Wherein, R, R, R, R and R are each independently a hydrogen atom, a halogen atom, an alkyl

1 2 3 4 5

Aromatic hydrocarbon which may have a substituent such as alkenyl group, alkynyl group, amino group, alkoxy group, hydroxyl group, cyano group, amino group, sulfonyl group, aromatic hydrocarbon group and heterocyclic group Group, a hydrocarbon group or a heterocyclic group, and R, R, R, R, R are the same

1 RR 2 RR 4 may be an alkyl group or an alkenyl group which may contain an aromatic ring. Aliphatic hydrocarbon group or aromatic hydrocarbon group such as alkyl group, An— represents Cl—, Br ′ ′, halogenide ion such as Γ, CF 2 SO 4 −, BF −, PF 4 −).

3 3 4 6

[16] The above R and R each independently represent a thiophen derivative, a furan derivative or a pyrrole derivative

twenty three

The fluorescent dye according to claim 15, which is one selected from the group consisting of a conductor, an imidazole derivative, an oxazole derivative, a thiazole derivative, a pyrazole derivative and a pyridine derivative.

[17] The fluorescent color according to claim 15, wherein R and R are each a phenyl group having a sulfonyl group.

twenty three

Raw.