JP2006234772A - Method of detecting protein and fluorescent dye used for same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of detecting protein capable of highly sensitive detection and easy operation and a fluorescent dye to be used for the same. <P>SOLUTION: The method of detecting protein labeled with the fluorescent dye in which the protein is detected by measuring the fluorescent light based on the second fluorescent wave length observed in the bonded state with the protein which is shorter than the first fluorescent wave length observed in the free state. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

  In recent years, the entire human genome has been clarified, and post-genomic research for gene therapy and gene diagnosis has been actively conducted. In particular, in proteome analysis that uses genome information to comprehensively and systematically analyze the properties and expression dynamics of all proteins contained in a single organism or cell, the amount of trace protein produced by gene expression is increased. Sensitivity needs to be detected and identified. In addition, since diseases such as infections caused by cancer and viruses each produce special proteins, these special proteins can be handled as disease markers and applied to the diagnosis and treatment of diseases. Sensitive proteins must be detected and identified with high sensitivity.

  As a high-sensitivity analysis method for proteins, for example, a sample protein is labeled with a fluorescent dye (Non-patent Document 1), separated by electrophoresis, and then fractionated using a mass spectrometer such as MALDI-TOF MS. A method for identifying the protein by measuring the molecular weight of the protein and performing a database search is used. Blotting methods such as Western blotting. Furthermore, protein chips using DNA chip technology are used for expression analysis and analysis of protein interactions (for example, Non-Patent Document 2). A protein chip uses a protein labeled with a fluorescent dye, and is used for protein expression analysis by attaching hydrophobic substances, ion exchangers, metal ions, etc., or by attaching antibodies etc. Used for analysis. By using a protein chip, simultaneous analysis of expression kinetics and interaction of various types of proteins can be performed easily and quickly.

In addition, the protein is divided by electrophoresis after the protein is placed on a gel and then moved on the chromatograph by passing electricity through electrodes set at both ends, and divided according to the difference in molecular weight and the like. Thereafter, fluorescent labeling is performed by immersing the gel in a fluorescent dye solution (for example, Patent Documents 1 and 2). However, since the fluorescence quenching occurs when the gel is dried, the quantification is performed in a wet state. However, the accurate quantification cannot be performed due to the thickness of the swollen gel.
Special table 2003-53946 gazette JP 2004-317297 A Michael Brinkley, Bioconjugate Chem., 1992, 3, 2-13 Paul Cutler, Proteomics, 2003, 3, 3-18

  However, when measuring changes in fluorescence intensity, there is a problem that the increase in fluorescence intensity is not reproducible, and if the fluorescence intensity is weak and the difference from the free state is small, it is difficult to detect proteins with high sensitivity. is there. When trying to measure a weak fluorescence intensity, the intensity of the excitation light must be increased, and there is a problem that the light source becomes larger or the sample becomes damaged. Furthermore, when measuring using a protein chip, there is also 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 small amount sample such as ml to μl, the sample is easy to dry, which is a very big problem. In addition, depending on the fluorescent dye, the temperature stability is low, and if the measurement takes time, a problem may occur in the quantitativeness.

  Therefore, the present invention has been made to solve the above problems, and to provide a protein detection method capable of highly sensitive detection and easy operation, and a fluorescent dye used therefor.

As a result of diligent efforts to solve the above-mentioned problems, the present inventor has found that a completely new protein detection method different from the detection method based on a change in fluorescence intensity in the past is possible, and has completed the present invention. .
That is, the protein detection method of the present invention is a protein detection method for detecting a protein labeled with a fluorescent dye, which is shorter than the first fluorescence wavelength observed in a free state and bound to the protein. Protein is detected by measuring fluorescence based on the second fluorescence wavelength observed in the state.

  According to the present invention, even when the fluorescence intensity in the bound state is weak, the second fluorescence is shorter than the first fluorescence wavelength observed in the free state and is observed in the state bound to the protein. Since the fluorescence of the wavelength is measured, for example, the second fluorescence wavelength value and the fluorescence intensity thereof may be measured, detection with higher sensitivity is possible as compared with the conventional detection method. Further, since the weak fluorescence intensity is measured, it is not necessary to increase the intensity of the excitation light. Further, even an unskilled inspector can easily make a determination. In addition, when the protein is added stepwise, the second fluorescence wavelength shifts to a short wavelength with an increase in the amount of protein binding, so the relationship between the shift value from the first fluorescence wavelength and the amount of protein. Proteins can also be quantified.

  Moreover, as one aspect of the detection method of the present invention, in the case of a detection method in which a protein in a sample is subjected to a separation means and the separated fraction is subjected to mass spectrometry, the second fluorescence wavelength is applied before the protein is subjected to the separation means. Proteins can be labeled with a first fluorescent dye that generates. Further, prior to or simultaneously with the labeling of the protein with the first fluorescent dye, the protein is labeled with the second fluorescent dye that does not shift to the short wavelength while being bound to the protein, and is then used as a separation means. Can also be provided.

  As another aspect of the detection method of the present invention, the protein and the first fluorescent dye that generates the second fluorescence wavelength are reacted in a solution, and the solution is spotted on a measurement substrate. A fluorescence image based on the second fluorescence wavelength from can be measured. Further, prior to or simultaneously with reacting the protein and the first fluorescent dye in the solution, the second fluorescent dye that does not shift to the short wavelength when the protein is bound to the protein is added to the protein and the solution. It can also be reacted.

  The first fluorescent dye preferably has an anionic group that electrostatically binds to the protein. The second fluorescent dye preferably has a covalent bond group that is covalently bonded to the protein. Examples of the covalent bond include an amide bond, an imide bond, a urethane bond, an ester bond, and a guanidine bond. The covalent bond group includes an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, and a carbodiimide group. And any one of active esterified carbonyl groups, more preferably any one of triazine group, carbodiimide group, and active esterified carbonyl group can be used.

  In the detection method of the present invention, an anionic fluorescent dye in which an anionic group that binds to a protein is bonded to an organic EL dye directly or via a linking moiety can be used. Here, as the anionic group, any of a carboxyl group, a sulfonyl group, a sulfate group, a phosphate group, and a combination thereof can be used. Further, the organic EL dye may be a compound containing a 5-membered ring compound having a conjugated system, and the 5-membered ring compound may contain one or more hetero atoms, selenium atoms or boron atoms. The organic EL dye may be a condensed polycyclic compound composed of the 5-membered ring compound and a 6-membered ring compound having a conjugated system, and the 5-membered ring compound is an azole derivative or an imidazole derivative. Is preferred.

  Since the anionic fluorescent dye of the present invention is not quenched even when the sample is dried, highly sensitive detection is possible even in a dry state. For example, an anionic fluorescent dye and a protein can be reacted in a solution, the solution can be spotted on a protein chip substrate, and imaged with an image scanner or the like for detection. Further, the anionic fluorescent dye used in the present invention can be used in a recently developed dry assay, and is a reagent that does not depend on the method of use. In addition, it is stable against heat and can withstand long-term storage at room temperature, so it is easy to handle.

  The reason why the above anionic fluorescent dye can be suitably used in the detection method of the present invention is as follows. According to the knowledge of the present inventor, even when a conventional fluorescent dye having an anionic group, such as methyl orange or orange G, was used, no shift in the fluorescence wavelength was observed. On the other hand, when the anionic fluorescent dye was used, the fluorescence wavelength was shifted to a shorter wavelength (blue shifted), and the fluorescence intensity was increased. Moreover, the absorption wavelength shifted to a longer wavelength (red shift), and the intensity decreased. Anionic fluorescent dyes electrostatically bind to positively charged groups of proteins, such as amino groups. However, when organic EL dyes are used in the color-developing part, organic EL dyes can easily release energy due to interactions with adjacent proteins. It is thought that. In addition, in the case of not having a bonding part composed of an anionic group, neither a blue shift of fluorescence wavelength nor an increase in fluorescence intensity was observed even when a coloring part composed of an organic EL dye was used. In the case of using a fluorescent dye that does not have a binding portion composed of an anionic group, it is considered that the fluorescent dye reacts only with a functional group on the surface of the protein to form a covalent bond. In contrast, fluorescent dyes having an anionic group binding moiety have a low molecular weight and low steric hindrance, so they bind not only to the amino group on the protein surface but also to the deep amino group by electrostatic bonding. It is also located in the deep part of protein (hydrophobic field), which is considered to cause a blue shift and an increase in fluorescence intensity.

The detection method of the present invention has the following effects.
That is, by measuring the second fluorescence wavelength value and its fluorescence intensity that are shorter than the first fluorescence wavelength observed in the free state and are bound to the protein, High sensitivity detection is possible. In addition, since the second fluorescence wavelength shifts to a shorter wavelength as the amount of protein binding increases, the protein is added stepwise to the fluorescent dye, and the protein is calculated from the fluorescence wavelength shift value from the first fluorescence wavelength. Can also be quantified. In addition, since the sample can emit fluorescence even in a dry state, protein detection can be performed simply and with high accuracy. Furthermore, the fluorescent dye used in the present invention has higher thermal stability than Cy3, Cy5 and Alexa dyes, and no fading is observed, so it is easy to handle and is cheaper than Cy3 and Cy5, so it is lower. Protein can be detected at low cost. In addition, since the fluorescent dye of the present invention shows the maximum quantum yield when dried, when the protein is divided by electrophoresis, the gel can be dried to reduce the thickness as much as possible. Accurate quantification can be performed.

Hereinafter, embodiments of the present invention will be described in detail.
The detection method of the present invention is a protein detection method for detecting a protein labeled with a fluorescent dye, which is shorter than the first fluorescence wavelength observed in a free state and is observed in a state bound to the protein. The protein is detected by measuring fluorescence based on the second fluorescence wavelength.
The fluorescence wavelength of the anionic fluorescent dye used in the present invention gradually shifts from the fluorescence wavelength in the free state to a shorter wavelength as the amount of binding to the protein increases, and the fluorescence wavelength does not further shift, and the fluorescence intensity also increases. It reaches saturation without increasing. Therefore, the presence or absence of protein in the sample can be confirmed and quantified using the fluorescence wavelength value in the intermediate state from the free state to the saturated state and the fluorescence intensity thereof. Moreover, protein can also be quantified from the relationship between the amount of added protein and the shift value from the fluorescence wavelength of a free state by adding protein to an anionic fluorescent dye in steps.

  The sample which is the subject of the present invention is not particularly limited as long as it contains a protein. Collagen which is a simple protein can also be labeled, and can be labeled after electrophoresis. Further, it can be used for detection of sugar chains and peptides having amino groups. In addition, antibody labeling can be handled in the same manner as before, and can be used for antigen-antibody reactions. For example, the antibody reaction can be observed by any technique including an antibody antigen chip and an evanescent wave immunoassay.

The detection method of the present invention can be applied to a sample in a solution state or a sample in a solid state.
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 a predetermined concentration of an anionic fluorescent dye is dissolved, and the fluorescence spectrum of the solution is measured using a fluorescence spectrophotometer or the like. The sample concentration is detected from the presence or absence of the fluorescence wavelength and the fluorescence intensity or the shift value of the fluorescence wavelength. Alternatively, when the second fluorescence wavelength is known in advance, the measurement wavelength can be fixed to the second fluorescence wavelength, and the sample concentration can be detected from the fluorescence intensity. Moreover, the method of adding the solution which melt | dissolved the anionic fluorescent dye to the sample solution containing protein can also be used.

  In the case of a solid state sample, for example, the following method can be used. A protein and an anionic fluorescent dye are reacted in a solution state, and then the solution is spotted and spotted on a measurement substrate such as a protein chip, and the chip is imaged using an image scanner or the like to detect a sample concentration. . Alternatively, an anionic fluorescent dye can be fixed beforehand on the sample, and then a sample solution containing the protein can be spotted on the measurement substrate.

  Here, the first fluorescence wavelength observed in a free state refers to a fluorescence wavelength observed when an anionic fluorescent dye is present alone in a solution or in a solid. On the other hand, the 2nd fluorescence wavelength observed in the state couple | bonded with protein is a fluorescence wavelength based on the anionic fluorescent dye couple | bonded with protein, and is shorter than a 1st 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.

  Anionic fluorescent dyes used in the detection method of the present invention include those in which an anionic group that binds to a protein is directly bonded to the organic EL dye, and those in which an anionic group is bonded to the organic EL dye through a linking moiety. It is. Here, as the anionic group, any of a carboxyl group, a sulfonyl group, a sulfate group, a phosphate group, and a combination thereof that electrostatically binds to a positively charged group such as an amino group of a protein can be used. It is preferable to use a group.

A compound represented by the general formula A ₁ -A ₂ (1) can be used for the connecting portion. Here, A ₁ is a first linking group that binds to the organic EL dye (color-developing part), and A ₂ is a second linking group that binds to an anionic group and also serves as a spacer group. The first linking group may be any one selected from the group consisting of a substituted or unsubstituted alkyl group, an ether group, a thioether group, a substituted or unsubstituted imino group, an amide group, and an ester group. preferable. The second linking group is preferably an alkylene group or an alkylene group containing a hetero atom in the main chain. As the alkylene group, a methylene group, an ethylene group, or a trimethylene group is preferably used. As the alkylene group containing a hetero atom in the main chain, an ethylene oxide group is preferably used.

Further, as the second linking group, any one of a triazine group, a carbodiimide group and an active esterified carbonyl group having an anionic group as a substituent, more preferably an active esterified carbonyl group can be used.
As the active esterified carbonyl group, N-hydroxy-succinimide ester or maleimide ester can be used, but N-hydroxy-succinimide ester is preferably used. By using N-hydroxy-succinimide, as shown in Formula I of Scheme 1 below, by using DCC as a condensing agent, an EL dye and a target molecule can be bonded by an amide bond via an N-hydroxy-succinimide ester. Join. Further, as shown in Formula II of Scheme 1, a triazine derivative can also be used for the carbonyl group subjected to active esterification. As the carbodiimide group, a carbodiimide reagent such as N, N′-dicyclohexylcarbodiimide (DCC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide can be used. The EL dye and the target molecule can be bound by an amide bond via a carbodiimide body (formula III). In addition, EL dyes in which a carbodiimide group or a triazine group is previously introduced into the molecule can be directly bonded to an amino group or an imino group in the biomolecule (formula IV). Here, R represents an aromatic hydrocarbon group, a hydrocarbon group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, which contains an anionic group as a substituent. In order to introduce a sulfonyl group into the active ester, for example, the method of formula V can be used.

  Here, the connecting portion ensures the connection between the coloring portion and the anionic group by the first binding group and the second binding group. Further, the presence of the second linking group ensures the physical distance between the coloring portion and the anionic group, and ensures the freedom of selection of the molecular skeleton of the coloring portion and the anionic group, while the anionic group is a protein. It has the effect of facilitating the bonding with the positively charged group in the deep part. Thereby, it becomes possible to selectively label only a specific protein. In addition, when a heteroatom such as a nitrogen atom is used for the first bonding group, the entire molecule can have a more rigid structure, and thus stacking of the coloring portions can be suppressed. In addition, by introducing oxygen atoms or the like, a flexible molecular structure can be obtained, and the stacking strength can be controlled.

  The organic EL dye used in the present invention is sandwiched in a solid state between a pair of an anode and a cathode, and can emit light by energy when holes injected from the anode and electrons injected from the cathode are recombined. If it is a pigment | dye, it will not specifically limit. For example, polycyclic aromatic compounds such as tetraphenylbutadiene and perylene, cyclopentadiene derivatives, oxadiazole derivatives, coumarin derivatives, distyrylpyrazine derivatives, acridone derivatives, quinacdrine derivatives, stilbene derivatives, phenothiazine derivatives, pyrazinopyridine derivatives, azole derivatives, Imidazole derivatives, carbazole derivatives, thiophene derivatives, and the like can be used.

  Specific examples of the organic EL dye include rubrene, anthracene, tetracene, pyrene, perylene, chrysene, decacyclene, coronene, tetraphenylbutadiene, tetraphenylcyclobutadiene, and pentaphenylcyclobutadiene as polycyclic aromatic compounds. it can. Examples of the cyclopentadiene derivative include 1,2,3,4-tetraphenyl-1,3-cyclopentadiene and 1,2,3,4,5-pentaphenyl-1,3-cyclopentadiene. As the oxadiazole derivatives, 2- (4′-t-butylphenyl) -5- (4′-biphenyl) 1,3,4-oxadiazole, 2,5-bis (4-diethylaminophenyl) 1, Mention may be made of 3,4-oxadiazole. Examples of coumarin derivatives include coumarin 1, coumarin 6, coumarin 7, and coumarin 30. Examples of the distyrylpyrazine derivatives include 2,5-bis- (2- (4-biphenyl) ethenyl) pyrazine, 2,5-bis- (4-ethylsteryl) pyrazine, 2,9-bis- (4-methoxysteryl). ) Pyrazine can be mentioned. Examples of acridone derivatives include acridone and its derivatives. Examples of quinacrine derivatives include quinacrine and derivatives thereof. Examples of the stilbene derivative include 1,1,4,4-tetraphenyl-1,3-butadiene and 4,4'-bis (2,2-diphenylvinyl) biphenyl. As the azole derivative, imidazole derivative, carbazole derivative, and thiophene derivative, those described in the general formula in this specification can be used.

  A preferable organic EL dye used in the detection method of the present invention is a compound containing a 5-membered ring compound having a conjugated system, wherein the 5-membered ring compound contains one or more heteroatoms, selenium atoms or boron atoms. Can be mentioned. More specifically, a monocyclic compound composed of a 5-membered ring compound having a conjugated system and a condensed polycyclic compound composed of a 6-membered ring compound having a conjugated system with the 5-membered ring compound can be exemplified. This is because even in the solid state, the quantum yield is large and strong fluorescence is exhibited. The 5-membered ring compound is preferably an azole derivative or an imidazole derivative. Furthermore, the azole derivative or imidazole derivative preferably has one or more quaternary ammonium groups. It is because water solubility can be improved.

  The condensed polycyclic compound described below can be used as an anionic fluorescent dye by binding to an anionic group via the above-mentioned linking part, but the condensed polycyclic compound to which an anionic group is directly bonded is It can be used as an anionic fluorescent dye. All of the following condensed polycyclic compounds can be suitably used in the detection method of the present invention, but are preferably a diazole derivative 3, an imidazole derivative 2, a thiadiazole derivative, a carbazole derivative, a thiazole derivative, and more preferably And an oxazolopyridine derivative (oxadiazolopyridine derivative).

ここで、式中、R₁、 R₂、 R₃、 R₄、 R₆、 R₇は、それぞれ独立に、水素原子、ハロゲン原子、ヒドロキシル基、シアノ基、あるいはスルホニル基などの置換基を有しても良い芳香族炭化水素基又は炭化水素基又は複素環基又はヘテロ原子を環内に含む芳香族基を示す。R₁、 R₂、 R₃、 R₄、 R₆、 R₇は同じでも異なっていてもよい。R'は芳香環を含んでも良いアルキル基又はアルケニル基等の脂肪族炭化水素基あるいは芳香族炭化水素基、An^-は、Cl^-、Br^-、I^-等のハロゲン化物イオン、CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-を示す。なお、以下の一般式においても、特に断らない限り同様である。 Below, the specific example of a condensed polycyclic compound is demonstrated.
(Monoazole derivative 1)

Here, in the formula, R ₁ , R ₂ , R ₃ , R ₄ , R ₆ , and R ₇ each independently have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, or a sulfonyl group. An aromatic group containing an aromatic hydrocarbon group, a hydrocarbon group, a heterocyclic group or a hetero atom in the ring. R ₁ , R ₂ , R ₃ , R ₄ , R ₆ and R ₇ 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 I ⁻ , CF ₃ SO _{3 ^{-, BF 4 -, PF 6}} - shows the. The same applies to the following general formulas unless otherwise specified.

ここで、式中、R₈、R₉は、それぞれ、水素原子、ハロゲン原子、ヒドロキシル基、シアノ基、スルホニル基などの置換基を有しても良い芳香族炭化水素基又は炭化水素基又は複素環基又はヘテロ原子を環内に含む芳香族基を示す。R₈、R₉は同じでも異なっていてもよい。なお、以下の一般式においても、特に断らない限り同様である。また、nは１以上の整数、好ましくは１〜５であり、以下の一般式中でも同様である。 (Monoazole derivative 2)

Here, in the formula, R ₈ and R ₉ are each an aromatic hydrocarbon group, a hydrocarbon group or a complex which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, or a sulfonyl group. An aromatic group containing a ring group or a hetero atom in the ring is shown. R ₈ and R ₉ may be the same or different. The same applies to the following general formulas unless otherwise specified. N is an integer of 1 or more, preferably 1 to 5, and the same applies to the following general formulas.

(Diazole derivative 1)

(Diazole derivative 2)

ここで、式中、R₁、 R₂、 R₃、 R₄は、それぞれ独立に、水素原子、ハロゲン原子、ヒドロキシル基、シアノ基、あるいはスルホニル基などの置換基を有しても良い芳香族炭化水素基又は炭化水素基又は複素環基又はヘテロ原子を環内に含む芳香族基を示す。R₁、 R₂、 R₃、 R₄、 R₆、 R₇は同じでも異なっていてもよい。R₂、 R₃は、置換基を有しても良い芳香族炭化水素基を用いることが好ましい。また、Xは、置換基を有しても良い窒素原子、硫黄原子、酸素原子、セレン原子又はボロン原子であり、特に断らない限り以下の一般式中でも同様である。 (Diazole derivative 3)

Here, in the formula, R ₁ , R ₂ , R ₃ , and R ₄ are each independently an aromatic group that may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, or a sulfonyl group. An aromatic group containing a hydrocarbon group, a hydrocarbon group, a heterocyclic group, or a hetero atom in the ring. R ₁ , R ₂ , R ₃ , R ₄ , R ₆ and R ₇ may be the same or different. R ₂ and R ₃ are preferably aromatic hydrocarbon groups which may have a substituent. X is a nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom which may have a substituent, and the same applies to the following general formulas unless otherwise specified.

(Diazole derivative 4)

ここで、N→Oは、窒素原子が酸素原子に配位結合している状態を示す。 (Diazole derivative 5)

Here, N → O indicates a state in which a nitrogen atom is coordinated to an oxygen atom.

(Diazole derivative 6)

(Diazole derivative 7)

(Diazole derivative 8)

ここで、式中、R₁₀、R₁₁は、それぞれ、水素原子、ハロゲン原子、ヒドロキシル基、シアノ基、あるいはスルホニル基などの置換基を有しても良い芳香族炭化水素基又は炭化水素基又は複素環基又はヘテロ原子を環内に含む芳香族基を示す。R₁₀、R₁₁は同じでも異なっていてもよい。また、R₁₂は、置換基を有してもよいオレフィン基又はパラフィン基であり、nは１から３の整数、好ましくは１である。なお、以下の一般式においても、特に断らない限り同様である。

Here, in the formula, R ₁₀ and R ₁₁ are each an aromatic hydrocarbon group or a hydrocarbon group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, or a sulfonyl group, or An aromatic group containing a heterocyclic group or a hetero atom in the ring is shown. R ₁₀ and R ₁₁ may be the same or different. R ₁₂ is an olefin group or a paraffin group which may have a substituent, and n is an integer of 1 to 3, preferably 1. The same applies to the following general formulas unless otherwise specified.

(Diazole derivative 9)

  Although it will not specifically limit if it is said diazole derivative, The oxadiazolo pyridine derivative represented by the following general formula can be used suitably.

  After synthesizing the carboxylic acid derivative, the oxazolopyridine derivative uses N, N′-dicyclohexylcarbodiimide (DCC) as a condensing agent by the reaction shown in the following scheme 2, for example, and includes an N-hydroxy-succinimide ester. It is preferable to use an ester derivative.

Scheme 1

Scheme 1

(Triazole derivative 1)

(Triazole derivative 2)

(Triazole derivative 3)

(Triazole derivative 4)

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

(Thiophene derivative 2)

ここで、式中、R₁₂,R₁₃,R₁₄はそれぞれ独立に、水素原子、直鎖、分岐または環状のアルキル基、置換または未置換のアリール基、あるいは置換または未置換のアラルキル基を表し、Ar₁およびAr₂は置換または未置換のアリール基を表し、さらに、Ar₁とAr₂は結合している窒素原子と共に含窒素複素環を形成してもよい。また、Y₁およびY₂は水素原子、ハロゲン原子、直鎖、分岐または環状のアルキル基、直鎖、分岐または環状のアルコキシ基、置換または未置換のアリール基、置換または未置換のアラルキル基、あるいは置換または未置換のアミノ基を表す。 (Thiophene derivative 3)
In the case of a thiophene derivative, it is a non-condensed compound, and a 2,3,4,5-tetraphenylthiophene derivative represented by the following general formula can also be used.

In the formula, each of R ₁₂ , R ₁₃ and R ₁₄ independently represents a hydrogen atom, a linear, branched or cyclic alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group. , Ar ₁ and Ar ₂ represent a substituted or unsubstituted aryl group, and Ar ₁ and Ar ₂ may form a nitrogen-containing heterocycle together with the nitrogen atom to which Ar ₁ and Ar ₂ are bonded. Y ₁ and Y ₂ are a hydrogen atom, a halogen atom, a linear, branched or cyclic alkyl group, a linear, branched or cyclic alkoxy group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, Alternatively, it represents a substituted or unsubstituted amino group.

ここで、式中、Ar₁〜Ar₆はそれぞれ独立に、置換または未置換のアリール基を表し、さらに、Ar₁とAr₂、Ar₃とAr₄およびAr₅とAr₆は結合している窒素原子と共に含窒素複素環を形成していても良い。 (Thiophene derivative 4)
In addition, 2,3,4,5-tetraphenylthiophene derivatives represented by the following general formula can also be used.

Here, in the formula, Ar _{1 to} Ar ₆ each independently represents a substituted or unsubstituted aryl group, and Ar ₁ and Ar ₂ , Ar ₃ and Ar _4, and Ar ₅ and Ar ₆ are bonded to each other. A nitrogen-containing heterocyclic ring may be formed together with the nitrogen atom.

  Further, imidazole can be used as the five-membered ring compound and an imidazole derivative represented by the following general formula can be used.

(Imidazole derivative 1)

(Imidazole derivative 2)

(Imidazole derivative 3)

(Imidazole derivative 5)

ここで、イミダゾール骨格は中央のベンゼン環R₈, R₉, R₁₀, R₁₁ の任意の位置に複数ユニットが結合していても良い。また、R₁₂は、置換基を有してもよいオレフィン基又はパラフィン基であり、nは１から３の整数、好ましくは１である。

Here, the imidazole skeleton may have a plurality of units bonded to any position of the central benzene ring R ₈ , R ₉ , R ₁₀ , R ₁₁ . R ₁₂ is an olefin group or a paraffin group which may have a substituent, and n is an integer of 1 to 3, preferably 1.

(Carbazole derivative)
Moreover, the carbazole derivative shown with the following general formula can also be used.

  Moreover, it is a 5-membered ring compound which has a conjugated system, and the monocyclic compound containing 1 or more types of hetero atoms, a selenium atom, or a boron atom can also be used. Although not particularly limited, for example, an azole derivative represented by the following general formula can be used.

ここで、式中、R₁、 R₄、 R₅は、それぞれ独立に、水素原子、ハロゲン原子、ヒドロキシル基、シアノ基、あるいはスルホニル基などの置換基を有しても良い芳香族炭化水素基又は炭化水素基又は複素環基又はヘテロ原子を環内に含む芳香族基を示す。R₁、 R₄、 R_５は同じでも異なっていてもよい。

Here, in the formula, R ₁ , R ₄ and R ₅ are each independently an aromatic hydrocarbon group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, or a sulfonyl group. Or the aromatic group which contains a hydrocarbon group, a heterocyclic group, or a hetero atom in a ring is shown. R ₁ , R ₄ and R ₅ may be the same or different.

The detection method of the present invention can be applied to any detection method as long as it is a detection method that measures the fluorescence of protein in a solution, solid or semi-solid state. The same applies to peptides, antibodies, sugars having amino groups, and the like.
For example, a protein in a sample is labeled with an anionic fluorescent dye, the labeled protein is used as a separation means, the molecular weight of a fraction is measured with a mass spectrometer such as MALDI-TOF MS, and a database search is performed to identify the protein. can do. Here, ion-exchange column HPLC, reverse phase partition HPLC, gel filtration HPLC, or electrophoresis can be used as the separation means. For electrophoresis, primary and secondary electrophoresis can be used. After electrophoresis, the gel can be dried for quantification.

  In addition, the following detection method can be achieved by using a covalent fluorescent dye and an anionic fluorescent dye having an anionic group introduced therein. Here, the covalent fluorescent dye is one whose fluorescence wavelength does not change even if the protein is labeled thereby. First, it is labeled with a covalent fluorescent dye. Thereafter, electrophoresis is performed to divide. Further, when the gel substrate after electrophoresis is labeled with an anionic fluorescent dye, the fluorescence wavelength changes. Since an anionic fluorescent dye can label an amino residue located in the deep part of a protein, a change in fluorescence wavelength is due to a difference in protein structure. Therefore, it is also possible to predict the protein structure from the change 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 exactly the same. Therefore, highly accurate quantification is possible.

  In addition, the present invention can be applied to a detection method using a protein chip as follows. 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 the second fluorescence wavelength from the measurement substrate can be measured. After the spotting, the protein is fixed on the substrate by allowing it to stand at a predetermined temperature, but incubation can be performed if necessary. On the protein chip, when capturing the protein labeled with this fluorescent dye, stable fluorescence is emitted under any circumstances, so that it is not necessary to handle it as nervously as before. Moreover, since fluorescence quenching does not occur even in a dry state, stable observation is possible even in a dry state. Moreover, the detection method using the above-mentioned covalent bond fluorescent dye and the anionic fluorescent dye into which an anionic group is introduced can also be used.

Hereinafter, the present invention will be described in more detail with reference to examples.
Synthesis Example 1
An example of synthesis of an active ester anionic fluorescent dye will be shown.
(1) Synthesis coloring portion 3 of the color portion 3 was synthesized according to Scheme 2 below.

1 with 50 mL three-necked flask oxadiazolopyridine carboxylic acid 1.0 g (0.0026 moL) and N- hydroxysuccinimide 2 0.30 g of (0.0026 Mol) were dissolved in DMF 20 mL. N, N'-dicyclohexylcarbodiimide 0.54 g (0.0026 moL) was added dropwise thereto over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform) to obtain 0.76 g of oxadiazolopyridine active ester 3 in a yield of 62%.

(2) Introduction of sulfonyl group The active ester 3 was reacted with taurine in DMF to be sulfonated 4 (Scheme 3).

Example 1.
(experimental method)
Dissolve sulfonyl 4 of active ester in pure water and add 0.1 M HEPES Buffer
(2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) (pH 7.3) is added, then BSA (Bovine Serium Albumin) is added, and the change in color tone is observed in the spectrum. The UV spectrum of 4 was measured, and then the fluorescence spectrum was measured to observe the interaction between the sulfonyl derivative 4 and BSA. The UV spectrum was measured by preparing 13 μM and 26 μM fluorescent reagents in a 2000 μL cell. In addition, the fluorescence spectrum was measured by preparing 3000 μL of a 26 μM solution in the cell and adding BSA to this to a predetermined concentration.

(result)
The color tone of the buffer solution containing the sulfonyl compound 4 changed from yellow to yellow-green when BSA was added (FIG. 1). FIG. 2 shows the UV spectrum of the sulfonyl compound 4 . From this result, it was found that the maximum absorption wavelength of the sulfonyl body 4 was 397 nm. Next, the fluorescence spectrum was measured using 397 nm as the excitation wavelength. The results are shown in FIG. Here, BSA is added to a concentration of 1.6 μM. The fluorescence spectrum showed a blue shift of 18 nm with the addition of BSA, and the fluorescence intensity increased about 5-fold. This is probably because the sulfonyl body 4 was electrostatically bonded to the amino group of BSA, and a blue shift was observed due to the interaction between the BSA surface and the sulfonyl body 4 . In addition, since the sulfonyl body 4 is located in the hydrophobic field of BSA, it is considered that the interaction with water was eliminated to some extent and the fluorescence intensity was increased.

Next, a sulfonyl body 4 26UM aqueous 2000μL was prepared and added in portions thereto to the BSA 0 to 15 nM in 8 times. The UV spectrum at that time is shown in FIG. The addition of BSA showed a light color effect and the peak wavelength was red shifted (7 nm red shift at 15 nM). From this, it is thought that the sulfonyl body 4 is located in the deep part (hydrophobic field) of BSA.

For comparison, the active ester form 3 was used in place of the sulfonyl form 4 , but no change in the peak wavelength of BSA and no increase in fluorescence intensity were observed. Active ester 3 binds to the protein via an amide bond formed by the nucleophilic substitution reaction between the active ester group and the amino group, but does not bind to the amino group located deep in the BSA due to steric hindrance such as a succinimide molecule. It is thought that it binds only to the surface amino groups. On the other hand, since the sulfonyl body 4 binds not only to the amino group on the BSA surface but also to the deep amino group by electrostatic bonding, it is also located in the deep part (hydrophobic field) of the BSA as described above. As a result, it is considered that a blue shift and an increase in fluorescence intensity occurred.

Example 2
(experimental method)
Next, it carried out by the same method as Example 1 except having used insulin (Inslin) for protein. Sulfonide 4 26.7 μM was prepared in 2000 μL in a fluorescent cell, and 0 to 232 μM of insulin was added in 6 portions.

(result)
The fluorescence spectrum when insulin is added is shown in FIG. As with BSA, a 19 nm blue shift and an increase in fluorescence intensity were observed. FIG. 6 shows the relationship between the insulin concentration in the cell when added and the peak wavelength FLmax at each concentration, and the relationship between the insulin concentration and the fluorescence intensity ΔInt. At the peak wavelength (the value obtained by subtracting the fluorescence intensity of only the sulfonyl 4 ). Is shown in FIG. As the insulin concentration increased, the peak wavelength FLmax linearly shifted to a lower wavelength. Moreover, the result that the fluorescence intensity ΔInt. Increased linearly with the increase in the insulin concentration was obtained. This shows that the concentration of insulin can be calculated from the change in fluorescence intensity or the change in peak wavelength.

Example 3
(experimental method)
Next, it carried out by the same method as Example 1 except having used lysozyme for protein. Sulfonide 4 26.7 μM was prepared in a fluorescent cell at 2000 μL, and lysozyme 0 to 46.6 μM was added in 4 portions.

(result)
The fluorescence spectrum when lysozyme was added is shown in FIG. Even when lysozyme was added, neither an increase in fluorescence intensity nor a blue shift in peak wavelength was observed. This indicates that the sulfonyl body 4 does not bind to lysozyme. This is considered to mean that the sulfonyl body 4 does not bind to the amino group of lysozyme because the amino group located on the surface of the protein has a different conformation depending on the type of protein. However, since sulfonyl body 4 binds to BSA and insulin, it is considered that sulfonyl body 4 can be used as a fluorescent reagent capable of selectively labeling proteins.

Comparative Example 1
The same procedure as in Example 1 was performed, except that methyl orange conventionally used was used. However, even when methyl orange was added, neither an increase in fluorescence intensity nor a blue shift in peak wavelength was observed.

It is a photograph which shows an example of the change of the color tone of the fluorescent dye in this invention. It is a figure which shows the change of UV spectrum of the fluorescent pigment | dye in Example 1 of this invention. It is a figure which shows the change of the fluorescence spectrum of the fluorescent pigment | dye in Example 1 of this invention. It is a figure which shows the change of UV spectrum of the fluorescent pigment | dye in Example 1 of this invention. It is a figure which shows the change of the fluorescence spectrum of the fluorescent pigment | dye in Example 2 of this invention. It is a graph which shows the relationship between the insulin concentration in Example 2 of this invention, and a fluorescence peak wavelength. It is a graph which shows the relationship between the insulin concentration and fluorescence intensity in Example 2 of this invention. It is a figure which shows the change of the fluorescence spectrum of the fluorescent pigment | dye in Example 3 of this invention.

Claims (12)

A protein detection method for detecting a protein labeled with a fluorescent dye,
A protein detection method for detecting a protein by measuring fluorescence based on a second fluorescence wavelength that is shorter than a first fluorescence wavelength observed in a free state and bound to a protein. The above detection method is a detection method in which a protein in a sample is subjected to separation means, and a separated fraction is subjected to mass spectrometry,
The detection 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 the separation means.   Prior to or simultaneously with the labeling of the protein with the first fluorescent dye, the protein is labeled with the second fluorescent dye that does not shift to the short wavelength while being bound to the protein, and is then provided to the separating means. The detection method according to claim 2.   The protein and the first fluorescent dye that generates the second fluorescence wavelength are reacted in a solution, the solution is spotted on a measurement substrate, and a fluorescence image based on the second fluorescence wavelength from the measurement substrate is displayed. The detection method of Claim 1 which measures.   Prior to or simultaneously with the reaction of the protein and the first fluorescent dye in the solution, the second fluorescent dye in which the first fluorescence wavelength does not shift to a short wavelength when bound to the protein is added to the protein and the solution. The detection method of Claim 4 made to react.   The detection method according to claim 2, wherein the first fluorescent dye has an anionic group that binds to a protein.   The detection method according to claim 3 or 4, wherein the second fluorescent dye has a covalent bond group that binds to a protein.   A fluorescent dye for protein detection comprising an anionic fluorescent dye containing an organic EL dye in which an anionic group that binds to a protein is bonded directly or via a linking moiety.   The fluorescent dye according to claim 8, wherein the anionic group is any one of a carboxyl group, a sulfonyl group, a sulfate group, a phosphate group, and a combination thereof.   The fluorescence according to claim 8 or 9, wherein the organic EL dye is a compound containing a 5-membered ring compound having a conjugated system, and the 5-membered ring compound contains one or more heteroatoms, selenium atoms, or boron atoms. Pigment.   The fluorescent dye according to claim 10, wherein the organic EL dye is a condensed polycyclic compound comprising the 5-membered ring compound and a 6-membered ring compound having a conjugated system. The fluorescent dye according to claim 10 or 11, wherein the 5-membered ring compound is an azole derivative or an imidazole derivative.

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