JPWO2006132030A1 - Novel compound, reagent for analysis of peptide or protein containing the compound, and analysis method using the analysis reagent - Google Patents

Abstract

Provided are a compound represented by the following formula I, which is useful as a reagent that can efficiently and easily analyze peptide and protein concentrations, and an analysis method using the reagent containing the compound. Formula I: ## STR1 ## wherein R1 is an optionally substituted aryl or heteroaryl group, and R3, R4, R5 and R6 are independently of each other a bond, hydrogen atom, carbon number 1 -10 alkyl group, C 1-10 alkoxy group, phenyl group (the phenyl group may be substituted with one or more groups selected from amino group, halogen and nitro group), amino group, Cyano group, nitro group, carboxyl group (or salt, ester, amide thereof), sulfonic acid (or salt, ester, amide), thiol group, hydroxyl group (or salt thereof), acyl group having 1 to 10 carbon atoms, halogen And n is an integer of 1 to 5. ]

Description

  The present invention relates to a novel compound, a reagent for analyzing a peptide or protein containing the compound, and an analysis method using the analysis reagent.

  Proteome research to investigate the state and cause of diseases by comprehensively examining the proteins in the body is underway, for example, research that tries to determine proteins that are markers of diseases like cancer marker proteins has accelerated rapidly Yes. In particular, protein surface and molecular recognition between protein surfaces are considered to be a common language in life phenomena, and much of the information from the outside of the cell is amplified and transmitted by interactions through the protein surface inside the cell membrane or inside the cell.

  In recent years, transcriptome analysis that comprehensively analyzes RNA expression has been actively performed by improving the DNA chip technology. However, this RNA expression profile does not necessarily match the protein expression profile, and the correlation is said to be 50% or less. For that purpose, in addition to comprehensive analysis of RNA, comprehensive analysis of proteins is important, and it is comparable to genome analysis such as two-dimensional electrophoresis, mass spectrometry, and protein chips, which have been remarkably developed recently. Attempts have been made to clarify the function of proteins using high-throughput analysis.

As typical methods for quantifying proteins in a sample, (a) absorptiometry, (b) Biuret method using Biuret reagent, (c) Lowry method combining phenol reagent and Biuret method, (d
) Bradford method, (e) ELISA method and the like. The principle, advantages, and disadvantages of each method are described below.

(A) Absorptiometric principle: A method for calculating a protein concentration using an absorption band near 280 nm caused by tyrosine or tryptophan in the protein. Since the tyrosine and tryptophan contents vary depending on the type of protein, the absorbance at 280 nm varies, but at a concentration of 1 mg / ml, A280 nm can be calculated as 1.0.
Advantages: The operation is simple and the sample can be collected after measurement.
Cons: Absorbance varies depending on the type of protein. Proteins that do not absorb at 280 nm (collagen, gelatin, etc.) cannot be measured. Furthermore, if a substance having absorption in the ultraviolet region is mixed, the determination becomes difficult.

(B) Biuret method Principle: When is a protein reacted with Cu ²⁺ solution under alkaline conditions, and utilizing the phenomenon that Cu ²⁺ is colored red by coordinate bonds with the nitrogen atom in the polypeptide chain, 540 nm Method for measuring the absorbance in water.
Advantages: Little difference in coloration rate due to protein differences. Easy to operate.
Cons: Low sensitivity, not suitable for low concentration samples.

(C) Principle of Lowry method: This is a method in which blue is colored by reacting with tyrosine, tryptophan and cysteine in protein using a phenol reagent in which phosphomolybdic acid and phosphotungstic acid are dissolved in an acidic solution. It is much more sensitive than the Biuret method due to the strong coloring effect derived from peptide bonds.
Pros: High sensitivity and the most commonly used method.
Disadvantages: Coloring is hindered by other reducing substances because it is colored by a reduction reaction. The operation is complicated and takes time to measure. There is a difference in color development rate by protein.

(D) When Coomassie Brilliant Blue G-250, which is a triphenylmethane blue pigment, binds to a protein in an acidic solution of Bradford method, the maximum absorption wavelength is shifted from 465 nm to 595 nm, and the color tone changes from red purple to blue. This is a method for quantifying proteins using this phenomenon.
Pros: Not easily affected by interfering substances. The operation is very simple.
Disadvantages: There is a difference in color development rate depending on the type of protein Further, the color development is hindered by the mixing of the surfactant.

(E) ELISA method An immunological method using an antibody that binds only to a specific protein.
Disadvantages: Since it is necessary to repeat the binding reaction between the target protein (antigen) and the antibody, it is impossible to obtain results quickly, such as taking more than 6 hours for the measurement.

  In addition to this, a method of analyzing a peptide or protein using a fluorometric method is known. The fluorometric method is a conventional method for analyzing various chemical substances, and has advantages such as high sensitivity, a small amount of sample, a large-scale apparatus, and no need for skilled techniques.

  There have been reports of protein quantification using a fluorometric method. For example, when fluorescamine reacts with a primary amine, it becomes a strong fluorescent substance that emits fluorescence at 495 nm, and a cyanine dye is used. Analysis is representative (Haugland, RP Handbook of Fluorescent Probes and Research Chemicals, 9th ed .; Molecular Probes Inc .: Leiden, 2002.).

  However, in this method, since the dye and the protein are reacted via a covalent bond, the reaction time is slow, the reactivity varies depending on the type of protein, the calibration curve is not a straight line, the measurement error due to the association between the dyes, and the Stokes shift is small. There is a problem.

US Pat. No. 5,616,502 Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 9th ed .; Molecular Probes Inc .: Leiden (2002) L. J. Jones, R. P. Haugland, V. L. Singer, Biotechniques, 34, 850 (2003) R. F. Pasternack, C. Fleming, S. Herring, P. J. Collings, J. DePaula, G. DeCastro, E. J. Gibbs, Biophys. J., 79, 550 (2000)

  Proteome research by comprehensive analysis of proteins is important, but conventional analysis methods have drawbacks such as low sensitivity and accuracy, complicated operation, and time.

  Therefore, in the present invention, a novel compound useful as a reagent that solves the above-described problems and can analyze peptide and protein concentrations with high sensitivity, efficiency, and simplicity, and a peptide using the reagent containing the novel compound Alternatively, an object is to provide a protein analysis method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have synthesized a novel fluorescent compound having a heterocyclic ring and an olefin conjugated system, and the compound has a highly sensitive, efficient and simple peptide and protein concentration. The present invention has been found to be useful as a reagent that can be analyzed in a simple manner.

  That is, the present invention includes the following inventions.

(1) A compound represented by the following formula I:

[Wherein, R ₁ represents an optionally substituted aryl group or heteroaryl group, and R ₃ , R ₄ , R ₅ and R ₆ are each independently a bond, a hydrogen atom, or a carbon number of 1 to 10 An alkyl group, an alkoxy group having 1 to 10 carbon atoms, a phenyl group (the phenyl group may be substituted with one or more groups selected from an amino group, a halogen and a nitro group), an amino group, a cyano group , Nitro group, carboxyl group (or salt, ester, amide thereof), sulfonic acid (or salt, ester, amide), thiol group, hydroxyl group (or salt thereof), acyl group having 1 to 10 carbon atoms, halogen and sugar N is an integer of 1-5. ]

(2) The compound according to (1), wherein R ₁ is an optionally substituted phenyl group or naphthyl group.

(3) R ₃ , R ₄ , R ₅ and R ₆ are independently selected from the group consisting of a bond, a hydrogen atom, and an alkyl group having 1 to 10 carbon atoms, and n is 1. (2) The compound according to (2),

(4) A reagent for peptide or protein analysis, comprising the compound described in any one of (1) to (3).

(5) A method for analyzing a peptide or protein, comprising mixing a sample containing a peptide or protein and the reagent according to (4), and measuring fluorescence or color development of the mixture.

(6) The analysis method according to (5), wherein the sample and the reagent are mixed in a liquid phase.

(7) The mixing of the sample and the reagent is performed by bringing a solution containing the reagent into contact with a gel containing the sample separated by gel electrophoresis ( The analysis method as described in 5).

  The conventional methods have drawbacks such as low sensitivity / accuracy, complicated operation, and time consuming. However, using the reagent and analysis method containing the novel compound of the present invention, the peptide in the sample and The presence or concentration of protein can be measured accurately and conveniently in a short time. In addition, since the method of the present invention measures changes in fluorescence and luminescence, it does not require a conventional expensive apparatus or skilled technique, and can easily perform accurate measurement without experience. In addition, the compound to be used as an analytical reagent can be easily synthesized from an inexpensive starting material, and since an expensive reagent is not used, the cost for analysis can be greatly reduced.

It is a schematic diagram of fluorescence emission by protein and pigment | dye complex formation. 3 is an absorption spectrum when various concentrations of BSA are added to Dye 1. It is a three-dimensional fluorescence spectrum when 200 μg / mL BSA is added to Dye 1. 2 is a fluorescence spectrum when various concentrations of BSA are added to Dye 1. FIG. It is a photograph observation figure when the pigment | dye 1 and various density | concentrations of BSA are added. It is a calibration curve in which the fluorescence intensity of Dye 1 is plotted against the BSA concentration. In Example 7, it is the electrophoresis photograph of BSA or IgG isolate | separated by SDS-PAGE. In Example 7, it is a figure which shows the relationship between the fluorescence intensity of a protein spot, and protein concentration.

Compounds useful as reagents of the present invention are represented by Formula I below. :

[Wherein, R ₁ represents an optionally substituted aryl group or heteroaryl group, and R ₃ , R ₄ , R ₅ and R ₆ are each independently a bond, a hydrogen atom, or a carbon number of 1 to 10 An alkyl group, an alkoxy group having 1 to 10 carbon atoms, a phenyl group (the phenyl group may be substituted with one or more groups selected from an amino group, a halogen and a nitro group), an amino group, a cyano group , Nitro group, carboxyl group (or salt, ester, amide thereof), sulfonic acid (or salt, ester, amide), thiol group, hydroxyl group (or salt thereof), acyl group having 1 to 10 carbon atoms, halogen and sugar N is an integer of 1-5. ]

  Examples of the aryl group include phenyl and a polycyclic aromatic group in which 2 to 4 benzene rings are condensed (for example, naphthyl, anthryl, pyrenyl). The heteroaryl group means an aryl group containing at least one heteroatom selected from the group consisting of N, O and S as a ring atom. These aryl group or heteroaryl group may be further condensed with another aromatic ring or heteroaromatic ring. Specific examples of the aryl group and heteroaryl group include pyridyl group, furyl group, dansyl group, coumarin, benzothiazol, fluorescein, rhodamine, azobenzene and the like.

  The aryl group or heteroaryl group is an alkyl group having 1 to 10 carbon atoms (preferably 1 to 6 carbon atoms), an alkoxy group having 1 to 10 carbon atoms (preferably 1 to 6 carbon atoms), a phenyl group (the phenyl group). Group may be substituted by one or more groups selected from amino group, halogen and nitro group), amino group, cyano group, nitro group, carboxyl group (or salt, ester, amide thereof), sulfonic acid (Or a salt, ester or amide thereof), a thiol group, a hydroxyl group (or a salt thereof), one selected from the group consisting of an acyl group having 1 to 10 carbon atoms (preferably 1 to 6 carbon atoms), halogen, and sugar. It may be substituted with the above groups. The aryl group or heteroaryl group is preferably substituted with a hydroxyl group for imparting water solubility and electron donating properties.

  The part of the compound that becomes the fluorescent group or the chromophoric group (which can absorb in the ultraviolet, visible, infrared, and fluorescence) forms a conjugated system with the olefin moiety of formula I-(CH = CH) n- Formula II:

It has the structure represented by these.

[Wherein R ₃ , R ₄ , R ₅ and R ₆ are each independently a bond, a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, a phenyl group (the phenyl group Group may be substituted by one or more groups selected from amino group, halogen and nitro group), amino group, cyano group, nitro group, carboxyl group (or salt, ester, amide thereof), sulfonic acid (Or a salt, ester or amide thereof), a thiol group, a hydroxyl group (or a salt thereof), an acyl group having 1 to 10 carbon atoms, a halogen, or a sugar. ]
Above all, the following formula:

The group represented by these is preferable.

  By connecting an alkenyl group and an aromatic group to the above structure, which is a fluorescent group or a color forming group, the conjugated system is extended, and a longer wavelength of the fluorescence / color generation wavelength is promoted.

  Compounds of formula I can be readily synthesized by known methods. For example, the following formula:

This compound can be produced by reacting 4-hydroxybenzaldehyde and 4- (dicyanomethylene) -2,6-dimethyl-4H-pyran in ethanol in the presence of a base (for example, an amine such as piperidine). it can.

  The reagent of the present invention contains the compound of formula I, but the form at the time of use is not particularly limited. For example, the reagent of formula I may be dissolved in water to form an aqueous solution. Alternatively, a filter paper, a silica sheet, an alumina sheet or the like may be impregnated with a solution of the compound of formula I and optionally dried.

  Next, a method for analyzing a peptide or protein (hereinafter also simply referred to as “protein”) using the reagent of the present invention will be described.

  The principle of the method of the present invention is shown in FIG. When the compound of formula I and the protein to be measured are mixed, they form a complex, and the compound of formula I emits fluorescence or color. This change in fluorescence and color development (for example, wavelength and intensity) has a correlation with the amount of protein, and thus, by measuring this change, qualitative analysis or quantitative analysis of protein can be performed.

  The mixing of the reagent of the present invention and the protein-containing sample may be performed in a liquid phase (for example, an aqueous solution), or a solid phase (for example, a filter paper, a silica sheet, an alumina sheet) impregnated with the reagent and the above-mentioned. This can be done by contacting with a solution containing the sample.

  When a protein is present in a sample, the compound of formula I forms a complex (charge transfer complex) with the protein by hydrophobic interaction and charge transfer interaction, whereby the intrinsic absorption wavelength and absorption of the compound of formula I Changes occur in intensity, fluorescence wavelength, or fluorescence intensity. In some cases, this change can be visually observed as a change in color tone, whereby the presence of the protein in the sample can be easily confirmed. Or you may measure using measuring instruments, such as a commercially available fluorometer and absorptiometer. The protein concentration can be accurately determined from the amount of change / displacement.

  In addition, the sample is subjected to electrophoresis such as agarose gel electrophoresis or polyacrylamide gel electrophoresis to separate proteins contained in the sample, and then present in the gel by contacting the analyzed reagent of the present invention with the gel after electrophoresis. It is also possible to qualitatively / quantitatively analyze the protein in the sample by staining the protein and capturing it as an image using a fluorescence image analyzer or the like.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

(Example 1: Synthesis of dye 1)

In a 50 ml three-necked flask, 0.70 g (5.81 mmol) of 4-hydroxybenzaldehyde, 1.0 g (5.81 mmol) of 4- (dicyanomethylene) -2,6-dimethyl-4H-pyran, 0.50 g (5.81 mmol) of piperidine, and 50 ml of ethanol In addition, the mixture was heated to reflux for 12 hours under an Ar stream. After evaporating the solvent under reduced pressure, the residue was purified by column chromatography (SiO2, CHCl3: MeOH = 10: 1 v / v) to obtain the target compound. Yield 30%.
¹ H NMR (400 MHz, CDCl ₃ , rt, TMS, d / ppm) 1.71 (s, 3H), 5.27 (s, 1H), 5.51 (s, 1H), 6.65 (d, 1H), 6.85 (d, 1H), 6.68 (d, 2H), 7.13 (d, 2H).

(Example 2: Synthesis of dye 2)

To a 50 ml three-necked flask, add 0.70 g (5.81 mmol) vanillin, 1.0 g (5.81 mmol) 4- (dicyanomethylene) -2,6-dimethyl-4H-pyran, 0.50 g (5.81 mmol) piperidine, 50 ml ethanol, Ar The mixture was heated to reflux for 12 hours under an air stream. After evaporating the solvent under reduced pressure, the residue was purified by column chromatography (SiO2, CHCl3: MeOH = 10: 1 v / v) to obtain the target compound. Yield 20%.
¹ H NMR (400 MHz, CDCl ₃ , rt, TMS, δ / ppm) 1.71 (s, 3H), 4.03 (s, 3H), 5.27 (s, 1H), 5.51 (s, 1H), 6.65 (d, 1H), 6.85 (d, 1H), 6.68 (d, 2H), 7.13 (d, 2H).

(Example 3: Synthesis of dye 3)

In a 50 ml three-necked flask, 0.70 g (5.81 mmol) of 4-hydroxy-1-naphthaldehyde, 1.0 g (5.81 mmol) of 4- (dicyanomethylene) -2,6-dimethyl-4H-pyran, 0.50 g (5.81 mmol) of piperidine Then, 50 ml of ethanol was added, and the mixture was heated to reflux for 12 hours under Ar flow. After evaporating the solvent under reduced pressure, the residue was purified by column chromatography (SiO2, CHCl3: MeOH = 10: 1 v / v) to obtain the target compound. Yield 25%.
¹ H NMR (400 MHz, CDCl ₃ , rt, TMS, δ / ppm) 1.71 (s, 3H), 5.27 (s, 1H), 5.51 (s, 1H), 6.65 (d, 1H), 6.85 (d, 1H), 6.59 (d, 1H), 7.21 (d, 1H), 7.35 (t, 1H), 7.38 (t, 1H), 8.08 (d, 1H).

(Example 4: Change in absorption spectrum due to complexation of dye 1 and protein (BSA))
Dye 1 was used as the reagent of the present invention. This was mixed with BSA solutions of various concentrations, and changes in the absorption spectrum were observed. The measurement conditions of the absorption spectrum are as follows.
Concentration: [Dye 1] = 1.0 x 10-5M
[BSA] = 0 to 200 μg / mL
Solvent: 0.9% NaCl aqueous solution Temperature: 25 ° C

  The measured absorption spectrum is shown in FIG. In FIG. 2, the curve (A) represents [BSA] = 0 μg / mL, and the curve (B) represents [BSA] = 200 μg / mL. As the concentration of BSA increased, the absorbance in the 400 nm to 500 nm absorption band increased slightly. Similar results were obtained for Dye 2 and Dye 3.

(Example 5: Change in fluorescence spectrum by complexing dye 1 and protein (BSA))
When analyzing the interaction between dye 1 and BSA by fluorescence spectrum measurement, in order to determine the optimum excitation wavelength, dye 1 and BSA (200 μg / mL) are mixed, and the three-dimensional fluorescence spectrum (excitation wavelength range) : 350 nm to 500 nm, fluorescence wavelength range: 400 nm to 750 nm). The result is shown in FIG. As the excitation wavelength goes to the longer wavelength side, a fluorescence spectrum having a maximum fluorescence wavelength at 590 nm was observed. In the measurement at 500 nm, since the scattered light is greatly covered with the fluorescence, the excitation wavelength in the subsequent fluorescence spectrum measurement was set to 470 nm. The same measurement was performed for dye 2 and dye 3, and the excitation wavelength was determined.

The fluorescence spectrum was measured under the following conditions.
Concentration: [Dye] = 1.0 x 10-5M
[BSA] = 0 to 200 μg / mL
Solvent: 0.9% NaCl aqueous solution Temperature: 25 ° C
Excitation wavelength: Dye 1 350nm, 470nm
Dye 2 350nm, 470nm
Dye 3 500nm

  Changes in the fluorescence spectrum when (0, 25, 50, 100, 200 μg / mL) BSA was added to Dye 1 were measured, and the results are shown in FIG. In FIG. 4, each curve represents the measurement result of the BSA concentration (0, 25, 50, 100, 200 μg / mL) in order from the bottom. As shown in FIG. 4, an increase in fluorescence intensity was observed with an increase in BSA concentration. The fluorescence intensity when BSA was added at 200 μg / mL increased about 20 times compared to the case of dye 1 alone. For dye 2 and dye 3, it was also confirmed that the fluorescence intensity increased as the BSA concentration increased.

  FIG. 5 shows a photograph observation when the ultraviolet light is irradiated to the solution before adding BSA to dye 1 (0 μg / ml) and after adding (100 μg / ml). It was observed that light was emitted in orange by adding BSA.

  FIG. 6 is a graph in which the value (I-Io) obtained by subtracting the fluorescence intensity (Io) at 590 nm of dye 1 alone from the fluorescence intensity (I) at the time of adding BSA to dye 1 is plotted against the BSA concentration. Shown in A good linear relationship with a correlation coefficient of 0.9974 was obtained. The detection limit was 100 ng / mL, which was found to be about 20 times more sensitive than the Bradfold method. A calibration curve was similarly prepared for Dye 2 and Dye 3, and it was found that there was a good linear relationship between fluorescence intensity and protein concentration.

Table 1 shows optical data before and after the interaction of each of the above dyes with BSA.

In all dyes, no shift in the maximum absorption wavelength was observed before and after the addition of BSA, and the molar extinction coefficient only increased slightly. On the other hand, in the fluorescence spectrum, although the shift of the maximum fluorescence wavelength was not observed, a large increase in fluorescence intensity was observed with the addition of BSA. Relative fluorescence intensity I / I ₀ (I ₀ : fluorescence intensity at 590 nm with dye alone, I: fluorescence intensity at 590 nm when BSA (200 mg / mL) was added to the dye) was calculated. In the case of 3, there was a 17-18 fold increase in fluorescence intensity and a large Stokes shift, indicating that even a very small amount of protein can be detected sensitively.

(Example 6: Effect on interfering substances)
The extent to which interfering substances such as inorganic salts, surfactants, and nucleic acids affect the interaction between the dye and protein was confirmed as follows.
(Measurement condition)
[Dye 3] = 1.0 × 10 ^-5 M
[BSA] = 1000 μg / mL
Solvent: 0.9% NaCl aqueous solution Temperature: 25 ° C
Excitation wavelength: Dye 3 500nm
(Compounds used as interfering substances)
Glycine, sodium chloride, ammonium sulfate, asparagine, sodium bicarbonate, zinc (II) chloride, Bicine, Bis-Tris, nickel chloride, sodium acetate, Tricine, sodium phosphate, guanidine / HCL, imidazole, calcium chloride, triethanolamine, Sodium citrate, HEPES, B-PER Reagent, Cobalt chloride (II), Nucleic acid (from testis), SDS, CHAPS, Tweem-20, Triton X-100, EDTA, EGTA, cysteine, glucose, melibiose, 2-mercapto Ethanol, thimerosal, acetone, acetonitrile, ethanol, methanol, Phenol Red, urea, glycerol, potassium thiocyanate, sucrose

(result)
In the state where the dye and BSA are mixed, the concentration of the nuclear material was measured when the fluorescence intensity changed by 10% by adding each of the above interfering substances. The observed concentration of interfering substance was in a large excess over that used under normal experimental conditions. Thus, it was found that the interaction between the dye and the protein is not affected by the interfering substance.

(Example 7: electrophoresis test)
As an application example of the synthesized dye, whether it can be used as a staining agent for detecting proteins separated by electrophoresis was examined as follows.
(Experimental conditions)
SDS-PAGE device: Sure Blot F1 Gel system (Astellas Pharma, Inc., Tokyo).
・ [Dye 3] = 0.1 mg / mL (or 0.2 mg / mL) in MeOH: H ₂ O = 1: 1 v / v
・ Immobilizing solution and cleaning solution: AcOH: MeOH: H ₂ O = 3: 10: 87
・ Detection device: a ProXpress imaging system (Perkin-Elmer, UK) or LS400 scanner (Tecan, USA))
(operation)
1) SDS-PAGE of BSA, Human IgG, Chymotrypsinogen or Transferrin was performed.
2) The gel was immersed in the immobilization solution for 30 minutes.
3) The gel was immersed in the dye solution for 30 minutes or 1 hour.
4) The gel was immersed in the washing solution for 30 minutes.
5) Fluorescence detection was performed (ex; 543 nm, em; 590 nm or ex; 480 nm, em; 530 nm).

(result)
An electrophoresis photograph of BSA or IgG separated by SDS-PAGE is shown in FIG. The protein was stained with a dye and succeeded in obtaining a beautiful electrophoretic image.
In FIG. 7, each number represents the following:
(1) IgG 0.02 μg, (2) IgG 0.1 μg, (3) IgG 0.4 μg, (4) IgG 1.1 μg, (1) IgG 3.3 μg, (6) BSA 0.01 μg, (7) BSA 0.02 μg, ( 8) BSA 0.1 μg, (9) BSA 0.4 μg, (10) BSA 1. 1 μg, (11) BSA 3.3 μg, (12) BSA 10.0 μg.
FIG. 8 shows the relationship between the fluorescence intensity of protein spots and the protein concentration. A good linear relationship was obtained, and no significant difference was observed in the slope of the calibration curve between proteins.

The protein analysis reagent and analysis method using the novel compound of the present invention are useful for highly sensitive, comprehensive and simple protein analysis in the fields of biochemistry, medicine, analytical chemistry and the like. Furthermore, it can be used not only for protein analysis but also for staining agents used when proteins are separated by electrophoresis, visualization imaging dyes for proteins in living cells, and the like.

Claims (7)

The following compound of formula I:
[Wherein, R ₁ represents an optionally substituted aryl group or heteroaryl group, and R ₃ , R ₄ , R ₅ and R ₆ are each independently a bond, a hydrogen atom, or a carbon number of 1 to 10 An alkyl group, an alkoxy group having 1 to 10 carbon atoms, a phenyl group (the phenyl group may be substituted with one or more groups selected from an amino group, a halogen and a nitro group), an amino group, a cyano group , Nitro group, carboxyl group (or salt, ester, amide thereof), sulfonic acid (or salt, ester, amide), thiol group, hydroxyl group (or salt thereof), acyl group having 1 to 10 carbon atoms, halogen and sugar N is an integer of 1-5. ] The compound according to claim 1, wherein R ₁ is an optionally substituted phenyl group or naphthyl group. R ₃ , R ₄ , R ₅ and R ₆ are independently selected from the group consisting of a bond, a hydrogen atom and an alkyl group having 1 to 10 carbon atoms, and n is 1. The compound according to claim 2.   A reagent for analyzing a peptide or protein comprising the compound according to any one of claims 1 to 3.   A method for analyzing peptide or protein, comprising mixing a sample containing peptide or protein and the reagent according to claim 4, and measuring fluorescence or color development of the mixture.   6. The analysis method according to claim 5, wherein the sample and the reagent are mixed in a liquid phase. 6. The mixing of the sample and the reagent is performed by bringing a solution containing the reagent into contact with a gel containing the sample separated by gel electrophoresis. Analysis method described.