WO2008023489A1

WO2008023489A1 - Squaric acid derivative compound, protein detection reagent comprising the compound, and protein detection method using the reagent

Info

Publication number: WO2008023489A1
Application number: PCT/JP2007/061199
Authority: WO
Inventors: Yoshio Suzuki; Kenji Yokoyama
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2006-08-21
Filing date: 2007-06-01
Publication date: 2008-02-28
Also published as: JPWO2008023489A1

Abstract

Disclosed is a novel squaric acid derivative which is useful as a reagent for detecting a peptide or protein at high sensitivity and in a simple manner. Also disclosed is a protein detection reagent comprising the compound. Further disclosed is a method for determining the concentration of a peptide or protein comprehensively and with efficiency by using the reagent. A novel compound having the structure represented by the formula (I) can be used as a reagent for the analysis of a protein. [Chemical formula 1] wherein R1 and R2 independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; m1 and m2 independently represent an integer ranging from 1 to 3; and n1 and n2 represent integers respectively satisfying the requirements represented by the formulae: n1=5–m1 and n2=5-m2.

Description

Specification

SQUAREIC ACID DERIVATIVE COMPOUND, PROTEIN DETECTION REAGENT CONTAINING THE COMPOUND, AND PROTEIN DETECTION METHOD USING THE REAGENT

Technical field

[0001] The present invention relates to a novel squaric acid derivative compound, a protein detection reagent containing the compound, and a protein detection method using the reagent.

Background art

[0002] With the progress of proteome research that comprehensively investigates proteins in the body and searches for disease states and causes, research to determine proteins that are markers of diseases such as cancer marker proteins is rapidly accelerating. is doing. In particular, molecular recognition on the protein surface and molecular recognition between the protein surfaces are considered to be a common language in biological phenomena, and much of the information from outside the cell interacts with the protein surface inside the cell membrane or inside the cell. Is amplified and transmitted. In recent years, transcriptome analysis that comprehensively analyzes RNA expression has been actively performed with the improvement of DNA chip technology. However, it is said that the correlation between the RNA expression profile and the protein expression profile is not necessarily the same, and is less than 50%. Therefore, in addition to comprehensive analysis of RNA, comprehensive analysis of proteins is important, and high-level analysis comparable to genome analysis such as two-dimensional electrophoresis, mass spectrometers, and protein chips, which have recently been remarkably developed. We are trying to elucidate protein functions using throughput analysis. In the future, in order to make use of the results in the field of medical care and health management, it will be important to establish a technique for easily and quickly analyzing proteins that serve as markers for diseases.

[0003] Typical methods for quantifying proteins in samples include (i) absorptiometry, (ii) Biuret method using Biuret reagent, (iii) Lowry method combining phenol reagent and Biuret method, (iv ) Bradford method. The principle, advantages and disadvantages of each method are described below.

[0004] (1) Spectrophotometric method

The principle of this method is that 280nm is attributed to tyrosine and tryptophan in the protein. The protein concentration is calculated using a nearby absorption band. Absorbance at 280nm fluctuates because the content of tyrosine and tributophan varies depending on the type of protein. Force A280nm can be calculated as 1.0 at a concentration of lmgZml. The advantage of this method is that the operation is simple and the sample can be collected after measurement. Disadvantages are that the absorbance varies depending on the type of protein, proteins that do not absorb at 280 nm (collagen, gelatin, etc.) cannot be measured, and contamination with substances that absorb in the ultraviolet region interferes with the determination. (Non-patent document 1).

[0005] (2) Biuret method

The principle of this method, when the protein is reacted with Cu ²⁺ solution under alkaline conditions, by utilizing the phenomenon that Cu 2 ⁺ is colored red by coordinate bonds with the nitrogen atom in the polypeptide chain, 5 40 nm The absorbance at is measured. The advantage of this method is that it is easy to operate with little difference in coloration rate due to protein differences. The disadvantage is that it is not suitable for low-concentration samples with low sensitivity (Non-patent Document 1).

[0006] (3) Lowry method

The principle of this method is to make blue color 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. This method is much more sensitive than the Biuret method because of the strong color development effect derived from peptide bonds. The advantage of this method is that it is most commonly used because of its high sensitivity. On the other hand, the coloration is caused by the reduction reaction, so that the color development is hindered by other reducing substances, the operation is complicated and it takes time to measure, and the color development rate varies depending on the protein. (Non-Patent Document 2).

[0007] (4) Bradford method

In acidic solution, when Coomassie Brilliant Blue G-2500, a trimethane blue dye, binds to protein, the maximum absorption wavelength shifts from 465 nm to 595 nm, and the color tone changes from red purple to blue. is there. This phenomenon is used to quantify proteins. The advantage of this method is that it is very easy to operate without being affected by interfering substances. Disadvantages are differences in coloration rate depending on the type of protein, and surface activity Coloring is disturbed by mixing of the agent (Non-patent Document 3).

[0008] (5) ELISA method

This method is an immunological technique using an antibody that binds only to a specific protein. The disadvantage of this method is that it is necessary to repeat the binding reaction between the target protein (antigen) and the antibody, so that it is impossible to obtain the results quickly, for example, it takes more than 6 hours for the measurement (Non-Patent Documents). Four).

On the other hand, the fluorometric method is a conventional method for analyzing various chemical substances. This method has the advantages of high sensitivity, small amount of sample, large-scale equipment, and no need for skilled techniques. In addition, since the colorimetric analysis method can be judged visually, the protein in the sample can be analyzed quickly and easily.

[0010] There have been reports on protein quantification using a fluorometric method. For example, fluoresamine, which reacts with primary amines, is a strong fluorescent substance that emits fluorescence at 495 nm, and analysis using cyanine dyes is typical. However, the conventional methods react the dye and protein via covalent bonds, so the reaction time is slow, the reactivity changes depending on the type of protein, the calibration curve is not linear, the measurement error due to the association between dyes, There is a problem that the task shift is small. A typical example of using a colorimetric analysis method is analysis using CBB. However, there are problems such as large measurement errors between proteins, unclear color changes, and protein recognition only under acidic conditions (Non-Patent Documents 5 to 8). Furthermore, conventional analysis reagents can only be applied to fluorescence analysis if they are fluorescent analysis reagents, or if they are colorimetric analysis reagents, they cannot be applied to colorimetric analysis. There is no reagent that can perform this analysis method.

[0011] Non-patent document 1: Masato Okada, Kasaki Kakizaki: Revised protein experiment note (above), pp29, Yodosha, 1 999

Non-Patent Document 2: Lowry, 0.H. et al. J. Biol. Chem., 193, 265-275 (1951).

Non-Patent Document 3: Bradford, M.M., Anal.Biochem., 72, 248-254 (1976).

Non-Patent Document 4: Biochemical Dictionary (3rd edition), Tokyo Chemical Doujin, 1998.

Non-Patent Document 5: RP Haugland, VL Singer, LJ Jones, TH Steinberg, US Pate nt 5616502 (1997).

Non-Patent Document 6: Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals, 9th ed .; Molecular Probes Inc .: Leiden, 2002.

Non-Patent Document 7: J. Jones, R. P. Haugland, V. and Singer, Biotechniques, 34, 850 (20 03).

Non-Patent Document 8: R. F. Pasternack, C. Fleming, S. Herring, P. J. Collings, J. DePaula, G. DeCastro, E. J. Gibbs, Biophys. J "79, 550 (2000).

Disclosure of the invention

Problems to be solved by the invention

[0012] An object of the present invention is to solve the above problems and provide a novel compound useful as a reagent capable of detecting a peptide or protein with high sensitivity and ease. Another object of the present invention is to provide a method for efficiently and comprehensively measuring peptide concentration or protein concentration.

Means for solving the problem

[0013] The present inventor has discovered that a novel compound synthesized based on squaric acid is useful as a dye and causes color change and fluorescence emission by binding to a protein. Then, the inventors have obtained the knowledge that the protein can be detected with high sensitivity and ease by using this squaric acid derivative, and the present invention has been completed.

That is, the present invention employs the following configurations 1 to 7.

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

[Chemical 1]

(In the formula, R1 and R2 each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Representation; ml and m2 are integers of 1 to 3; nl = 5—ml, n2 = 5—m2. 2. The compound according to 1, wherein the compound represented by the above formula (I) is a compound represented by the following formula (Π):

[Chemical 2]

3. A protein detection reagent comprising the compound according to claim 1 or 2.

4. The protein detection reagent according to 3, wherein the reagent is obtained by dissolving the compound in a solvent.

5. The protein detection reagent according to 3, wherein the reagent is obtained by immobilizing the compound on a carrier.

6. A test sample is brought into contact with the protein detection reagent according to any one of 3 to 5, and a change in color of the protein detection reagent or fluorescence generated from the protein detection reagent is detected. A method for detecting a protein in a test sample.

7. The detection method according to 6, wherein the protein is detected by a method selected from the group consisting of fluorometry, absorptiometry, and test strip photometry.

The invention's effect

[0015] According to the present invention, a squaric acid derivative useful as a reagent for protein analysis is provided.

This compound reacts instantly just by mixing with protein, and fluorescence emission and color change occur at the same time. Therefore, if quantitative detection of protein is possible, visual quantitative analysis can be performed without force. Therefore, protein detection can be performed quickly and easily without the need for expensive equipment and skilled techniques.

Brief Description of Drawings

[0016] FIG. 1 shows a schematic diagram of fluorescence emission and color change due to complex formation between the analytical reagent and protein.

FIG. 2 shows absorption spectra when various concentrations of BSA are added to the analytical reagent. FIG. 3 shows changes in the color of the analytical reagent solution before and after the addition of BSA. A shows reagent only, B shows reagent + BSA.

FIG. 4 is a graph plotting absorbance at 600 nm against BSA concentration.

[Figure 5] Shows the fluorescence spectra of reagents when various concentrations of BSA are added to the reagents for analysis.

FIG. 6 is a graph plotting the fluorescence intensity at 610 nm against the BSA concentration.

[FIG. 7] A stained image of the protein separated by SDS-PAGE. A shows the case using this analytical reagent, and B shows the case using the CBB method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] The squaric acid derivative of the present invention is a novel compound represented by the following general formula (I).

[Chemical 3]

(In the formula, R1 and R2 each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; ml and m2 are integers of 1 to 3; nl = 5—ml, n2 = 5—m2 As the squaric acid derivative represented by the above formula (I), for example, a compound represented by the following formula (Π) may be mentioned.

[Chemical 4]

The squaric acid derivative represented by the formula (I) of the present invention is a kind of so-called cyanine dye, and is useful as a dye because it has a chromophore containing a squarate ring. And this These dye compounds are particularly suitably used as protein detection reagents.

[0020] In order to use the novel compound of the present invention as a reagent for protein detection, a test sample is brought into contact with a squaric acid derivative represented by the above formula (I), and the color of the squaric acid derivative is adjusted. Changes or fluorescence resulting from the squaric acid derivative is detected. This detection method can be performed by a method such as a fluorescence photometry method, an absorptiometry method, a test paper photoelectric photometry method, or the like.

[0021] Hereinafter, the reagent for protein detection using the squaric acid derivative of the present invention will be described in detail.

1. Reagent for protein analysis

In the present invention, a squaric acid derivative represented by the general formula (I) is used as a protein detection reagent.

The present invention is based on the fact that when a compound having the structure of formula (I) and a protein are brought into contact with each other, they form a complex by hydrophobic interaction, thereby causing a color change and fluorescence emission. ing. By measuring this dye change or fluorescence intensity, qualitative analysis and quantitative analysis of proteins can be performed.

[0023] The present inventor has selected (i) a squaric acid as a site that forms a complex with a protein and simultaneously causes a change in fluorescence and a change in color tone by a solvatochromism effect, and (ii) extends a conjugate system. In order to promote a high molar extinction coefficient and a high fluorescence quantum yield, an olefin group is introduced at the succinic acid site, a phenol group is introduced at the end of the olefin group, and (iii) water solubility is further improved. The structure of formula (I) above was designed by selecting hydroxyl groups to donate and donate electrons.

[0024] The solvatochromism effect usually refers to a phenomenon in which a spectrum changes due to a change in the type of solvent in a solution state. However, in the present invention, the free state force is not limited to that of a protein. It also means that the spectrum changes due to the change in the environment surrounding the dye molecule to the bound state of.

[0025] Preferable specific examples of the compound having a structure represented by the formula (I) include a compound represented by the above formula (Π).

[0026] Since these squaric acid derivatives have the structure of the formula (I), they can bind to proteins by hydrophobic interaction to form a complex. Therefore, protein It is suitably used as a detection reagent.

As shown in FIG. 1, the reagent for protein analysis of the present invention has a structure of the formula (I), so that the coloration in the free state and the coloration in the bound state with other substances are changed. Is born. The change in color is considered to be stabilized by entering the energy level force of the π-electron orbital of the dye molecule into the hydrophobic field of the protein, and the absorption wavelength of light shifts to the longer wavelength side. The structure represented by the formula (I) is considered to cause a change in color tone such as yellow → green, yellow → red, and green → blue due to binding with a protein. Further, as shown in FIG. 1, the reagent for protein analysis is non-fluorescent in the free state, but emits fluorescence in the bound state with other substances. Regarding fluorescence emission, it is thought that the fluorescence quantum yield increases when the dye enters the hydrophobic field of the protein.

[0028] For example, the compound represented by the formula (Π) is red in a free state and is non-fluorescent, but when it is complexed with a protein, it is blue and fluoresces. .

[0029] The compound having the structure of the formula (I) is, for example, 3,4-dihydroxy-3 cyclobutene 1,2 dione and the corresponding aromatic compound dissolved in a toluene η-butanol mixed solvent and then refluxed under an argon atmosphere for 24 hours. (See, for example, KT Arun and D. Ramaiah, J. Phys. Chem. A, 109, 5571-5578 (2005)). After the solvent is distilled off under reduced pressure, purification is carried out using operations such as recrystallization, reprecipitation, or column chromatography.

For example, the compound represented by the formula (Π) can be produced by reacting phenol and squaric acid in a suitable solvent.

[0030] 2. Protein detection method

The protein detection reagent of the present invention forms a complex by hydrophobic interaction with a protein, and changes color and generates fluorescence by the solvatochromism effect. Accordingly, the protein in the sample can be detected by contacting the analytical reagent with a sample that may contain the protein and detecting the resulting color change or fluorescence. The reaction between the reagent for analysis and protein has a high molar extinction coefficient and fluorescence quantum yield, so that even a trace amount of protein can be detected. In the present invention, “detection” of protein is not only a detection of the presence or absence of protein in a sample. It also includes quantitative detection of protein.

[0031] The protein to be analyzed using the present analytical reagent is not particularly limited as long as it includes a plurality of linked amino acids and can bind to the present analytical reagent by hydrophobic interaction. Absent. Examples of proteins to be analyzed include peptides, proteins, protein complexes (eg, glycoproteins, protein-nucleic acid complexes, labeled proteins) and the like.

[0032] The sample is not particularly limited as long as it is suspected of containing a protein, and may be either a liquid sample or a solid sample. Specific examples include a solution that may contain a protein, a gel obtained by electrophoresis of a protein, a membrane to which a protein has been transferred (such as a PVDF membrane), cells or tissues, feces and urine, and the like.

[0033] The analysis reagent used may be used after being dissolved in an appropriate solvent (NaCl solution, HEPES buffer solution, Tris buffer solution, MES buffer solution, methanol, ethanol, DMSO, etc.). It may be used by being fixed to an appropriate carrier (membrane, test paper, etc.).

[0034] Protein detection typically involves contacting a sample with the present analytical reagent and detecting a change in color and Z or fluorescence emission of the analytical reagent. In the present invention, “contact” means that the protein present in the sample and the reagent for analysis are brought into close proximity so that a complex can be formed. For example, mixing a liquid sample with a solution of the analysis reagent, infiltrating the liquid sample into a carrier (membrane, test paper, etc.) on which the analysis reagent is fixed, and applying the analysis reagent to a solid sample Application | coating and operations, such as immersing a solid sample in the reagent for this analysis, are included.

[0035] Conditions for bringing the sample into contact with the present analysis reagent can be appropriately selected by those skilled in the art in consideration of the type and amount of the sample to be used, the contact form, and the like. For example, when it is estimated that the amount of protein contained in a sample is about ~ 1000 μg / mL, 10 to: LOO μΜ, preferably 15 to 80 μΜ (for example, about 20 to 30 μ 例えば) Use this analytical reagent. The contact is carried out at room temperature (about 20-30 ° C.) for about 1-30 minutes, preferably about 1-15 minutes.

[0036] After contacting the sample with the present analytical reagent, the sample is separated using a method known in the art. Detects color change and Z or fluorescence emission of analysis reagents. The color change can be detected, for example, by visual observation, absorptiometry (using a spectrophotometer, a plate reader, etc.), reflection spectrum analysis, or the like. Fluorescence emission can be detected by, for example, a fluorescence method (using a fluorimeter, a fluorescence plate reader, a fluorescence scanner, etc.).

[0037] After the reaction, the bound analytical reagent and protein can be desorbed by, for example, a heating operation or alkalinizing the solvent (for example, pHIO or higher).

[0038] The protein detection method according to the present invention can detect a protein with high sensitivity, speed, and without using a complicated device. Since this analytical reagent binds to a specific protein at a certain ratio, it can be easily quantified by creating a calibration curve. In addition, unlike the antibody that specifically binds to a specific protein, this analytical reagent can bind to any protein, so it is possible to comprehensively analyze multiple types of proteins. Therefore, this detection method is useful in the fields of biochemistry, medicine, and analytical chemistry.

Example

Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0040] (Example 1)

In this example, a squaric acid derivative for use as a protein analysis reagent (dye) was synthesized. This dye was synthesized according to the following reaction formula.

[0041] [Chemical 5]

Specifically, to a 50 ml three-necked flask, 40 mL of a mixed solvent of phenol (0.2 g, 1.7 mmol), squaric acid (0.3 lg, 0.9 mmol), n-butanol: toluene = 1: 3νΖν was added, and argon was added. The mixture was refluxed for 24 hours under an air stream. After evaporating the solvent under reduced pressure, the residue was purified by large thin layer chromatography (S102, acetone: AcOEt = l: lvZv) to obtain a brown solid. [0043] Yield 55%

1H-NMR (400 MHz, Acetone—d6, TMS, r.t., d / ppm) 5.5 to 5.8 (m, 4H).

ESI (+) [M + Na] ⁺ = 353

[0044] (Example 2)

In this example, characteristic evaluation (absorption spectrum measurement) of the dye synthesized in Example 1 was performed.

The absorption spectrum was measured using an absorptiometer (UV-165 0PC, manufactured by Shimadzu Corporation) under the following conditions:

Concentration: [Dye] = 25 M

[BSA] = 0 to: L000 μg / mL

Solvent: 0.9% NaCl aqueous solution

Measurement temperature: 25 ° C

[0045] Fig. 2 shows the absorption spectra of the dyes in order of decreasing force when BSA at concentrations of 0, 60, 120, 250, 500, and 1000 gZm was added. Absorbance increased with increasing BSA concentration. Furthermore, the maximum fluorescence wavelength shifted from 530 nm to 600 nm with increasing BSA concentration. Due to this large shift in the maximum absorption wavelength, the color of the dye solution changed from red to blue before (A) and after (B) addition of BSA, as shown in Fig. 3.

[0046] FIG. 4 shows the results of plotting the absorbance at 600 nm against the BSA concentration. A good linear relationship represented by the following formula was obtained between the absorbance at 600 nm and the BSA concentration.

y = 0. 0004x + 0. 0101

R ² = 0. 997

[0047] (Example 3)

In this example, characteristic evaluation (fluorescence spectrum measurement) of the dye synthesized in Example 1 was performed.

The fluorescence spectrum measurement was performed using a fluorometer (FP-6500 manufactured by JASCO Corporation) under the following conditions:

Concentration: [Dye] = 25 M [BSA] = 0 to: LOOO μg / mL

Solvent: 0.9% NaCl aqueous solution

Measurement temperature: 25 ° C

Excitation wavelength: 595nm

[0048] Fig. 5 shows the results of the measurement of changes in fluorescence spectrum when BSA was added to the dye at a concentration of 0, 60, 120, 250, 500, 1000 (g / mL). As shown in FIG. 5, an increase in fluorescence intensity was observed with an increase in BSA concentration. The fluorescence intensity when BSA was added at 1000 / z gZmL increased about 4500 times compared to the fluorescence intensity of the dye alone.

[0049] When the fluorescence intensity at 610 nm when BSA was added to the dye was plotted against the BSA concentration, a good linear relationship was obtained as shown in FIG. The detection limit was 100 ηgZmL, which was found to be about 20 times more sensitive than the Bradfold method.

[0050] (Example 4)

In this example, the characteristics (electrophoresis experiment) of the dye synthesized in Example 1 were evaluated. Electrophoresis experiments were performed under the following conditions:

Concentration: [Dye] = 0. lmgZmL (in 30% EtOH)

[BSA] = 0 ~ 5 μgZ uel

Immobilizing solution and washing solution; H 2 O: MeOH: AcOH = 87: 10: 3v / v

2

Electrophoresis (SDS—PAGE) system: Sureblot Fl Gel System

[0051] After completion of the electrophoresis, the gel was taken out and washed in a fixed solution for 30 minutes. Thereafter, the gel was immersed in the staining solution for 30 minutes and then washed in the washing solution for 30 minutes.

[0052] The results are shown in FIG. The protein in the gel run by electrophoresis formed a complex with the analytical reagent, and a red band as shown in Fig. 7A was observed.

[0053] Furthermore, when compared with the CBB method (Bradford method) (B in Fig. 7), which is a protein stain for electrophoresis currently on the market, it is clear that the detection sensitivity is higher than that of CBB. It became. Industrial applicability

[0054] The present invention provides a novel squaric acid derivative useful as a protein analysis reagent. Reagents for protein analysis containing this compound can be mixed with protein. Since it reacts instantaneously and fluorescence emission and color change occur at the same time, it is possible to perform visual quantitative analysis without force if high-sensitivity detection of proteins is possible. Therefore, protein detection can be performed quickly and easily without the need for expensive equipment and skilled techniques.

Claims

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

[Chemical 1]

(In the formula, Rl and R2 each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; ml and m2 are integers of 1 to 3; nl = 5—ml, n2 = 5—m2 .)

[2] The compound force represented by the above formula (I): The compound according to claim 1, which is a compound represented by the following formula (Π):

[Chemical 2]

[3] A protein detection reagent comprising the compound according to claim 1 or 2.

[4] The reagent for protein detection according to [3], wherein the reagent is obtained by dissolving the compound in a solvent.

[5] The reagent for protein detection according to [3], wherein the reagent is obtained by immobilizing the compound on a carrier.

[6] A test sample is brought into contact with the protein detection reagent according to any one of claims 3 to 5, and a color change of the protein detection reagent or fluorescence generated from the protein detection reagent is detected. A method for detecting a protein in a test sample, which comprises detecting the protein.

[7] The detection method according to [6], wherein the protein is detected by a method selected from the group consisting of fluorometry, absorptiometry, and test strip photometry.