JP4616194B2 - Biotin compound - Google Patents

Description

  The present invention relates to a biotin compound which is a synthetic intermediate compound of a noble metal compound used in a method for measuring surface plasmon resonance.

  Certain in vivo enzymes have serine, threonine, and tyrosine residues at specific sites such as active centers and allosteric sites, and these hydroxyl groups are phosphorylated or dephosphorylated by enzymes called kinases. Thus, the enzyme activity is regulated. In addition, there is an enzyme whose activity is regulated by phosphorylation (or dephosphorylation) of an amino group or imino group of lysine, arginine, or histidine, or a carboxyl group of aspartic acid or glutamic acid.

  As a metabolic system regulated by such phosphorylation-dephosphorylation, inhibition of glycogen synthesis and its degradation system are well known. This metabolic system is cascade-controlled and regulated mainly by phosphorylation-dephosphorylation.

  In recent years, it has become clear that this phosphorylation-dephosphorylation has an important role in metabolic systems related to diseases.

  For example, cell canceration is said to be caused by abnormal phosphorylation-dephosphorylation. In other words, progression and arrest of the cell cycle are controlled by phosphorylation (or dephosphorylation) of various enzymes (proteins), and this phosphorylation (or dephosphorylation) involves cyclin and cyclin-dependent kinase (CDK). However, when such a mechanism is damaged, phosphorylation (or dephosphorylation) is disturbed, and as a result, cell overgrowth is triggered.

  In addition, protein kinase C is involved in histamine degranulation, which causes allergic diseases such as atopic dermatitis and hay fever, and neurofibrillary tangles occurring in the brain of Alzheimer's disease patients are phosphorylated. It has been shown to be due to tau protein.

  Therefore, grasping the state of phosphorylation / dephosphorylation of proteins may be useful not only for gene expression search and enzyme activity evaluation in living tissue cells but also for diagnosis and treatment of diseases.

  However, conventional methods for identifying phosphorylated proteins (or dephosphorylated proteins) have various drawbacks.

  For example, the enzyme immunization method has an advantage that it can be analyzed even if a target protein sample is a trace amount, but it is difficult to obtain a sufficient amount of the necessary antibody. Moreover, when the target protein is several kDa or less, an antibody that binds to a phosphorylation site in the protein cannot be prepared.

In addition, a method of detecting specific binding to a protein by using phosphoric acid labeled with a radioisotope ³² P is conceivable. However, the handling of the radioisotope requires a caution, and the waste liquid Management and processing are required.

  Furthermore, since the charges differ between phosphorylated protein and dephosphorylated protein, application of two-dimensional electrophoresis is also conceivable. However, in particular, when analyzing a biological sample, it is very difficult to identify a spot because the sample contains many kinds of proteins. In addition, if a radioisotope is used to identify this spot, the above-described problems arise.

  In addition to these techniques for identifying phosphorylated (or dephosphorylated) proteins, surface plasmon resonance (as a general method for searching for compounds (proteins, etc.) that specifically bind to specific compounds (ligands, etc.) Technology using Surface Plasmon Resonance (hereinafter referred to as “SPR”) has been developed (see FIG. 1). This technique is described in detail as follows.

  When light is totally reflected at an interface having a different refractive index, light called an evanescent wave is generated on the total reflection surface. In addition, a kind of electron density wave generated at the metal-dielectric interface called surface plasmon is generated on the metal surface. Since this surface plasmon is resonantly excited by adjusting the angle of the incident light so that the phase velocities of the two (evanescent wave and surface plasmon) coincide, it is possible to greatly increase the electromagnetic field on the metal surface. Become. At this time, the energy of the incident light is lost due to the excitation of the surface plasmon, so that the intensity of the reflected light is reduced.

  The incident angle at which this absorption occurs and the wavelength of the incident light change very sharply according to the state of the metal surface, particularly within several hundred nm. In other words, the intensity of reflected light reacts sensitively due to changes in the state of the metal surface such as the presence or absence of a compound. Therefore, if a test sample is allowed to act after binding a ligand to the metal surface, it interacts with the ligand. The intensity of reflected light changes depending on the presence or absence of the compound to be used. Therefore, as shown in FIG. 1, the presence or absence of a compound that interacts with the ligand or the like is determined by comparing the reflected light intensity with and without the addition of the sample to be sampled on the metal surface. be able to. Furthermore, it is conceivable to apply this technique to bioimaging. That is, it may be possible to capture the localization of a compound that interacts with a specific compound as an image in a cell or a living tissue.

  As an example of such a technique, the one described in JP-T-11-512186 can be cited. According to this document, when a running buffer → test sample → running buffer is continuously applied to a noble metal film as shown in FIG. 1, the incident angle at which SPR is observed is the time as shown in FIG. Change. In this technique, this change over time is measured, and the relationship between the minimum and maximum reflectance and the relationship between the refractive index and time is determined, thereby dissociating the compound supported on the noble metal film from the compound in the test sample. It is said that the constant, binding constant, and concentration of the compound in the sample can be determined.

  In addition, JP-A-10-505910 discloses a method in which N- (5-amino-1-carboxypentyl) iminodiacetic acid is bonded to a noble metal film via carboxymethylated dextran and nickel is further coordinated. A technique for performing SPR measurement using the same is disclosed. Since this nickel complex shows a specific affinity for a peptide having two adjacent histidine residues, it is called a His-tag, and a peptide having a dihistidine residue is detected from the test sample. Can be detected.

As a method for measuring surface plasmon resonance, the application of Raman spectroscopy can be considered. Raman spectroscopy is based on measuring scattered light (Raman scattered light) at a frequency other than the same frequency (ν ₀ ± ν _i ) in the scattered light generated by irradiating a substance with monochromatic light at a constant frequency ν ₀ . This is a technology for obtaining information on compounds. Specifically, the Raman frequency ν _i is equal to the vibration frequency between the vibrational and rotational energy levels of the molecules and crystals that make up the material, so information for determining, identifying, and quantifying the energy level of the material The source (see “Chemical Dictionary”, Tokyo Kagaku Dojin).

  However, since this Raman scattered light is very weak, it has been conventionally measured after being enhanced using the surface plasmon resonance effect (for example, Japanese Patent Laid-Open No. 9-257578). That is, in general, light does not couple with electron waves (plasmons), but coupling occurs on the surface of metal particles. Therefore, in this technique, the weak Raman band intensity is enhanced by causing a metal to be present in the vicinity of the measurement sample.

  However, the effect of enhancing the Raman band intensity according to such a conventional technique is not always satisfactory. This is because the surface plasmon resonance effect is higher as the distance between the target molecule and the metal is shorter. However, even if the substrate (metal) 2 and the sample 3 are brought close to each other as shown in FIG. This is because the molecule does not touch the metal. Such a situation is the same when a metal is added to an aqueous solution sample.

  By the way, in Hironori Takeda et al., RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Vol.17, Issue 18, pp.2075-2081 (2003), a compound that specifically binds to a phosphate group (phosphate monoester group) is used. Thus, a method for obtaining the molecular weight of a phosphorylated compound is described. However, this document neither describes nor suggests the technical idea of bringing a metal close to the target compound via this specific binding compound or the idea of applying the compound to the measurement of surface plasmon resonance.

  Under the circumstances described above, the problem to be solved by the present invention is to detect a phosphorylated peptide (protein) in a test sample and to determine whether or not the peptide is phosphorylated. It is an object of the present invention to provide a synthetic intermediate compound of a noble metal compound having a substituent (phostag) specific to a phosphate monoester group and having a high coordination ability on the surface.

  In order to solve the above problems, the inventors of the present invention have made extensive studies on a metal complex that can be coordinated to a phosphate group (phosphate monoester group) bound to a protein. Coordination binding ability to two hydroxyl groups in phosphate ion or phosphate monoester is extremely high. As a result, it strongly coordinates to phosphate group (phosphate monoester group) bound to peptide, It was found that the SPR can be measured using this substituent because the mixed sample can be specifically bound to the phosphorylated peptide to form a complex.

  That is, the first surface plasmon resonance measurement method according to the present invention is a surface plasmon resonance measurement method in which a noble metal compound is arranged on the bottom surface of a prism, and the reflected light is detected by irradiating the prism with light. As the noble metal compound, one having a substituent represented by the following formula (I) on the side opposite to the side in contact with the prism is used, and the noble metal compound is tested on the side having the substituent (I). It is characterized by adding a sample.

[Wherein, X represents a linker group. ]

  The second surface plasmon resonance measurement method according to the present invention is characterized in that Raman spectroscopy is performed by adding a noble metal compound having a substituent represented by the above formula (I) on the surface to a test sample. And

  In the noble metal compound, the substituent of formula (I) is bonded to the noble metal surface via a linker group. Some such linker groups utilize biotin and streptavidin or avidin. Since biotin has a very high affinity for streptavidin or avidin, a compound of the following formula (IX) or (X) containing a substituent of formula (I) is a surface of a noble metal having streptavidin or avidin, etc. Can be easily combined.

  That is, the biotin compound of the present invention is represented by the following formula (IX).

[Wherein Y is a C1-C6 alkylene group, amino group, ether group, thioether group, carbonyl group, thionyl group, ester group, amide group, urea group, thiourea group; amino group, ether group, thioether group, carbonyl group A C1-C6 alkylene group having at one end a group selected from the group consisting of a group, thionyl group, ester group, amide group, urea group, thiourea group; amino group, ether group, thioether group, carbonyl group, thionyl group, ester A C1-C6 alkylene group having the same or different groups selected from the group consisting of a group, an amide group, a urea group, and a thiourea group at both ends; or two or more groups selected from the group consisting of these groups are linear A bonded group. ]

  Another biotin compound of the present invention is characterized in that zinc ions are coordinated to the biotin compound (IX).

  According to the method for measuring surface plasmon resonance (SPR) according to the present invention, it is possible to easily determine the presence or absence of phosphorylated peptide (protein) even in a test sample containing various compounds such as a biological sample. . In addition, the amount and concentration of the phosphorylated peptide can be determined. It can also be determined whether the peptide is phosphorylated. Therefore, it is very useful in that it can be applied to disease diagnosis and the like by applying the method of the present invention to a biological sample or the like.

  In addition, the biotin compound of the present invention can be easily bound to streptavidin or a compound having avidin. Therefore, the biotin compound of the present invention is similarly useful as a synthetic intermediate compound for synthesizing noble metal compounds and the like used in the method of the present invention.

  First, the SPR measurement method according to the present invention will be exemplified.

  What is necessary is just to measure SPR in the 1st method of this invention using a well-known apparatus. For example, the apparatus disclosed in US Pat. No. 5,313,264 can be used. Such SPR measurement methods are (1) no labeling operation such as introduction of fluorescent chromophores, (2) high sensitivity to relatively high molecular weight molecules, (3) thiol groups and disulfide groups. Since the molecule having a bond is easily bonded to the surface of a noble metal compound such as gold, it has an advantage that substituents can be introduced to the surface at a high density, so that it is extremely suitable for detection of phosphorylated peptides.

The measurement principle is as described above with reference to FIGS. 1 and 2, and the SPR measurement device will be described in detail as follows. In FIG. 1, when the light is irradiated from the prism side to the noble metal film so as to be totally reflected, there is an incident angle at which the reflected light intensity decreases due to SPR. Since the angle at this time changes sharply according to the amount of the compound bonded to the noble metal film, that is, the mass change, the SPR measurement apparatus measures the mass change from the reflected light intensity (resonance unit, 1RU = 1 pg / mm). ² ) can be displayed as.

  Therefore, after introducing a substituent showing specific binding ability to the phosphorylated peptide to the noble metal film, the test sample is acted to acquire data, and the test sample is compared with the data in the steady state. It becomes possible to grasp the presence or absence and the amount of phosphorylated peptide in it.

  In the second method of the present invention, a conventional Raman spectroscopy apparatus can be used as it is. However, the noble metal compound (noble metal particles) of the present invention is allowed to act on the test sample. As a result, the peak of the phosphorylated peptide is not only shifted by the binding of the noble metal compound, but also its strength is enhanced by the surface plasmon resonance effect, compared to the case where the noble metal compound of the present invention is not allowed to act. . Therefore, whether or not the peptide is phosphorylated can be determined by comparison with the Raman spectrum when the noble metal compound of the present invention is not allowed to act.

  The test sample used in the second method of the present invention is an aqueous solution or an aqueous dispersion. This is because the noble metal compound of the present invention is bound to the phosphorylated peptide. Note that the test sample may be, for example, a biological sample as it is, but it is preferable to purify a peptide to be judged whether or not it is phosphorylated. This is because in Raman spectroscopy, a satisfactory spectrum may not be obtained due to the presence of impurities.

  The substituent used in the noble metal compound of the present invention is extremely excellent in specific binding ability to a phosphorylated peptide and has the following structure.

[Wherein, X represents a linker group. ]

  In the above formula (I), the reason for selecting Zn as the coordination metal is that the coordination ability to the phosphate group (phosphate monoester group) of the phosphorylated protein is extremely high.

  The “linker group” is a bond between the noble metal part and the main skeleton (main part that interacts with phosphorylated protein. Hereinafter referred to as “phos tag” or “Phos-tag”) in the noble metal compound of the present invention. Facilitates the production of the noble metal compound according to the present invention, increases the degree of freedom of the substituent (I), and facilitates the coordination between the substituent (I) and the phosphate group bound to the peptide. Has the effect of

  The “linker group” is not particularly limited as long as it has the above-described action. For example, a sugar chain, a C1-C6 alkylene group, an amino group (—NH—), an ether group (—O—), a thioether group ( -S-), carbonyl group (-C (= O)-), thionyl group (-C (= S)-), ester group, amide group, urea group (-NHC (= O) NH-), thiourea group (-NHC (= S) NH-), conjugate of biotin and streptavidin, conjugate of biotin and avidin; amino group, ether group, thioether group, carbonyl group, thionyl group, ester group, amide group, urea A sugar chain having at one end a group selected from the group consisting of a group and a thiourea group; and an amino group, an ether group, a thioether group, a carbonyl group, a thionyl group, an ester group, an amide group, a urea group, a thiourea group, biotin, Streptavidin conjugate, biotin and C1-C6 alkylene group having at one end a group selected from the group consisting of conjugates with vidin; amino group, ether group, thioether group, carbonyl group, thionyl group, ester group, amide group, urea group, thiourea group A C1-C6 alkylene group having both the same or different groups selected from the group; and a group in which two or more groups selected from the group consisting of these groups are linearly bonded.

  The end of the linker group that is bonded to the noble metal portion is preferably a thioether group. This is because such a noble metal compound can be easily produced using a thiol compound or a disulfide compound.

  In the present invention, the “sugar chain” refers to a general saccharide linked in a linear or branched chain, and examples thereof include dextran in which D-glucose is polymerized by a glucoside bond. In addition to the above-mentioned action of the linker group, this sugar chain is highly compatible with a biological sample because of its high hydrophilicity, and can easily synthesize a branched chain. There is also an advantage that more main skeletons can be bonded.

  Here, the “C1-C6 alkylene group” means a linear or branched divalent aliphatic hydrocarbon group having 1 to 6 carbon atoms, such as methylene, ethylene, propylene, tetramethylene, hexa Examples include methylene, methylethylene, methylpropylene, dimethylpropylene, etc., preferably a C1-C4 alkylene group, more preferably a C1-C2 alkylene group.

  The length of the linker group described above is not particularly limited, but is preferably within 200 nm, more preferably within 100 nm. This is because the shorter the length of the linker group, the more sensitively the phosphorylated peptide can be detected.

  In addition, in the substituent (I), it is possible to introduce a methyl group or the like into the pyridine ring as having the same effect as the present invention, but such an equivalent is also within the scope of the present invention. Shall be included.

  Further, the position of the linker group in the substituent (I) according to the present invention is not particularly limited, and may be present at the position shown in the following substituent (I ′).

  The substituent (I ′) and the substituent (I) are completely equivalent, and it is not always clear which substituent will be synthesized, but it is considered that it is actually a mixture of the two. The group (I ′) is also included within the scope of the present invention.

  In the present invention, the “noble metal” refers to gold, silver, platinum, rhodium, ruthenium, palladium, osmium, iridium, and in the present invention, it is preferable to use any of gold, silver, platinum, or rhodium. It can be suitably used. This is because gold has been demonstrated to exhibit the surface plasmon resonance effect well.

  The shape of the noble metal compound according to the present invention is preferably a film shape in the first method. This is because it can be directly applied to a surface plasmon measuring apparatus. The thickness of the noble metal film is not particularly limited, but is preferably 10 to 100 nm, and usually about 50 nm.

  On the other hand, the second method is preferably in the form of particles. In the present invention, since the noble metal compound is bound to the phosphorylated peptide contained in the test sample dissolved or dispersed in the test sample, it is necessary to disperse the noble metal compound in the test sample. Because there is. Incidentally, the average particle diameter is preferably in the range of 30 to 50 nm. This is because if it is less than 30 nm, the effect of enhancing Raman scattered light may not be sufficient, and if it exceeds 50 nm, particles may aggregate excessively during measurement. However, since the said range is an average particle diameter, the particle | grains outside the said range may exist.

  The method for measuring the average particle size is not particularly limited, but it is considered that the particle size to be measured is in the range of several nanometers to several tens of nanometers, so that a laser scattering method suitable for particle size measurement of fine particles can be applied ( The average particle size may be calculated after measuring the particle size distribution with a laser scattering photometer.

  The noble metal compound of the present invention can be easily produced by a method characterized by including Scheme 1, but the production method is not limited to the following.

[Wherein, X represents the linker group described above, and R ¹ and R ² represent a reactive group for forming a —X-phostag group on the surface of the noble metal film. ]

In the above scheme 1, first, R ¹ which is a reactive group for forming the linker group X is substituted on the noble metal. Here, the type of bond between the reactive group R ¹ and the noble metal is not necessarily clear, and the type of bond is not particularly limited. For example, it is known that a thiol compound or a disulfide compound spontaneously adsorbs on a noble metal surface to form a monomolecular film called a self-assembled monomolecular film. Therefore, when the reactive group R ¹ and the noble metal are bonded via a sulfur atom, the type of bond between the noble metal and the sulfur atom in the above formula is not particularly limited, and is linked by some interaction. To do.

When the final target product is a noble metal film, the noble metal to be substituted with the reactive group R ¹ may be cut into a shape suitable for the SPR measuring device to be used after extending the raw noble metal to an appropriate thickness. In the case of particles, after manufacturing by applying a known metal particle manufacturing method, particles that are too large or too small may be removed using a membrane filter or the like corresponding to the desired particle size.

In Scheme 1 above, it is known that when the reactive group R ¹ is bonded to the surface of the noble metal via a sulfur atom, the reaction between the thiol compound (eg, R ¹ —SH) and the noble metal proceeds very easily. The reaction conditions etc. may follow conventional methods. For example, both can be condensed only by bringing the noble metal surface into contact with the solution of the thiol compound.

Subsequently, in order to bind the phostag precursor via the linker group X, a tetrakis (pyridin-2-ylmethyl) -1,3-diaminopropan-2-ol derivative (compound (VI)) having a substituent R ² is obtained. React. The types of R ¹ and R ² in the step of reacting R ¹ and R ² in Scheme 1 above, the solvent, the reaction temperature, other reagents, the purification method, etc. are mainly determined by the type of X. For example, in the case of the X by combining R ¹ and R ² by an amide bond, as the combination of R ¹ and R ² are groups and activated with a terminal amino group (primary amino group) And a combination with a carboxy group. The general reaction conditions in this case may be those generally used in the field of synthetic organic chemistry. Thus, a noble metal compound having a substituent (VII) on the surface can be obtained.

  Moreover, the noble metal compound which has a substituent (VII) can be manufactured also with the following method.

[Wherein X ″ represents a linker group in which the noble metal side is a sulfur atom, and X ′ represents a portion other than the terminal sulfur atom in X ″ (provided that X ″ represents only a sulfur atom) , X ′ will simply represent a covalent bond). ]

  As described above, since the reaction between the thiol compound and the noble metal proceeds very easily, tetrakis (pyridin-2-ylmethyl) -1,3-diaminopropane-2-substituted with a group having a thiol group at the terminal is used. By using all derivatives, a precursor compound of a noble metal compound having a phostag on the surface can be synthesized.

  Finally, a noble metal compound having a phos tag on the surface can be obtained by adding a metal salt to the noble metal compound having a substituent (VII). For example, zinc nitrate (II) or zinc acetate (II) may be added. However, when zinc acetate (II) is added, the following compound once coordinated with acetic acid is obtained.

  This compound is more stable and convenient for storage than the substituent (I), but is equivalent to the substituent (I) and can be used in the same manner as the substituent (I). That is, at the time of SPR measurement, a phosphorylated peptide can be detected because a phosphate monoester group is coordinated with acetic acid in an exchangeable manner.

  In the above scheme 1, a raw material compound (compound (VI)) for bonding a phos tag to a noble metal can be produced by the following scheme 2.

[Wherein R ² has the same meaning as described above. “Hal” represents a halogen atom, preferably a bromine atom. ]

  A commercially available compound (II) (1,3-diamino-2-propanol) as a raw material compound can be used. In addition, since the compound (III) and the compound (V) have a relatively simple structure, they can be used commercially or synthesized by methods known to those skilled in the art.

  In scheme 2, first, compounds (II) and (III) are subjected to a condensation reaction in the presence of a catalyst to obtain compound (IV). In this reaction, compound (III) may be introduced step by step, but compound (IV) can also be obtained by one step reaction by using 3 equivalents or more of compound (III).

  In Scheme 2, a reductive amination reaction is performed as a condensation reaction. The solvent used in that case can be used without particular limitation as long as it can substantially dissolve the compounds (II) and (III) and does not inhibit the reaction. For example, methanol, ethanol, isopropanol Alcohols such as: ethers such as diethyl ether, tetrahydrofuran, dioxane, etc .; water; or a mixed solvent thereof.

  In the reductive amination reaction, the compounds (II) and (III) can be first condensed in the presence of concentrated hydrochloric acid as a catalyst and then reduced with a general reducing reagent.

  For the reaction temperature and reaction temperature, suitable conditions may be adopted depending on the type of raw material compound and the like, for example, the reaction is carried out at 20-80 ° C. for 12-100 hours.

  After completion of the reaction, the solvent and the like are distilled off under reduced pressure, water is added, extraction is performed with a water-insoluble solvent, the oil phase is dried over anhydrous magnesium sulfate and the solvent is distilled off under reduced pressure. Next, the residue can be purified by a known method such as silica gel column chromatography to obtain compound (IV).

  The method for obtaining compound (IV) is not limited to the method shown in Scheme 2. For example, compound (IV) may be synthesized from compound (II) and a halogen compound.

Next, compound (VI) can be obtained by reacting compound (V). This reaction can employ a general tertiary amine synthesis reaction. For example, the condensation is carried out in the presence of a base in a solvent. In this step, a protective group may be introduced and deprotected as appropriate depending on the type of R ² . Alternatively, after performing the steps using a compound having a place of inert substituents of the compound (V) Medium R ^2, by converting the inactive substituent to R ² by a functional group transformation, the compound (VI ) May be synthesized. For example, a compound having a nitro group as an inert substituent may be used, and after the step, the nitro group may be converted to an amino group which is a reactive group.

  As a substituent that can be used in the method according to the present invention, the lower complex compound (VIII) can be used instead of the substituent (I).

[Wherein, X has the same meaning as described above. R ^{3 to} R ⁵ represent an electron-donating substituent at the 4- or 6-position on the pyridine ring. ]

  The substituent (VIII) used in the method of the present invention is excellent in coordination to zinc because the pyridine nitrogen is electrically rich due to the electron donating substituent introduced at an appropriate substitution position. As a result, it is easy to manufacture and has stability. Those equivalent to the substituent (I) can be used.

  Hereinafter, production examples and test examples will be shown to describe the present invention in more detail, but the scope of the present invention is not limited thereto.

Production Example 1 Production of Phostag-Sensor Chip for SPR Analysis A sensor chip (BIOCRE, Sensor Chip CM5) coated with carboxymethyldextran was set in an SPR measuring device (BIOCRE, BIOCORE J).

10 mM HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid containing 5 × 10 ⁻³ % (v / v) Tween 20,0.20 M sodium nitrate and 10 μM zinc nitrate as a running buffer ) -Sodium hydroxide aqueous solution (pH 7.4) was used. The temperature of the sensor chip was 25 ° C., and the running buffer flow rate was 30 μL / min. After confirming that the value of surface plasmon resonance was stable, mixing of EDC (1-ethyl-3,4-dimethylaminopropylcarbodiimide, 200 mM) and NHS (N-hydroxysuccinimide, 50 mM) as a carboxyl group activator The carboxyl group of the sensor chip was activated by adding an aqueous solution for 6 minutes.

  Next, N, N, N′-tri (2-pyridylmethyl) -N ′-[5-N ″-(2-aminoethyl) carbamoyl-2-pyridylmethyl] is used to support the phos tag on the sensor chip. A 50% (v / v) acetonitrile solution (10 mM) of 1,3-diaminopropan-2-ol was added for 6 minutes. Thereafter, an aqueous monoethanolamine solution (1.0 M) was added for 6 minutes in order to block the remaining activated carboxyl groups.

  By the above operation, a sample flow path (flow cell A) having a sensor unit combined with a phos tag was produced.

Comparative production example 1
In the production example 1 described above, the sample channel (flow cell B) having the reference portion to which the phos tag is not bonded is obtained by using the same method except that the phos tag is supported, including activation of the carboxyl group and blocking thereof. Prepared in parallel with A.

Test example 1
As analysis samples, (i) β-casein (pentaphosphorylated protein, SIGMA), (ii) dephosphorylated β-casein, and (iii) bovine serum albumin (BSA, New England BioLabs) were used. Dephosphorylated β-casein was prepared by incubating a mixed solution of β-casein 10 mg / mL (50 μL), potato-derived acid phosphatase (SIGMA) and 0.20 M MES-NaOH (pH 6.8, 50 μL) for 12 hours at 38 ° C. Was prepared. Each sample was dissolved in the running buffer used in Production Example 1 to obtain a sample solution having a sample concentration of 1.5 μM.

  SPR measurement was performed on the sample solution. Specifically, for each of flow cells A and B prepared in Production Example 1 and Comparative Production Example 1 and stabilized with a running buffer, each sample solution was flowed at a temperature of 25 ° C. and a flow rate of 30 μL / min. It was allowed to bind for 15 minutes and then dissociated by running only running buffer for 15 minutes. After measurement of each sample solution, 25 mM monopotassium phosphate-25 mM disodium phosphate aqueous solution (pH 6.86) for 6 minutes, 0.20 M ethylenediaminetetraacetate aqueous solution (pH 7.4) for 6 minutes, and the above running buffer for 5 minutes. Then, the sensor chip was reactivated (removal of remaining bound substances).

  The SPR measurement results of each sample (i) to (iii) are shown in FIGS. 3 to 5, respectively, and the difference between the RU value of the flow cell A and the RU value of the flow cell B in the measurement result of each sample is shown in FIG. . According to the results of FIGS. 4 to 6, the changes in the flow cells A and B are almost the same, and it can be seen that dephosphorylated β-casein and BSA do not specifically bind. On the other hand, the results of FIGS. 3 and 6 revealed that phosphorylated β-casein specifically binds to the sensor portion to which the phostag is bound (maximum binding amount: 3150 RU). Therefore, according to the method of the present invention, it was demonstrated that only phosphorylated peptides can be detected.

Test example 2
As analysis samples, (iv) phosphorylated SRC peptide (monophosphorylated peptide, ANA SPEC) and (v) SRC peptide (non-phosphorylated peptide, ANA SPEC) were used. Each sample was dissolved in the running buffer used in Production Example 1 to obtain a sample solution having a sample concentration of 1 to 15 μM.

  The sample solution was subjected to SPR measurement using the flow cells A and B prepared in Production Example 1 and Comparative Production Example 1. Specifically, in Test Example 1 above, the binding time and dissociation time of the sample were 5 minutes, the sensor chip was reactivated (removal of the remaining bound material) was 0.40 M phosphate buffer (pH 7.0) for 5 minutes, and 0.20. M SPR measurement was performed under the same conditions as in Test Example 1 except that an aqueous solution of disodium ethylenediaminetetraacetate (pH 7.4) was flowed for 5 minutes and the running buffer of Production Example 1 was flowed for 5 minutes. The results are shown in FIG.

  From the results, the non-phosphorylated peptide shows almost no change even at a concentration of 15 μM, whereas the phosphorylated peptide has a concentration of 15 μM as well as 1 and 5 μM, I was able to clearly understand its existence. Therefore, according to the present invention, it has been clarified that it is possible to clearly determine whether or not phosphate is bound even for peptides having the same amino acid sequence.

Production Example 2 Production of Phostag-sensor chip for SPR analysis Streptavidin-sensor chip (BIOCRE, Sensor Chip SA) with streptavidin bonded to the surface is used as an SPR measuring device (BIOCRE, BIOCORE J). I set it.

10 mM HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid containing 5 × 10 ⁻³ % (v / v) Tween 20,0.20 M sodium nitrate and 10 μM zinc nitrate as a running buffer ) -Sodium hydroxide aqueous solution (pH 7.4) was used. The temperature of the sensor chip was 25 ° C., and the running buffer was allowed to flow at a flow rate of 30 μL / min until the surface plasmon resonance value was stabilized.

  Next, in order to carry the phos tag on the sensor chip, N, N, N′-tri (2-pyridylmethyl) -N ′-[5-N ″ -2- (6-D- Biotinamide hexacarboxyamidoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropan-2-ol (a compound having the following structure) 1.0 mM was added to the above running buffer solution. The temperature was 25 ° C., the flow rate was 30 μL / min, and the binding time was 6 minutes.

  The above compound is coordinated to zinc ions in the running buffer to become a phos tag, and biotin at the end shows extremely high affinity for streptavidin, so that the phos tag is supported on the sensor chip.

Comparative production example 2
A sensor chip to which no phos tag was bonded was produced using the same method as in Production Example 2 except that the phos tag was supported.

Test example 3
As an analysis sample, β-casein (5 × 10 ⁻³ % (v / v) Tween 20, 0.20 M sodium nitrate and 10 μM zinc nitrate in 10 mM HEPES-sodium hydroxide aqueous solution (pH 7.4)) dissolved in running buffer ( Pentaphosphorylated protein (SIGMA) was used. The sample concentration was 1.5 μM.

  For the analysis sample, SPR measurement was performed at a temperature of 25 ° C., a flow rate of 30 μL / min, a binding time of 15 minutes, and a dissociation time of 10 minutes. After the measurement, the sensor chip was reactivated (remaining of bound substances) by flowing 0.40M phosphoric acid aqueous solution for 6 minutes, 0.20M ethylenediaminetetraacetic acid aqueous solution (pH 8.0) for 6 minutes, and running buffer for 5 minutes. Removed).

  The measurement results are shown in FIG. According to the result, since the RU increased by 2056 by flowing the analysis sample, it can be seen that the phosphorylated protein is bound to the noble metal film of the present invention (the noble metal sensor chip to which the phostag is bound). In addition, under the same conditions as described above, experiments were performed using bovine serum albumin, which is a non-phosphorylated protein, instead of phosphorylated protein, but RU did not change at all. Therefore, it was clarified that the noble metal film of the present invention can detect only the protein to which phosphoric acid is bound.

  Moreover, although the experiment was performed on the sensor chip manufactured in Comparative Manufacturing Example 2 under the same conditions, the RU did not change at all.

It is a schematic diagram which shows one method for measuring SPR. Adjust the wavelength and angle of incident light so that SPR is measured (decrease in reflected light intensity), add a test sample to the noble metal film to which the ligand is bound, and observe the change in incident light intensity Is. In the figure, 1 is a prism, 2 is a noble metal film, 3 is a ligand, 4 is a protein contained in the test sample, and shows an interaction with the ligand. In the SPR measurement shown in FIG. 1, it is a figure which shows typically the time-dependent change of the SPR angle when running buffer-> test sample-> running buffer is made to act continuously. This shows that when the compound in the test sample is bonded to the noble metal film, the incident angle at which the SPR is measured changes. It is the result of having performed SPR measurement about the sample containing (beta) -casein phosphorylated. It is the result of having performed SPR measurement about the sample containing beta-casein dephosphorylated. It is the result of having performed SPR measurement about the sample containing bovine serum albumin (BSA) which is a general protein which is not phosphorylated. It is a figure which shows the difference of the RU value of the flow cell B from the RU value of the flow cell A in the measurement result shown to FIGS. It is the result of having performed SPR measurement about the sample containing a phosphorylated peptide (P-p60csrc) and a non-phosphorylated peptide (p60csrc). It is the result of having performed SPR measurement about the sample containing (beta) -casein phosphorylated.

Claims (4)

A biotin compound represented by the following formula (IX).
[Wherein Y is a C1-C6 alkylene group, amino group, ether group, thioether group, carbonyl group, thionyl group, ester group, amide group, urea group, thiourea group; amino group, ether group, thioether group, carbonyl group C1-C6 alkylene group having at one end a group selected from the group consisting of a group, thionyl group, ester group, amide group, urea group, thiourea group; amino group, ether group, thioether group, carbonyl group, thionyl group, ester A C1-C6 alkylene group having the same or different groups selected from the group consisting of a group, an amide group, a urea group, and a thiourea group at both ends; or two or more groups selected from the group consisting of these groups are linear A bonded group. ]   A biotin compound in which zinc ions are coordinated to the biotin compound according to claim 1. A biotin compound represented by the following formula:
A biotin compound in which zinc ions are coordinated to the biotin compound according to claim 3.