RU2315771C2 - Method for labeling phosphorylated peptides, method for selective adsorption of phosphorylated peptides, chelating compounds used in such methods, method for preparing chelating compounds and parent compounds for preparing chelating compounds - Google Patents

Abstract

FIELD: organic chemistry, peptides.

SUBSTANCE: invention proposes a method for labeling phosphorylated peptides, method for adsorption of phosphorylated peptides and compounds possessing high coordination capacity with respect to phosphorylated peptides that are used in such methods, and compounds used as parent substances. Chelating compound is represented by the formula (I):

wherein X represents linker group; Y represents fluorescent group or biotin as labeling group. Compound of the formula (I) provides easy detection and identification of phosphorylated peptide in a sample prepared from a living body.

EFFECT: improved methods of preparing and analysis.

13 cl, 9 dwg, 8 ex

Description

Field of Invention

The present invention relates to a method for labeling phosphorylated peptides, a method for selectively adsorbing phosphorylated peptides, to complex compounds used in such methods, to a method for producing complex compounds and to compounds used as starting materials in a preparation method.

BACKGROUND OF THE INVENTION

In vivo enzymes are known that contain a serine, threonine or tyrosine residue at a specific site corresponding to the active site or allosteric site. The enzymatic activity of such enzymes is regulated by phosphorylation or dephosphorylation of hydroxyl groups in such residues by an enzyme called kinase, etc. Enzymes are also known whose enzymatic activity is regulated by phosphorylation or dephosphorylation of the amino group or the imino group of lysine, arginine or histidine, or the carboxyl group of aspartic acids or glutamic acids.

One example of metabolic systems regulated by the above phosphorylation-dephosphorylation is a system for suppressing glycogen synthesis and its decomposition. First of all, there is a regulation of the activation pathway of such a metabolic system by phosphorylation-dephosphorylation processes.

Recent studies have clearly shown that phosphorylation-dephosphorylation plays a significant role in disease-related metabolic systems.

For example, it is believed that one of the causes of cell carcinogenesis is abnormal phosphorylation-dephosphorylation. Specifically, the development and arrest of the cell cycle is regulated by phosphorylation or dephosphorylation of various enzymes, i.e. proteins. Cyclin and cyclin-dependent kinase (CDK) are relevant factors in phosphorylation or dephosphorylation. If the mechanism associated with cyclin and CDK is impaired, phosphorylation or dephosphorylation may be uncontrolled, thereby triggering the mechanism of abnormal cell proliferation.

In addition, it is known that protein kinase C is associated with histamine degranulation, which causes allergic diseases such as atopic dermatitis and hay fever, and that phosphorylated tau protein causes neurofibrillary plexus in the brain in patients with Alzheimer's disease.

In light of the foregoing, an understanding of the phosphorylation-dephosphorylation conditions of proteins provides a useful tool for various measurements, not only in studying gene expression in living tissue cells and determining the enzymatic activity of cells, but also in the diagnosis of diseases or medical treatment.

Conventional methods for identifying phosphorylated proteins or dephosphorylated proteins have various disadvantages.

For example, although enzyme immunoassay is preferable for analyzing a target protein sample in very small amounts, it is very difficult to obtain sufficient target protein antibodies. In addition, in the case when the level of the target protein is several kDa or lower, it is impossible to obtain antibodies that reliably bind to the site in the protein where phosphorylation occurs.

A method is proposed for detecting a protein specifically bound by phosphoric acid using phosphoric acid labeled with a radioactive isotope ³² P. However, special care should be taken when handling radioactive isotopes, and the relevant rules for the introduction and elimination of liquid radioactive isotope waste should be observed.

The idea of using two-dimensional electrophoresis is proposed, taking into account the fact that differentiation of electric charges between phosphorylated proteins and dephosphorylated proteins occurs. However, it is very difficult to determine the band or spot of a phosphorylated or dephosphorylated protein when analyzing a sample obtained from a living organism, because The sample contains many proteins. In addition, the use of a radioactive isotope to determine a band or spot is associated with the above problems.

Document Morio YASHIRO, et al. [Preparation and Study of Dinuclear Zinc (II) Complex for the Efficient Hydrolysis of the Phosphodiester Linkage in a Diribonucleotide], Journal of the Chemical Society, Chemical Communications, pp. 1793-1794 (1995), discloses a zinc complex. The zinc complex has such a function that two zinc ions in the complex decompose the phosphoric acid group, namely the phosphoric acid diester, from the dinucleotide. However, the zinc complex, as disclosed in this document, has a simple catalyst function. The specified document does not disclose the ability of the zinc complex to coordinate coordinated with the phosphoric acid group. The experiments conducted by the inventors showed that the dissociation constant of the zinc complex relative to the phosphoric acid group located between the two nucleosides in the form of a sandwich, namely a phosphoric acid diester, is extremely high. In other words, the zinc complex has a low coordination ability with respect to the phosphoric acid diester group.

Also, Hidekazu ARII, et al. [A novel diiron complex as a functional model for hemerythrin], Journal of Inorganic Biochemistry, 82, pp. 153-162 (2000), discloses an iron complex having a structure similar to that of zinc. The iron complex, however, is a product synthesized as a model of hemeritrin, namely, a carrier protein that carries oxygen molecules. As in the case of the above document, this document also does not disclose and does not even contain any remote assumptions regarding the coordination relationship of the iron complex with the phosphoric acid monoester group.

Description of the invention

In light of the foregoing, the aim of the present invention is to overcome the problems of the prior art. Another objective of the present invention is to provide a method for labeling phosphorylated peptides, namely proteins, for easier detection and a method for selectively adsorbing phosphorylated peptides for purification and the like.

Another objective of the present invention is the provision of compounds with high coordination ability in relation to phosphorylated peptides that can be used in the method of labeling and in the method of selective adsorption, a method for producing compounds and compounds that are the starting materials used in the method of obtaining.

In accordance with one aspect of the present invention, there is provided a method for labeling a phosphorylated peptide with a complex compound represented by formula (I):

where X represents a linker group and Y represents a tagging group.

In accordance with another aspect of the present invention, there is provided a method for selectively adsorbing a phosphorylated peptide using the above complex compound.

In accordance with a further aspect of the present invention, the above complex compound is provided.

In accordance with a further aspect of the present invention, there is provided a process for the preparation of compound (I) comprising Scheme 1.

Scheme 1

where R ¹ and R ² each represents a reactive group to form a linker group X and Y represents a labeling group.

In accordance with a further aspect of the present invention, there is provided a compound represented by formula (II):

where R ¹ represents a reactive group, with the exception of the aminomethyl group, hydroxymethyl group, amino group and carboxyl group.

According to the labeling method of the present invention, a phosphorylated peptide, namely a protein, can be easily detected. Thus, the present invention is useful for diagnosing diseases or the like. using samples obtained from a living organism or the like.

In addition, since compound (I) of the present invention exhibits a unique coordination bond with two hydroxy groups in the phosphoric acid monoester or phosphoric ion group, compound (I) of the present invention is useful as a compound that can be used in the method of the present invention. Also, compound (I) of the present invention is useful for purifying or concentrating a phosphorylated peptide and for obtaining chemical information on a phosphorylated peptide.

These and other objectives, features and advantages of the present invention will be more apparent upon reading the following detailed description of the invention and the accompanying drawings.

Brief Description of the Drawings

Figure 1 presents the result of a study of the zinc complex of the present invention, obtained using a MALDI-TOF mass spectrometer.

Figure 2 shows the electrophoresis gels stained with the zinc complex (A) of the present invention and other traditional dye (B). This illustration clearly demonstrates that only the phosphorylated peptide can be identified by the method of the present invention, while using the traditional method, all peptides are stained.

Figure 3 presents the result of the study of the zinc complex of the present invention, obtained using a MALDI-TOF mass spectrometer, and also presents an enlarged picture of a certain area that is obtained as a result of the study.

Figure 4 presents the result of the study of the zinc complex of the present invention, obtained using a MALDI-TOF mass spectrometer, and also presents an enlarged picture of a certain area obtained as a result of the study.

Figure 5 presents the result of the study obtained using the MALDI-TOF mass spectrometer, the starting compound used to obtain the compound of the present invention containing biotin as a labeling group, and also shows an enlarged picture of a certain area obtained from the study.

Figure 6 presents the result of the study, obtained using a MALDI-TOF mass spectrometer, the starting compound used to obtain the compound of the present invention containing biotin as a labeling group, and also presents an enlarged picture of a certain area obtained from the study.

Figure 7 presents the result of the study of the zinc complex of the present invention, obtained using a MALDI-TOF mass spectrometer, and also presents an enlarged picture of a certain area obtained as a result of the study.

On Fig presents the result of a study of the zinc complex of the present invention, obtained using a MALDI-TOF mass spectrometer.

Figure 9 shows the electrophoresis gels stained with the zinc complex (A) of the present invention and other traditional dye (B). This illustration clearly demonstrates that only the phosphorylated peptide can be identified by the method of the present invention, while using the traditional method, all peptides are stained.

The best way of carrying out the invention

The main distinguishing feature of the method of the present invention is that a phosphorylated peptide can be easily identified by the formation of a complex compound in which a complex compound containing a labeling group represented by formula (I) is specifically associated with a phosphorylated peptide.

To date, various metal complexes capable of binding to a phosphoric acid group have been known. However, a compound that is similar to the compound represented by formula (I) and which contains a labeling group was not known. The inventors of the present invention have found that the use of the complex compound represented by formula (I) is advantageous for providing easy detection and identification of a phosphorylated peptide even in a sample obtained from a living organism containing many different peptides, and thus the present invention has been created.

The following describes a method for labeling phosphorylated peptides in accordance with one embodiment of the present invention.

First get a sample containing essentially all possible types of peptides that make up the tissue cell that you want to explore. This preparation is carried out in accordance with the traditional method widely used in biochemistry.

Then the peptides contained in the sample are separated. The separation method is not particularly limited, and a conventional separation method such as electrophoresis can be used.

In electrophoresis, the gel after electrophoresis is immersed in a solution containing the complex compound represented by formula (I) for labeling a phosphorylated peptide, and then the phosphorylated peptide is determined by a detection method depending on the type of labeling group.

The solvent used in the solution containing the complex compound represented by formula (I) is not particularly limited provided that it does not interfere with the detection of the phosphorylated peptide. Examples of such a solvent are water, including a buffer, and a solution containing a salt other than the buffer; alcohols such as methanol and ethanol; and a mixed solvent containing these components. Preferably, an aqueous solvent is used mainly to prevent denaturing of the peptide.

The following describes the compound (I) used in the above method.

where X represents a linker group and Y represents a tagging group.

In formula (I), Zn is selected as the coordination metal, since Zn has a high coordination ability with respect to the phosphoric acid group in the phosphorylated protein, namely the phosphoric acid monoester.

A linker group in the present description and claims means a group capable of binding to the main skeleton and to the labeling group. The linker group promotes the preparation of compound (I) and inhibits the action of the labeling group, which impedes the coordination of compound (I) with the phosphoric acid group associated with the peptide. Given this circumstance, the linker group can be a coordination bond directly linking the main skeleton to the tagging group, if it is easy to obtain the starting compound in which the tagging group is directly linked to the main skeleton, in the synthesis of compound (I) or if the tagging group has such a small size, which can easily prevent the effect of preventing the coordination of compound (I) with the phosphoric acid group.

The type of linker group in the present description and the claims is not particularly limited, provided that the linker group has the above functions. Examples of linker groups are: C1-C6 alkylene group, amino group (-NH-), ether group (-O-), 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-); A C1-C6 alkylene group containing at one end a group selected from the group consisting of an amino group, an ether group, a thioether group, a carbonyl group, a thionyl group, an ester group, an amide group, a urea group and a thiourea group; A C1-C6 alkylene group containing at the opposite ends two groups selected from the group consisting of an amino group, an ether group, a thioether group, a carbonyl group, a thionyl group, an ester group, an amide group, a urea group and a thiourea group, where the groups located at opposite ends are the same or different from each other; and a group in which two or more two groups are selected from the group consisting of an amino group, an ether group, a thioether group, a carbonyl group, a thionyl group, an ester group, an amide group, a urea group and a thiourea group, and a C1-C6 alkylene group are linearly connected.

C1-C6 alkylene group means a divalent aliphatic hydrocarbon group containing 1-6 carbon atoms, which is linear or branched, for example methylene, ethylene, propylene, tetramethylene, hexamethylene, methylene, methylpropylene and dimethylpropylene group, preferably represents a C1-C4 alkylene group and more preferably represents a C1-C2 alkylene group.

The labeling group in the present description and the claims is not particularly limited provided that it is a group traditionally used in biochemistry. However, a compound containing a radioisotope is not preferred in terms of handling it. Examples of the labeling group are a fluorescent group, a group containing a nitric oxide radical, and biotin.

A fluorescence group is a substituent capable of stably generating fluorescence with a relatively long wavelength, and the group commonly used in biochemistry can be used indefinitely as a fluorescent group, regardless of whether the compound is soluble in water or in oil. Examples of fluorescent groups are aminomethylcoumarin and its derivatives, fluorescein and its derivatives, tetramethylrodamine and its derivatives, anthraniloyl and its derivatives, nitrobenzoxadiazole and its derivatives and dimethylaminonaphthalene and its derivatives.

A group containing a nitric oxide radical is a group containing a stable radical, and is capable of detecting a phosphorylated peptide by electron spin resonance (ESR). As a rule, a biological molecule does not exhibit electron spin resonance, because it does not contain an unpaired electron. On the other hand, since the peptide associated with the compound (I) containing the nitric oxide radical coordinated with it exhibits electron spin resonance, a phosphorylated peptide can be identified.

Biotin has a specific and high affinity for avidin derived from albumin and streptavidin derived from actinomycetes. In light of this, the complex compound can specifically bind to the enzyme via biotin and avidin or streptavidin by binding of avidin or streptavidin to compound (I) containing biotin as a labeling group, and then binding to the biotinylated enzyme. The phosphorylated peptide can be identified using an enzyme such as alkaline phosphatase, peroxidase and luciferase, and using a staining agent, depending on the type of enzyme. For example, if the phosphorylated peptide is first coupled using the complex compound of the present invention, after which the complex compound is coupled using alkaline phosphatase as an enzyme via streptavidin, then nitrosine tetrazolium and 5-bromo-4-chloro-3-indolylphosphate are added as staining agents and the mixture is allowed to react for several hours, the phosphorylated peptide turns purple, which makes it possible to identify the phosphorylated peptide. In addition, streptavidin labeled with a fluorescent pigment such as rhodamine is commercially available. The use of streptavidin labeled with a fluorescent pigment makes it possible to identify a phosphorylated peptide by a known method for analyzing a fluorescence image.

The following describes a method for the selective adsorption of phosphorylated peptides in accordance with an embodiment of the present invention.

According to a method for the selective adsorption of phosphorylated peptides, biotin or a similar substance capable of specific binding to a specific compound is used as a labeling group. For example, an agarose gel with associated streptavidin is commercially available. An agarose gel with a complex compound of the present invention bound thereto can be prepared by combining it with a complex compound containing biotin as a labeling group for interaction. The phosphorylated peptide in the mixed sample can be selectively adsorbed into the complex compound of the present invention by applying the mixed sample to an agarose gel. After selective adsorption, the addition of phosphoric acid buffer or the like, capable of desorption of the phosphorylated peptide from the complex compound of the present invention, enables exclusive production of the phosphorylated peptide. In the same way, the use of an agarose gel is beneficial for purification or concentration of a phosphorylated peptide without the use of electrophoresis. Also, magnetic beads with associated streptavidin are commercially available. The use of magnetic beads also makes it possible to purify or concentrate the phosphorylated peptide. In addition, plates with associated streptavidin for measuring surface plasmon resonance (SPR) are commercially available. A plate with a complex compound of the present invention associated with it for measuring surface plasmon resonance can be obtained by applying to the plate a complex compound of the present invention containing biotin as a labeling group. The presence or absence of a phosphorylated peptide in a sample can be determined by measuring the surface plasmon resonance (SPR) of the plate.

You can synthesize a compound equivalent to compound (I) in which a methyl group or a similar group is introduced into the pyridine ring to provide essentially the same action and effects as in the described embodiment of the present invention. Such a compound equivalent to compound (I) is included in the scope of the present invention.

The position of the (-X-Y) group in the compound of the present invention is not particularly limited. Group (-X-Y) may be located on the site, as shown in compound (I ').

Compound (I) and compound (I ') are substantially equivalent. Although it is not clear which compound is synthesized, compound (I) or compound (I '), which is really understood, is that a mixture of compound (I) and compound (I') is synthesized. It is not necessary to indicate that the compound (I ') is included in the scope of the present invention.

The complex compound represented by formula (I) can be easily prepared by a process comprising Scheme 1.

Scheme 1

where X and Y are defined above and R ¹ and R ² each represents a reactive group to form a linker group X.

In the above scheme, the labeling group Y is introduced into the main skeleton through the linker group X by reacting the reactive groups R ¹ and R ² .

The type of groups R ¹ , R ² , solvent, reaction temperature, a reagent different from the above, the purification method and other factors are mainly determined by the type of group X. For example, if a labeling group is introduced through an amino group (secondary or tertiary amino group), as combinations of R ¹ and R ² , a combination of a group containing an amino group (primary or secondary amino group) at its remote end and a leaving group, such as a halogen atom. The condensation of R ¹ and R ² in the presence of basic groups in a solvent is an example of a general reaction condition. In case R ¹ is an active group, a tagging group can be easily entered.

Compound (I) can then be synthesized by adding a metal salt to a solution containing compound (IV). As a metal salt, zinc (II) nitrate or zinc (II) acetate can be added. When zinc (II) acetate is added, a compound of formula (V) is provided below, wherein the acetic acid is temporarily coordinated.

Compound (V) is more chemically stable than compound (I) and is therefore useful for storage. Compound (V) is equivalent to compound (I) and can be used in the same way as compound (I). Specifically, by adding compound (V) to a mixture containing a peptide, phosphorylated peptide can be detected since the phosphoric acid monoester group is coordinated interchangeably to compound (I) instead of acetic acid.

Compound (II), i.e. the starting compound for compound (I) can be synthesized in accordance with scheme 2 below.

Scheme 2

where R ^{1 is} defined above and "Hal" represents a halogen atom and, preferably, bromine.

Compound (VI), i.e. 1,3-diamino-2-propanol, as a starting material compound, may be commercially available. In addition, since compound (VII) and compound (IX) both have a relatively simple structure, compounds (VII) and (IX) may be commercially available or can be synthesized by a method well known to those skilled in the art.

In Scheme 2, first, compound (VI) and compound (VII) are reacted with each other in the presence of a condensation catalyst to give compound (VIII). Such an interaction can be carried out stepwise by the administration of compound (VII). Alternatively, compound (VIII) can be prepared in one step using 3 or more equivalents of compound (VII) relative to compound (VI).

In Scheme 2, reductive amination is carried out as a condensation reaction. The solvent used in reductive amination is not particularly limited provided that the solvent is able to substantially dissolve compound (VI) and compound (VII) and does not inhibit the amination. For example, alcohols such as methanol, ethanol and isopropanol; ethers, such as diethyl ether, tetrahydrofuran and dioxane; water or a mixed solvent containing two or more of these components can be used as a solvent.

Reductive amination can be carried out using a conventional reducing agent after condensation of compound (VI) and compound (VII) in the presence of concentrated hydrochloric acid as a catalyst.

Optimum conditions regarding the reaction temperature and reaction time may be selected, optionally, depending on the type of the starting compound or other factors. For example, the reaction can be carried out at a reaction temperature of from 20 to 80 ° C. for a time of 12 to 100 hours.

After completion of the reaction, a solvent and the like. distilled off under reduced pressure before water is added. After adding water, the resulting mixture was extracted with a solvent insoluble in water, and the organic layer was dried over anhydrous magnesium sulfate or the like. Then, the solvent was distilled off under reduced pressure. After that, the residue is purified by a well-known method, such as silica gel column chromatography, to give compound (VIII).

The method for preparing compound (VIII) is not limited to the method shown in Scheme 2. Alternatively, compound (VIII) can be synthesized using, for example, compound (VI) and a halogen-containing compound.

Compound (II) can then be synthesized by reacting compound (VIII) with compound (IX). This interaction can be carried out in a known manner for the synthesis of tertiary amines. For example, compound (VIII) and compound (IX) are condensed in the presence of a base in a solvent. In the condensation step, a protecting group can be introduced and cleaved, depending on the type of R ¹ , as necessary. Alternatively, compound (II) can be synthesized by performing a condensation step using a compound containing an inactive substituent group instead of using R ¹ in compound (IX), and by replacing the inactive substituent with R ¹ by converting functional groups. For example, the condensation step is carried out using a compound containing a nitro group as an inactive substituent, and replacing the nitro group with an amino group as a reactive group.

The following complex compound (X) can be used in the method of the present invention instead of complex compound (I).

where X, Y are defined above, and R ³ - R ⁵ each represents a group of an electron-donor substituent at position 4 or 6 of the pyridine ring.

The complex compound (X) used in the method of the present invention is electrically enriched with pyridine nitrogen through an electron-donating substituent group introduced at the appropriate position for substitution. Therefore, the complex compound (X) used in the method of the present invention is highly coordinated to zinc, thus providing the possibility of obtaining the complex compound (X) in a simple manner while ensuring stability.

The method for using complex compound (X), the method for producing complex compound (X) and the starting compound for complex compound (X) are essentially the same as indicated for complex compound (I).

In the above methods and compound (I), it is preferable to use biotin as a labeling group in the preparation of the complex compound. The use of biotin is preferred, since biotin is not difficult to handle, and it has been widely used because it exhibits various staining reactions. The use of biotin is effective for easy identification of a phosphorylated peptide.

Below are examples of obtaining and experimental examples for a more detailed description of the present invention. However, the present invention is not limited to the examples given.

Examples

Production Example 1-1: Methyl 6-bromomethyl nicotinate

To a solution of methyl 6-methylnicotinate (50 g, 331 mmol) in carbon tetrachloride (625 ml) was added N-bromosuccinimide (59 g, 331 mmol). Then 1.0 g of benzoyl peroxide was added, the reaction of the mixture was carried out at a temperature of from 40 to 50 ° C for 24 hours when irradiated with light from a searchlight.

After cooling the reaction mixture, the precipitated crystals were separated by filtration. The filtrate was washed with an aqueous solution containing sodium bicarbonate and concentrated. The residue obtained by concentration was purified by silica gel column chromatography to obtain 37 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 3.96 (3H, s, OCH ₃ ), 4.58 (2H, s, CH ₂ Br), 7.54 (1H, d, Py), 8 30 (1H, dd, Py); 9.17 (1H, d, Py).

Production Example 1-2: N, N, N'-tri (2-pyridylmethyl) -1,3-diaminopropan-2-ol

To a solution of 1,3-diaminopropan-2-ol (32.6 g, 362 mmol) in methanol (2400 ml) was added 60 ml of concentrated hydrochloric acid. Then, 2-pyridinaldehyde (116.3 g, 1.09 mol) was added dropwise, and then sodium cyanoborohydride (50.16 g, 798 mmol) was added. Upon completion of the addition, the reaction of the mixture was carried out at room temperature for 3 days.

After adding concentrated hydrochloric acid to the solution and adjusting the pH of the solution to 6, the resulting solution was concentrated to some extent. Then added 0.1 N. an aqueous solution of sodium hydroxide to adjust the pH of the solution to 7, and then was extracted with chloroform. The extracts were collected and dried, the resulting substance was concentrated. The residue obtained by concentration was purified by silica gel column chromatography to obtain 34 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.59-2.83 (4H, m, CH ₂ ), 3.86-4.01 (7H, m, NCH ₂ Py, CH), 7, 15 (3H, dd, Py), 7.23-7.32 (3H, m, Py), 7.56-7.65 (3H, m, Py), 8.53 (3H, dd, Py).

Production Example 1-3: N, N, N'-tri (2-pyridylmethyl) -N '- (5-methoxycarbonyl-2-pyridylmethyl) -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -1,3-diaminopropan-2-ol (18.2 g, 50 mmol) obtained in Production Example 1-2 in anhydrous dimethylformamide (150 ml) potassium carbonate (13.8 g, 100 mmol) was added, and then a solution of methyl 6-bromomethyl nicotinate (11.5 g, 50 mmol) obtained in Production Example 1-1 in anhydrous dimethylformamide (75 ml) was added dropwise. After completion of the dropwise addition, the mixture was reacted at 50 ° C. for 1 hour.

Upon completion of the reaction, the solution was cooled. Then the cooled solution was poured into 750 ml of water and the pH of the solution was adjusted to 8 by adding 1 N. hydrochloric acid solution. After extraction with ethyl acetate, the extracts were collected, washed with water and brine, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography to obtain 21.5 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.58-2.73 (4H, m, CH ₂ ), 3.83-3.95 (12H, m, OCH ₃ , NCH ₂ Py, CH) 7.10-7.14 (3H, m, Py), 7.34 (3H, d, Py), 7.50-4.60 (4H, m, Py), 8.17 (1H, dd, Py), 8.50 (3H, d, Py), 9.09 (1H, d, Py).

Production Example 1-4: N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-aminoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -N '- (5-methoxycarbonyl-2-pyridylmethyl) -1,3-diaminopropan-2-ol (9.7 g, 18.9 mmol), obtained in Production Example 1-3, ethylene diamine (22.7 g, 378 mmol) was added dropwise in methanol (100 ml). After completion of the dropwise addition, the mixture was reacted at room temperature for 3 days.

Upon completion of the reaction, the solution was concentrated and the residue obtained by concentration was purified by silica gel column chromatography to obtain 9.72 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.54-2.71 (4H, m, CH ₂ ), 2.94 (2H, t, CH ₂ N), 3.49 (2H, dt, CH ₂ N), 3.80-3.99 (9H, m, NCH ₂ Py, CH), 7.12 (3H, ddd, Py), 7.35 (3H, d, Py), 7.45 ( 1H, d, Py), 7.58 (3H, ddd, Py), 8.02 (1H, ddd, Py), 8.49 (3H, ddd, Py), 8.89 (1H, d, Py) .

Production Example 1-5: N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (7-nitro-2,1,3-benzoxadiazol-4-ylaminoethyl) carbamoyl-2- pyridylmethyl] -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-aminoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropan-2-ol (200 mg, 0.37 mmol) obtained in Production Example 1-4, sodium bicarbonate (336 mg, 4.0 mmol) was added in acetonitrile (20 ml), then 4-chloro-7-nitro-2,1,3-benzoxadiazole was added (73.8 mg, 0.37 mmol) The mixture was reacted at room temperature for 2 hours.

Upon completion of the reaction, the solution was concentrated. Then, 50 ml of dichloromethane and 50 ml of water were added, and the organic layer and the aqueous layer were separated. The organic layer was dried over anhydrous sodium sulfate, concentrated, and the obtained residue was purified by silica gel column chromatography to obtain 71.2 mg of the target compound.

^l H-NMR (CDCl ₃ , 300 MHz): δ 2.46-2.69 (4H, m, CH ₂ ), 3.65-3.95 (13H, m, NCH ₂ CH ₂ N, NCH ₂ Py , CH), 6.06 (1H, d, Ar), 7.08-7.13 (3H, m, Py), 7.32 (3H, d, Py), 7.42 (1H, d, Py ), 7.56 (3H, ddd, Py), 7.96 (1H, dd, Py), 8.44-8.48 (3H, m, Py), 8.19 (1H, d, Ar), 8.83 (1H, d, Py).

Production Example 1-6: A Solution Containing the Zinc Complex of the Present Invention

A 50 μM aqueous solution was obtained containing the compound obtained in Production Example 1-5, then 2 equivalents of zinc nitrate were added to the solution. Thus, a solution containing the zinc complex of the present invention was obtained.

The zinc complex was identified in accordance with the following method. Namely, the compound obtained in Production Example 1-5 was dissolved in phosphoric acid buffer (pH = 6.86) to obtain a 50 μM solution, then 2 equivalents of zinc nitrate were added to the solution. The zinc complex of the present invention in solution showed the following structure, determined using a MALDI-TOF mass spectrometer (matrix: 2 ', 4', 6'-trihydroxyacetophenone, wave type: reflector, accelerating voltage: 20,000 V, grid voltage: 57,500% , laser: 2500, scans, average: 128, pressure: 5.05e-07).

The measurement results obtained using a MALDI-TOF mass spectrometer are presented in figure 1. As shown in FIG. 1, a molecular ion peak was observed at 926.2 (exact mass: 926.11). It is believed that the appearance of a molecular ion peak at 910.2 is caused by the elimination of oxygen in the oxadiazole group.

Production Example 2-1: N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-D-biotinamidoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropane-2 ol

To a solution of D-biotin (137 mg, 0.56 mmol) in dimethylformamide (10 ml) was added 1,1'-carbonyldiimidazole (116 mg, 0.72 mmol). The mixture was reacted at room temperature for 12 hours. The solution was then cooled with ice, after which a solution of N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-aminoethyl) carbamoyl-2-pyridylmethyl] -1,3- was added dropwise. diaminopropan-2-ol (260 mg, 0.48 mmol) obtained in Production Example 1-4 in dimethylformamide (3 ml). The cooling bath was removed and the reaction was carried out at room temperature for 2 hours.

Upon completion of the reaction, the solution was poured into 50 ml of water, and then 50 ml of chloroform was extracted twice. Then, the extracts were concentrated, the crude product was purified by silica gel column chromatography to obtain 265 mg of the target compound.

¹ H-NMR (CDC1 ₃ , 300 MHz): δ 1.27-1.47 (2H, m, CH ₂ ), 1.50-1.75 (4H, m, CH ₂ ), 2.13-2 26 (2H, m, COCH ₂ ), 2.52-2.74 (5H, m, NCH ₂ , SCH ₂ ), 2.79-2.88 (1H, m, SCH ₂ ), 3.01- 3.12 (1H, m, SCH), 3.43-3.65 (4H, m, NCH ₂ CH ₂ N), 3.80-4.02 (9H, m, NCH ₂ Py, CHO), 4 22-4.28 (1H, m, NCH), 4.42-4.49 (1H, m, NCH), 5.83 (1H, br s, NHCO), 6.60 (1H, br. s, NHCO), 7.07-7.18 (3H, m, Py), 7.31-7.38 (3H, m, Py), 7.44 (1H, d, Py), 7.59 ( 3H, ddd, Py), 8.03 (1H, dd, Py), 8.15 (1H, br s, NHCO), 8.42-8.58 (3H, m, Py), 8.94 ( 1H, d, Py).

Production Example 2-2: Solution Containing Zinc Complex of the Present Invention

The compound obtained in Production Example 2-1 was dissolved in phosphoric acid buffer (pH = 6.86) to obtain a 3 mM solution, then 2 equivalents of zinc nitrate were added to the solution.

The zinc complex of the present invention in solution showed the following structure, determined using a MALDI-TOF mass spectrometer (matrix: 2 ′, 4 ′, 6′-trihydroxyacetophenone).

The measurement results obtained using a MALDI-TOF mass spectrometer are shown in FIG. 3. As shown in FIG. 3, a molecular ion peak was observed at 989.6 (exact mass: 989.19).

Experimental example 1: Electrophoresis in 10% native polyacrylamide gel

First, electrophoresis gels, a pH buffer for electrophoresis, and a staining solution were prepared to dissolve the samples under the following conditions.

Concentrating Gel:

125 mM Tris-hydrochloric acid buffer (pH = 6.8)

4.5% (w / v) polyacrylamide (acrylamide: bisacrylamide = 30: 1)

Separating gel:

375 mM Tris-hydrochloric acid buffer (pH = 8.8)

10% (w / v) polyacrylamide (acrylamide: bisacrylamide = 30: 1)

pH of electrophoresis buffer (pH = 8.3):

25 mm Tris

190 mm glycine

Staining solution for dissolving samples (3 × concentrated solution):

195 mM Tris-hydrochloric acid buffer (pH = 6.8)

10% (w / v) glycerin

0.1% (w / v) bromophenol blue (BPB) used in electrophoresis as a staining marker

Then 2 μg of each of 1: bovine serum albumin, 2: β-casein (phosphorylated), and 3: β-casein (dephosphorylated) were dissolved in a staining solution to obtain samples. Appropriate samples were applied to the gel. A direct current of 4 mA was then reported until the stain marker leaked.

The gel was immersed in a solution containing a zinc complex (50 μM) obtained in Production Example 1-6 for about 30 minutes. Then the gel was removed from the solution and photographed by irradiation with a UV lamp. Then the gel was stained with Coomassie brilliant blue in accordance with the traditional method of staining and the stained gel was photographed. A gel stained with a solution containing a zinc complex is indicated as “gel A”, and a gel stained with Coomassie brilliant blue is indicated as “gel B”, both of these gels are shown in FIG.

As can be seen from figure 2, in accordance with the method of the present invention, β-casein associated with phosphoric acid can be separately identified. Thus, it is obvious that the method of the present invention is advantageous for identifying exclusively phosphorylated peptides in samples obtained from living organisms.

Production Example 3-1: N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- 2- (6-D-biotinamidohexacarboxyamidoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropane -2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-aminoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropan-2-ol (113 mg, 0.21 mmol) obtained in Production Example 1-4 in acetonitrile (10 ml) was added dropwise a solution of 5- (N-succinimidyloxycarbonyl) pentyl-D-biotinamide (95 mg, 0.21 mmol) in dimethyl sulfoxide (2 ml )

After the mixture was reacted at room temperature for 6 hours, the reaction mixture was concentrated and the crude product obtained by concentration was purified by silica gel column chromatography to obtain 136 mg of the target compound (yield: 76%).

¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.25-1.32 (2H, m, CH ₂ ), 1.38-1.47 (4H, m, CH ₂ ), 1.56-1 67 (5H, m, CH ₂ ), 1.68-1.77 (1H, m, CH ₂ ), 2.11-2.22 (4H, m, COCH ₂ ), 2.58 (2H, dd , J = 8.0 and 13.3 Hz, NCH), 2.66 (1H, dd, J = 3.9 and 13.3 Hz, NCH), 2.68 (1H, dd, J = 3.9 and 13.3 Hz, NCH), 2.71 (1H, dd, J = 3.0 and 13.3 Hz, SCH), 2.88 (1H, dd, J = 5.2 and 13.0 Hz, SCH), 3.07-3.21 (3H, m, NCH, SCH), 3.45-3.59 (4H, m, NCH ₂ ), 3.82-3.91 (8H, m, NCH ₂ Py), 3.94 (1H, tt, J = 3.9 and 8.0 Hz, OCH), 4.28-4.32 (1H, m, NCH), 4.46-4.50 (1H, m, NCH), 5.62 (1H, br s, NHCO), 6.40 (1H, br s, NHCO), 6.62 (1H, t, J = 5.7 Hz, NHCO), 7 10-7.14 (3H, m, Py), 7.23 (1H, t, J = 6.0 Hz, NHCO), 7.32-7.36 (3H, m, Py), 7.43 (1H, d, J = 8.2 Hz, Py), 7.56-7.61 (3H, m, Py), 8.06 (1H, dd, J = 2.3 and 8.2 Hz, Py ), 8.17 (1H, t, J = 5.0 Hz, NHCO), 8.46-8.50 (3H, m, Py), 8.96 (1H, d, J = 2.3 Hz, Py).

¹³ C-NMR (CDCl ₃ , 125 MHz): δ 25.0 (CH ₂ ), 25.5 (CH ₂ ), 26.2 (CH ₂ ), 27.7 (CH ₂ ), 27.8 (CH ₂ ), 29.0 (CH ₂ ), 35.6 (CH ₂ CO), 36.1 (CH ₂ CO), 39.0 (CH ₂ NH), 39.3 (CH ₂ NH), 40.7 (CH ₂ S), 41.0 (CH ₂ NH), 55.6 (CH ₂ S), 59.2 (CH ₂ N), 60.2 (CHNH), 60.7 (CH ₂ Py), 60 8 (CH ₂ Py), 61.0 (CH ₂ Py), 61.8 (CHNH), 67.4 (CHOH), 122.1 (Py), 122.8 (Py), 123.2 (Py ), 128.5 (Py), 135.6 (Py), 136.5 (Py), 148.0 (Py), 149.0 (Py), 159.3 (Py), 159.4 (Py) 162.6 (Py); 163.9 (NCON); 166.3 (CONH); 173.4 (COM); 174.9 (CONH).

Production Example 3-2: Solution Containing Zinc Complex of the Present Invention

200 nmol of the compound obtained in Production Example 3-1 was dissolved in phosphoric acid buffer (pH = 6.86) to obtain a 3 mM solution containing the compound, then 2 equivalents of zinc nitrate were added to the solution. Thus, a solution containing the zinc complex of the present invention was obtained.

The solution was analyzed using a MALDI-TOF mass spectrometer (matrix: 2 ′, 4 ′, 6′-trihydroxyacetophenone) and a zinc complex was identified. The measurement results obtained using a MALDI-TOF mass spectrometer are presented in FIG. 4. As shown in FIG. 4, a molecular ion peak was observed at 1102.3 (exact mass: 1102.27).

Experimental Example 2

A suspension (0.3 ml) containing streptavidinagarose (amount of biotin bound group: 60-120 nmol / ml) and buffer (Sigma-Aldrich Co.) were loaded into a centrifugal filter unit (volume: 0.5 ml, filter pore diameter: 0.22 μm). The filter unit was centrifuged to separate at a centrifuge acceleration of 2000 g for 15 seconds, after which the suspension was filtered. 5.0 mM Tris-Acetate buffer (pH = 7.4, 0.30 ml) was loaded into the filter unit, then separation was performed by centrifuging the filter unit under centrifugal acceleration at 2000 g for 15 seconds and filtering for washing. This stage of centrifugal separation and filtration for washing was repeated 5 times.

5.0 mM Tris-acetate buffer (pH = 7.4, 0.30 ml) with 120 nmol / ml of the compound obtained in Production Example 3-1 and 500 nmol / ml of zinc acetate dissolved in it was loaded into the filter unit. The filter unit was brought into equilibrium for 5 minutes and then separated by centrifugation at 2000 g for 15 seconds and filtered. Then, 5.0 mM Tris-acetate buffer (pH = 7.4, 0.30 ml) was loaded into the filter unit and centrifugal separation was carried out at 2000 g for 15 seconds and filtered for washing. The centrifugal separation and filtration step for washing is cyclically repeated 5 times. Then, 5.0 mM Tris-acetate buffer (pH = 7.4, 0.30 ml), in which 10 nmol / ml zinc acetate was dissolved, was loaded into the filter unit, and centrifugal separation was carried out at 2000 g for 15 seconds and filtered flushing. The centrifugal separation and filtration step for washing is cyclically repeated 5 times.

5.0 mM Tris-acetate buffer (pH = 7.4, 0.30 ml) was loaded into the filter unit with the unphosphorylated p60c peptide (p60c-src peptide 521-533, 14 nmol / ml) and phosphorylated P- peptide dissolved in it p60c (O-phosphoryl p60c-src peptide 521-533, 12 nmol / ml), as samples. The filter unit was brought into equilibrium for 5 minutes and then separated by centrifugation at 2000 g for 15 seconds and filtered. The resulting filtrate was recovered as fraction 1.

Then, 5.0 mM Tris-acetate buffer (pH = 7.4, 0.50 M NaNO ₃ , 0.30 ml) was loaded into the filter unit and centrifugal separation was carried out at 2000 g for 15 seconds and filtered (3 times). The filtrate obtained after the first filtration was recovered as fraction 2, the filtrate after the second filtration was recovered as fraction 3, and the filtrate after the third filtration was recovered as fraction 4.

In addition, 1.0 mM buffer containing phosphoric acid-sodium hydroxide (pH = 7.4, 0.50 M NaNO ₃ , 0.30 ml) was loaded into the filter unit, and centrifugal separation was carried out at 2000 g for 15 seconds and filtration (2 times). The filtrate obtained after the first filtration was recovered as fraction 5, and the filtrate after the second filtration was recovered as fraction 6.

The amount of peptide in the appropriate fractions 1-6 was determined by high performance liquid chromatography (HPLC). Indicators of isolation / extraction of the corresponding peptides relative to the total amount are presented in table 1.

Table 1 Faction No. one 2 3 four 5 6 p60s (%) 67 24 9 0 0 0 R-p60s (%) 0 0 0 0 83 17

HPLC analysis conditions:

column: Capcell Pak C18 type UG80, 4.6 mm (diameter)

mobile phase: acetonitrile: water = 14:86 (vol./about.), 0.1% (vol./about.) trifluoroacetic acid

flow rate: 1 ml / min

column temperature: 40 ° C

detection method: UV 266 nm

retention time: P-p60c (5.3 minutes), p60c (13.4 minutes)

The above results show that the complex compound of the present invention can selectively adsorb a phosphorylated peptide, and that the use of the complex compound of the present invention is beneficial for the selective isolation of a phosphorylated peptide in a mixed sample of a phosphorylated peptide and a non-phosphorylated peptide.

Comparative Example 1

A suspension (0.3 ml) containing streptavidinagarose (amount of biotin bound group: 60-120 nmol / ml) and buffer (Sigma-Aldrich Co.) were loaded into a centrifugal filter unit (volume: 0.5 ml, filter pore diameter: 0.22 μm). The filter unit was centrifuged at 2000g for 15 seconds, then the suspension was filtered. Then, 5.0 mM Tris-acetate buffer (pH = 7.4, 0.30 ml) was loaded into the filter unit and centrifugal separation was carried out at 2000 g for 15 seconds and filtered for washing. The centrifugal separation and filtration step for washing is cyclically repeated 5 times.

A method similar to that of Experimental Example 2 was carried out using samples equivalent to those used in Experimental Example 2 using a filter unit (the complex compound of the present invention was not used in this comparative example). Thus, fractions 7-12 were obtained.

HPLC for the analysis of fractions 7-12 was carried out in the same manner as in experimental example 2. The results of the analysis are presented in table 2.

table 2 Faction No. 7 8 9 10 eleven 12 p60s (%) 75 twenty 5 0 0 0 R-p60s (%) 83 17 0 0 0 0

The results presented in table 2 show that the separation of the phosphorylated peptide from the non-phosphorylated peptide was not successful, even with a method similar to that of experimental example 2, since the complex compound of the present invention was not used.

Production Example 4-1: 2-acetoxymethyl-4-nitropyridine

200 ml of acetic anhydride was heated to 100 ° C, after which 2-methyl-4-nitropyridine N-oxide (25.0 g, 162 mmol) was gradually added. After addition, the mixture was gradually heated and maintained at 130 ° C for 20 minutes. Then the reaction mixture was cooled to 80 ° C, after which 200 ml of ethanol was added dropwise to stop the reaction.

Then the reaction mixture was concentrated, the residue was taken up in water (500 ml) and the pH of the mixture was adjusted to 9 by adding sodium bicarbonate. After extraction with ethyl acetate 2 times (500 ml for the first extraction and 200 ml for the second extraction), the resulting organic layers were washed with 200 ml of water and 200 ml of saturated saline. After concentration of the organic layer, the resulting crude product was purified by silica gel column chromatography to obtain 7.68 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.23 (3H, s, COCH ₃ ), 3.36 (2H, s, CH ₂ Py), 7.97 (1H, dd, Py), 8 07 (1H, d, Py); 8.90 (1H, d, Py).

Production Example 4-2: 2-hydroxymethyl-4-nitropyridine

To 2-acetoxymethyl-4-nitropyridine (7.68 g, 39.2 mmol) obtained in Production Example 4-1, 30 ml of a 10% hydrochloric acid solution was added. The reaction of the mixture was carried out at 50 ° C for 30 minutes. After cooling the reaction mixture, the solution was poured into 200 ml of water and the pH of the mixture was adjusted to 9 by adding sodium bicarbonate. The mixture was extracted with 100 ml of ethyl acetate (three times) and the resulting organic layers were concentrated. The residue obtained by concentration was purified by silica gel column chromatography to give 4.70 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 3.36 (1H, t, OH), 4.94 (2H, d, CH ₂ Py), 7.95 (1H, ddt, Py), 8, 09 (1H, dt, Py); 8.86 (1H, d, Py).

An example of obtaining 4-3: 2-bromomethyl-4-nitropyridine

To a suspension of 2-hydroxymethyl-4-nitropyridine (1.00 g, 6.49 mmol) obtained in Production Example 4-2 in anhydrous ether (40 ml) was added dropwise phosphorus tribromide (0.88 g, 3, 24 mmol) at 0 ° C. After completion of the dropwise addition, the reaction of the mixture was carried out at 0 ° C. for 2 hours, after which the reaction was carried out at 20 ° C. for 64 hours.

The reaction mixture was poured into 250 ml of ice-cold water and the pH of the aqueous layer was adjusted to 8 by adding sodium bicarbonate. Then the ether layer and the aqueous layer were separated, the aqueous layer was extracted twice with 100 ml of ethyl acetate. The organic layers were collected and dried over anhydrous sodium sulfate. Then, the solvent was distilled off, the crude product was purified by silica gel column chromatography to obtain 0.80 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 4.66 (2H, s, CH ₂ Py), 7.96 (1H, dd, Py), 8.19 (1H, dd, Py), 8, 88 (1H, dd, Py).

Production Example 4-4: N, N, N'-tri (2-pyridylmethyl) -N '- (4-nitro-2-pyridylmethyl) -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -1,3-diaminopropan-2-ol (2.65 g, 7.28 mmol) obtained in Production Example 1-2 in anhydrous dimethylformamide (40 ml) was added anhydrous potassium carbonate (2.02 g, 14.6 mmol) and the mixture was heated to 55 ° C. Then, a solution of 2-bromomethyl-4-nitropyridine (1.58 g, 7.28 mmol) obtained in Production Example 4-3 was added dropwise in anhydrous dimethylformamide (20 ml) at the same temperature, i.e. 55 ° C. After which the reaction of the mixture was carried out at 55 ° C for 1.5 hours and the mixture was cooled.

The reaction mixture was poured into 200 ml of water and the pH of the mixture was adjusted to 8 by adding 1N. hydrochloric acid solution. After extraction with 400 ml of ethyl acetate (3 times), the organic layer was washed with 250 ml of saturated saline. After drying over anhydrous magnesium sulfate, the organic layer was concentrated. Then the solvent was distilled off, the crude product was purified by silica gel column chromatography to obtain 3.30 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.60-2.78 (4H, m, NCH ₂ ), 3.84-3.96 (6H, m, CH ₂ Py), 3.96- 4.03 (1H, m, OCH), 4.07 (2H, s, CH ₂ Py), 7.09-7.14 (3H, m, Py), 7.34 (3H, t, Py), 7.55-7.62 (3H, m, Py), 7.80 (1H, dd, Py), 8.22 (1H, d, Py), 8.49-8.52 (3H, m, Py ), 8.76 (1H, dd, Py).

Production Example 4-5: N, N, N'-tri (2-pyridylmethyl) -N '- (4-azide-2-pyridylmethyl) -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -N '- (4-nitro-2-pyridylmethyl) -1,3-diaminopropan-2-ol (1.01 g, 2.00 mmol), obtained in Production Example 4-4, in anhydrous tetrahydrofuran (17 ml) was added dropwise at room temperature sodium azide (156 mg, 2.40 mmol) in water (3.3 ml). The reaction mixture was heated to 50 ° C and reacted at the same temperature, i.e. 50 ° C, for 17 hours.

Then the mixture was cooled, the aqueous layer was extracted twice with 10 ml of chloroform. The extracts were collected and concentrated, and the obtained crude product was purified by silica gel column chromatography to obtain 980 mg of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.58-2.74 (4H, m, NCH ₂ ), 3.83-3.92 (8H, m, CH ₂ Py), 3.92- 4.01 (1H, m, OCH), 6.74 (1H, dd, Py), 7.09-7.14 (3H, m, Py), 7.20 (1H, d, Py), 7, 32-7.36 (3H, m, Py), 7.55-7.60 (3H, m, Py), 8.39 (1H, d, Py), 8.49-8.52 (3H, m , Py).

Production Example 4-6: N, N, N'-tri (2-pyridylmethyl) -N '- (4-amino-2-pyridylmethyl) -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -N '- (4-azide-2-pyridylmethyl) -1,3-diaminopropan-2-ol (980 mg, 1.98 mmol) obtained in Production Example 4-5, 100 mg of platinum oxide was added in ethanol (20 ml). The reaction of the mixture was carried out at a hydrogen pressure of 30 kPa for 2 hours. Then, the catalyst (platinum oxide) was removed by filtration, the filtrate was concentrated, and the obtained crude product was purified by silica gel column chromatography to obtain 690 mg of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.43-2.71 (4H, m, NCH ₂ ), 3.66-3.92 (8H, m, CH ₂ Py), 3.92- 4.01 (1H, m, OCH), 6.35 (1H, dd, Py), 6.63 (1H, d, Py), 7.08-7.14 (3H, m, Py), 7, 27-7.39 (3H, m, Py), 7.54-7.62 (3H, m, Py), 8.10 (1H, d, Py), 8.49-8.50 (3H, m , Py).

Production Example 4-7: N, N, N'-tri (2-pyridylmethyl) -N '- (4-D-biotinamido-2-pyridylmethyl) -1,3-diaminopropan-2-ol

62.4 mg of D-biotin (0.26 mmol) was suspended in 5 ml of dichloromethane, then 5.2 mg of 4-dimethylaminopyridine (0.04 mmol) and 35.8 μl of triethylamine (0.26 mmol) were added at room temperature. To the resulting mixture was added dropwise at room temperature a solution of N, N, N'-tri (2-pyridylmethyl) -N '- (4-amino-2-pyridylmethyl) -1,3-diaminopropan-2-ol (100 mg, 0.21 mmol) obtained in Production Example 4-6 in dichloromethane (2 ml). After completion of the addition, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (49.0 mg, 0.26 mmol) was added dropwise at room temperature. The reaction mixture was heated and the reaction was carried out at the boil under reflux for 5 hours. D-Biotin (6.0 mg, 0.02 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (10.0 mg, 0.05 mmol) and 2 ml of dichloromethane were added to the reaction mixture, followed by the reaction was carried out under reflux for 6 hours.

Then the reaction mixture was cooled and poured into 40 ml of water. After extraction with 30 ml of chloroform (3 times), the organic layers were washed with 30 ml of water (two times) and 30 ml of saturated saline. Then the organic layer was dried over anhydrous magnesium sulfate and concentrated, the resulting crude product was purified by HPLC to obtain 62.5 mg of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 1.37-1.45 (2H, m, CH ₂ ), 1.53-1.71 (4H, m, CH ₂ ), 2.19-2 25 (2H, m, COCH ₂ ), 2.62-2.75 (5H, m, NCH ₂ , SCH ₂ ), 2.87-2.93 (1H, dd, SCH ₂ ), 3.09- 3.15 (1H, m, SCH), 3.67-3.80 (9H, m, OCH, NCH ₂ Py), 4.28-4.32 (1H, m, NCH), 4.48-4 52 (1H, m, NCH), 5.15-5.40 (2H, m, NHCO), 5.89 (1H, broad s, NHCO) 6.48-6.50 (1H, m, Py ), 6.72-6.76 (1H, m, Py), 7.13-7.17 (3H, m, Py), 7.27-7.29 (1H, m, Py), 7.37 (2H, d, Py), 7.56-7.66 (3H, m, Py), 8.00 (lH, dd, Py), 8.49-8.50 (3H, m, Py).

This compound was analyzed using a MALDI-TOF mass spectrometer using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. The measurement result is presented in figure 5. As can be seen from FIG. 5, a molecular ion peak of 696.2 proton adduct ions (M ⁺ +1) was observed.

Production Example 5-1: Methyl 6-Aminohexanoate Hydrochloride

5.51 g of concentrated sulfuric acid was added to a suspension of 6-aminohexanoic acid (10.0 g, 76.2 mmol) in methanol (300 ml). The mixture was heated and its interaction was carried out for 8 hours, while distilling off the water formed during the esterification reaction with methanol, and 60 ml of methanol was added four times in turn. The reaction mixture was cooled and the pH was adjusted to 6.2 by adding a 28% solution of sodium methylate in methanol. The solvent was distilled off, the residue was taken up in 500 ml of water, and the pH of the mixture was adjusted to 10.3 by adding sodium carbonate. After extraction with 350 ml of dichloromethane (3 times), the extract was dried over anhydrous magnesium sulfate. Then, the solvent was distilled off to obtain methyl 6-aminohexanoate.

The resulting methyl 6-aminohexanoate was dissolved in 40 ml of dioxane, then 7.1 ml of a 4-n-hydrochloride solution of dioxane was added. After this, the mixture was stirred at room temperature for 30 minutes, the solvent was distilled off, then ether was added to the residue. Precipitated crystals were isolated by filtration and dried under reduced pressure to obtain 3.3 g of the target compound.

¹ H-NMR (DMSO-d ₆ , 300 MHz): δ 1.26-1.36 (2H, m, CH ₂ ), 1.48-1.61 (4H, m, CH ₂ ), 2.31 (2H, t, CH ₂ CO), 2.73 (2H, t, CH ₂ N), 3.59 (3H, s, CH ₃ ), 8.1 (3H, br s, NH ₃ ).

Production Example 5-2: 5-carbomethoxypentyl-D-biotinamide

To a solution of methyl 6-aminohexanoate hydrochloride (1.00 g, 5.50 mmol) obtained in Production Example 5-1 in dimethylformamide (40 ml) was added triethylamine (1.20 g, 11.8 mmol) and 4-dimethylaminopyridine (137 mg, 1.1 mmol). In addition, D-biotin (1.34 g, 5.50 mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.42 g, 7.42 mmol) were added. The reaction of the mixture was carried out at a temperature of from 35 to 45 ° C for 23 hours.

The reaction mixture was poured into 300 ml of water and extracted with 150 ml of chloroform (5 times). Then, the extracts were dried over anhydrous magnesium sulfate, and the solvent was distilled off. The resulting crude product was purified by silica gel column chromatography to give 1.04 g of the target compound.

¹ H-NMR (DMSO-d ₆ , 300 MHz): δ 1.18-1.65 (12H, m, CH ₂ ), 2.04 (2H, t, CH ₂ CO), 2.28 (2H, t, CH ₂ CO), 2.57 (1H, d, CH ₂ S), 2.82 (1H, dd, CH ₂ S), 3.00 (1H, dt, NHCO), 3.06-3, 12 (1H, m, CH ₂ S), 3.58 (3H, s, OCH ₃ ), 4.09-4.15 (1H, m, CHN), 4.28-4.32 (1H, m, CHN), 6.35 (1H, br s, NHCO), 6.41 (1H, br s, NHCO), 7.72 (1H, t, NHCO).

An example of obtaining 5-3: 5-carboxypentyl-D-biotinamide

In a mixture of 25 ml of tetrahydrofuran and 25 ml of methanol was dissolved 5-carbomethoxypentyl-D-biotinamide (800 mg, 2.15 mmol) obtained in Production Example 5-2, then sodium hydroxide solution (2.33 g) was added dropwise at room temperature. 58.3 mmol) in water (12.5 ml). Upon completion of the addition, the mixture was heated to 40 ° C and the reaction was carried out for 2 hours.

After cooling, the reaction mixture was concentrated, then 60 ml of water was added. Then added 1 N. a solution of hydrochloric acid and the pH of the mixture were adjusted to 2.0. Precipitated crystals were isolated by filtration and washed with water. The resulting crystals were dried in vacuo to give 724 mg of the target compound.

¹ H-NMR (DMSO-d ₆ , 300 MHz): δ 1.17-1.67 (12H, m, CH ₂ ), 2.04 (2H, t, CH ₂ CO), 2.19 (2H, t, CH ₂ CO), 2.57 (1H, d, CH ₂ S), 2.82 (1H, dd, CH ₂ S), 3.00 (1H, dt, NHCO), 3.06-3, 12 (1H, m, CH ₂ S), 4.13 (1H, t, CHN), 4.30 (1H, t, CHN), 6.36 (1H, br s, NHCO), 6.43 ( 1H, broad s, NHCO), 7.74 (1H, t, NHCO), 11.95 (1H, broad s, CO ₂ H).

Production Example 5-4: N, N, N'-tri (2-pyridylmethyl) -N '- [4- (6-D-biotinamidohexacarboxyamido) -2-pyridylmethyl] -1,3-diaminopropan-2-ol

To a solution of 5-carboxypentyl-D-biotinamide (99.0 mg, 0.27 mmol) obtained in Production Example 5-3, anhydrous dimethylformamide was added triethylamine (38.1 μl, 0.27 mmol) and 4-dimethylaminopyridine ( 5.7 mg, 0.04 mmol), then a solution of N, N, N'-tri (2-pyridylmethyl) -N '- (4-amino-2-pyridylmethyl) -1,3-diaminopropane-2 was added dropwise -ol (100 mg, 0.21 mmol) obtained in Production Example 4-6 in anhydrous dimethylformamide (1 ml). After this, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (51.7 mg, 0.27 mmol) was added. The reaction of the mixture was carried out at 40 ° C for 24 hours. 8.9 μl of triethylamine (0.06 mmol), 22.8 mg of 5-carboxypentyl-D-biotinamide (0.06 mmol) and 12.2 mg of 1-ethyl-3- (3-dimethylaminopropyl) hydrochloride were added to the reaction mixture. carbodiimide (0.06 mmol). The reaction of the mixture was carried out at 40 ° C for 6 hours.

After cooling the reaction mixture, the mixture was poured into 150 ml of water, then sodium chloride was added to saturate the solution. After extraction with 40 ml of chloroform (5 times), the organic layers were washed with 150 ml of water (twice) and 150 ml of saturated saline. The extract was then dried over anhydrous magnesium sulfate and the solvent was distilled off. The resulting crude product was purified by HPLC to give 37.5 mg of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 1.30-1.80 (12H, m, CH ₂ ), 2.14-2.25 (4H, m, COCH ₂ ), 2.60-2 76 (5H, m, NCH ₂ , CH ₂ S), 2.87 (1H, dd, CH ₂ S), 3.10-3.25 (3H, m, CH ₂ NHCO, SCH), 3.68 -3.82 (9H, m, NCH ₂ Py, OCH), 4.28-4.33 (1H, m, NCH), 4.45-4.52 (1H, m, NCH), 5.27 ( 1H, broad s, NHCO), 5.50 (1H, broad s, NHCO), 5.60 (1H, broad s, NHCO), 6.22 (1H, broad s, NHCO), 6, 52-6.57 (1H, m, Py), 6.72-6.75 (1H, m, Py), 7.16 (3H, t, Py), 7.27-7.30 (1H, m , Py), 7.37 (2H, d, Py), 7.57-7.66 (3H, m, Py), 7.99 (1H, d, Py), 8.46-8.52 (3H , m, Py).

This compound was analyzed using a MALDI-TOF mass spectrometer using CHCA as a matrix. The measurement result is presented in Fig.6. As can be seen from FIG. 6, a molecular ion peak of 809.5 proton adduct ions (M ⁺ +1) was observed.

Production Example 6-1: 2-bromomethyl-3-hydroxypyridine

To a 25% solution of hydrogen bromide in acetic acid (12.8 g, 39.6 mmol) was added 3-hydroxy-2-hydroxymethylpyridine hydrochloride (5.0 g, 54.7 mmol). The reaction of the mixture was carried out at 110 ° C for 1 hour. An additional amount of a 25% solution of hydrogen bromide in acetic acid (4.90 g, 15.1 mmol) was added dropwise and the mixture was reacted for 2 hours.

After cooling, the reaction mixture was poured into 50 ml of water and the pH of the mixture was adjusted to 8 by adding a saturated aqueous solution of sodium bicarbonate. After extraction with 50 ml of dichloromethane (four times), the dichloromethane layers were concentrated. The crude product after concentration was purified by silica gel column chromatography to obtain 1.36 g of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 5.31 (2H, s, CH ₂ Br), 7.20-7.32 (2H, m, Py), 8.18-8.21 (1H , m, Py), 8.55 (1H, br s, OH).

Production Example 6-2: N, N, N'-tri (2-pyridylmethyl) -N '- (3-hydroxy-2-pyridylmethyl) -1,3-diaminopropan-2-ol

In dimethylformamide (15 ml) was dissolved N, N, N'-tri (2-pyridylmethyl) -1,3-diaminopropan-2-ol (1.00 g, 2.75 mmol) obtained in Production Example 1-2, and 2-bromomethyl-3-hydroxypyridine (633 mg, 3.37 mmol) obtained in Production Example 6-1, then potassium carbonate (760 mg, 5.50 mmol) was added. The reaction of the mixture was carried out at 50 ° C for 2 hours and the mixture was cooled.

The reaction mixture was poured into 100 ml of water and the pH of the mixture was adjusted to 8 by adding 1N. hydrochloric acid solution. After extraction with 100 ml of ethyl acetate (3 times), the organic layers were washed with 100 ml of water and 100 ml of saturated saline and dried over anhydrous sodium sulfate. The resulting crude product was purified and collected by HPLC to give 660 mg of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 2.51-2.69 (4H, m, NCH ₂ ), 3.71-4.08 (9H, m, CH ₂ Py, OCH), 7, 05-7.14 (4H, m, Py), 7.15-7.19 (1H, t, Py), 7.23-7.28 (3H, m, Py), 7.55 (2H, dt , Py), 7.64 (1H, dt, Py), 7.91 (1H, dd, Py), 8.48-8.55 (3H, m, Py).

Production Example 6-3: N, N, N'-tri (2-pyridylmethyl) -N '- (3-D-biotinyloxy-2-pyridylmethyl) -1,3-diaminopropan-2-ol

To a solution of N, N, N'-tri (2-pyridylmethyl) -N '- (3-hydroxy-2-pyridylmethyl) -1,3-diaminopropan-2-ol (100 mg, 0.21 mmol) obtained in Production Example 6-2, in anhydrous dimethylformamide (5 ml) was added sodium hydride (dispersion in mineral oil) (9.0 mg, 0.23 mmol) at 3 ° C. After the temperature rose again to room temperature, the mixture was reacted at room temperature for 30 minutes, then a solution of N-succinimidyl D-biotinate (79.8 mg, 0.23 mmol) in anhydrous dimethyl sulfoxide (1 ml) was added dropwise.

After completion of the dropwise addition, the reaction of the mixture was carried out at room temperature for 3.5 hours, and the mixture was poured into 100 ml of water. After extraction with 300 ml of chloroform (3 times), the chloroform layers were dried over anhydrous sodium sulfate. Then, the solvent was distilled off, the crude product was collected and purified by HPLC to obtain 63.6 mg of the target compound.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 1.43-1.56 (2H, m, CH ₂ ), 1.60-1.85 (4H, m, CH ₂ ), 2.53 (2H , t, COCH ₂ ), 2.58-2.68 (4H, m, NCH ₂ ), 2.71-2.77 (1H, m, SCH ₂ ), 2.91 (1H, dd, SCH ₂ ) , 3.13-3.19 (1H, m, SCH), 3.76-3.99 (9H, m, OCH, NCH ₂ Py), 4.28-4.36 (1H, m, NCH), 4.47-4.53 (1H, m, NCH), 5.16 (1H, br s, NHCO), 5.93 (1H, d, NHCO) 7.08-7.14 (3H, m, Py), 7.20 (1H, dd, Py), 7.33-7.42 (4H, m, Py), 7.52-7.62 (3H, m, Py), 8.42 (1H, dd, Py), 8.51 (3H, m, Py).

Production Example 6-4: A Solution Containing the Zinc Complex of the Present Invention

To a solution containing 200 nmol N, N, N'-tri (2-pyridylmethyl) -N '- (3-D-biotinyloxy-2-pyridylmethyl) -1,3-diaminopropan-2-ol obtained in Production Example 6 -3, in acetonitrile (66.7 μl) 40.1 μl of an aqueous solution of zinc nitrate (100 mM) was added, then 560 μl of phosphoric acid buffer (pH = 6.86) was added. The solution was analyzed using a mass spectrometer using 2 ', 4'-6'-trihydroxyacetophenone (THAP) as a matrix and the zinc complex was identified. The measurement results obtained using a MALDI-TOF mass spectrometer are presented in Fig.7. As shown in FIG. 7, a molecular ion peak was observed at 919.1 (exact mass: 919.13).

Production Example 7-1: N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-dansylaminoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropan-2-ol

In anhydrous acetonitrile (9 ml), N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "-2- (aminoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropane-2 was dissolved -ol (1.1 g, 2.1 mmol) obtained in Production Example 1-4, then dansyl chloride (840 mg, 3.1 mmol) was added.The reaction mixture was heated and reacted at 50 ° C. for 3 hours. Then the reaction mixture was concentrated, the crude product was purified by silica gel column chromatography to obtain 1.30 g of the target compound.

^l H-NMR (CDCl ₃ , 500 MHz): δ 2.57 (1H, dd, CH ₂ ), 2.60 (1H, dd, CH ₂ ), 2.67 (1H, dd, CH ₂ ), 2 69 (1H, dd, CH ₂ ), 2.85 (6H, s, NCH ₃ ), 3.17 (2H, dd, NCH ₂ CH ₂ N), 3.50 (2H, dd, NCH ₂ CH ₂ N), 3.88 (8H, dd, NCH ₂ Py), 3.83-3.92 (1H, m, OCH), 6.31 (1H, m, SO ₂ NH), 7.11 (1H, d, Ar), 7.13 (3H, ddd, Py), 7.35 (3H, d, Py), 7.36 (1H, d, Py), 7.42 (1H, m, CONH), 7 46 (1H, dd, Ar), 7.49 (1H, dd, Ar), 7.60 (2H, dt, Py), 7.60 (1H, dt, Py), 7.90 (1H, dd , Py), 8.23 (1H, dd, Ar), 8.27 (1H, d, Ar), 8.49 (2H, ddd, Py), 8.50 (1H, ddd, Py), 8, 51 (1H, dd, Ar); 8.80 (1H, d, Py).

¹³ C-NMR (CDCl ₃ , 125 MHz): 39.9 (NCH ₂ CH ₂ N), 42.7 (NCH ₂ CH ₂ N), 45.3 (NCH ₃ ), 59.0 (CH ₂ ), 59.1 (CH ₂ ), 60.6 (CH ₂ Py), 60.7 (CH ₂ Py), 61.3 (CH ₂ Py), 67, l (CHO), 115.2 (Ar), 118 6 (Ar), 122, l (Py), 122.8 (Py), 123.2 (Ar), 123.2 (Py), 128.1 (Ar), 128.5 (Ar), 129, 5 (Ar), 129.6 (Ar), 129.9 (Py), 130.6 (Ar), 134.5 (Ar), 135.4 (Py), 136.5 (Py), 136.6 (Py), 147.6 (Py), 148.9 (Py), 149.0 (Py), 152.0 (Ar), 159.1 (Py), 159, l (Py), 162.8 ( Py), 166.2 (CONH).

Production Example 7-2: A Solution Containing the Zinc Complex of the Present Invention

200 nmol of N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-dansylaminoethyl) carbamoyl-2-pyridylmethyl] -1 was dissolved in phosphoric acid buffer (pH = 6.86), The 3-diaminopropan-2-ol obtained in Production Example 7-1 to obtain a 3 mM solution, then 2 equivalents of zinc nitrate was added to the solution.The solution was analyzed using a MALDI-TOF mass spectrometer using THAP as a matrix, and identified zinc complex The measurement results obtained using a MALDI-TOF mass spectrometer are shown in Fig. 8. As shown in Fig. 8, a molecular ion peak was observed at 996.2 (i.e. actual mass: 996.16).

Production Example 8-1: N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-N-5-fluoresceinylthioureidoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropane -2-ol

N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2-aminoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropane-2 was dissolved in anhydrous dimethylformamide (150 ml) -ol (950 mg, 1.76 mmol) obtained in Production Example 1-4, then anhydrous pyridine (38 ml) was added, then a solution of fluorescein-5-isothiocyanate (750 mg, 1.92 mmol) in anhydrous was added dropwise. dimethylformamide (150 ml) and the reaction mixture was carried out at room temperature for 3 hours, then the reaction mixture was concentrated, the crude product was purified by silica gel column chromatography to obtain 961 mg of cete evogo compound.

¹ H-NMR (DMSO-d ₆ , 300 MHz): δ 2.33-2.43 (2H, m, CH ₂ ), 2.50-2.61 (2H, m, CH ₂ ), 3.38 -3.57 (4H, m, NCH ₂ CH ₂ N), 3.67-3.92 (9H, m, NCH ₂ Py, CHO), 6.52-6.67 (6H, m, Ar), 7.16-7.23 (3H, m, Py), 7.17 (1H, d, Ar), 7.34-7.41 (3H, m, Py), 7.52 (1H, d, Py ), 7.69 (3H, dt, Py), 7.68-7.77 (1H, m, Ar), 8.14 (1H, dd, Py), 8.24 (1H, d, Ar), 8.41-8.46 (3H, m, Py); 8.78 (1H, m, NHCO); 8.92 (1H, d, Py).

Experimental Example 3: Electrophoresis in 10% native polyacrylamide gel

First, gels for electrophoresis, a pH buffer for electrophoresis and a staining solution for dissolving the samples under the same conditions as described in experimental example 1 were prepared.

Then, 2 μg each of 1: bovine serum albumin, 2: human serum albumin, 3: carbonic anhydrase, 4: β-galactosidase, 5: α-casein (phosphorylated), 6: α-casein (dephosphorylated), 7: β- casein (phosphorylated), 8: β-casein (dephosphorylated), 9: pepsin (phosphorylated), and 10: pepsin (dephosphorylated) were dissolved in a staining solution to obtain samples. Appropriate samples were applied to the gel. A direct current of 40 mA was then reported until the stain marker leaked.

The resulting gel was immersed for 30 minutes in 10 mM Tris-acetate buffer (pH = 7.4) containing 1 μM N, N, N'-tri (2-pyridylmethyl) -N '- [5-N "- (2- N-5-fluoresceinylthioureidoethyl) carbamoyl-2-pyridylmethyl] -1,3-diaminopropan-2-ol obtained in Production Example 8-1 and 100 μM zinc acetate. The gel was then removed from the solution. This gel was washed twice with 100 ml 10 mM Tris Acetate Buffer (pH = 7.4) for 10 minutes, then a fluorescent gel image was obtained using a photograph using a FLA-5000 fluorescence image analyzer (Fuji Photo Film Co., Ltd.) at an excitation wavelength of 47 3 nm using a 510 nm fluorescence detection filter, the gel was then stained with Coomassie brilliant blue according to the traditional staining method and the colored gel was photographed. The gel stained with a solution containing zinc complex is indicated as “Gel A” and the gel stained with Coomassie brilliant blue is indicated as "gel B", both of these gels are shown in Fig.9.

As can be seen from figure 9, in the case of staining in the traditional way, all peptides were stained, as shown in the example of gel B. On the other hand, only 5: α-casein (phosphorylated), 7: β-casein (phosphorylated), and 9: pepsin (phosphorylated), all associated with phosphoric acid, were each identified individually, as shown in gel A, which was treated with the solution containing the zinc complex of the present invention. Thus, it is obvious that the method of the present invention is advantageous for identifying exclusively phosphorylated peptides in samples obtained from living organisms.

The basis of this application is Japanese patent application No. 2003-56068, filed March 3, 2003, No. 2003-113707, filed April 18, 2003 and No. 2003-356934, filed October 16, 2003, the contents of which are included in this application by reference.

Although the present invention has been described in detail by way of examples, it should be understood that various changes and modifications are obvious to those skilled in the art. Therefore, if such changes and modifications are not a departure from the scope of the present invention defined hereinafter, they should be construed as being included in the scope of the present invention.

Claims (11)

1. The method of labeling a phosphorylated peptide with a complex compound of the formula (I)

where X represents a linker group, and Y represents a fluorescent group or biotin as a labeling group. 2. The method according to claim 1, where the complex compound is a compound containing biotin as a labeling group. 3. A method for adsorption of a phosphorylated peptide using a complex compound of the formula (I)

where X represents a linker group, and Y represents a fluorescent group or biotin as a labeling group. 4. The method according to claim 3, where the complex compound is a compound containing biotin as a labeling group. 5. The complex compound of the formula (I)

where X represents a linker group, and Y represents a fluorescent group or biotin as a labeling group. 6. The complex compound according to claim 5, where the labeling group is biotin. 7. The compound of formula (II)

where R ¹ represents a (amino (C ₁ -C ₆ alkyl) carbamoyl group or hydroxy group. 8. The precursor compound of compound (I) represented by formula (IV)

where X represents a linker group, and Y represents a fluorescent group or biotin as a labeling group. Priority on points: 03.03.2003 according to claims 1, 3, 5 and 8, except for the sign when Y represents biotin as a labeling group, claim 7; 04/18/2003 according to claims 1, 3, 5 and 8 for the case when Y represents biotin as a labeling group, claims 2, 4 and 6; 10.16.2003 according to claims 1-6 and 8 for the case of variation of the values of radicals.

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