KR100877644B1 - Linker compound, ligand complex and process for producing them - Google Patents

Abstract

The present invention provides a novel linker compound capable of efficiently forming metal-sulfur bonds by suppressing non-specific hydrophobic interactions as much as possible and also making it possible to easily adjust the length to disulfide groups provided for metal bonds. And novel ligand complexes, ligand carriers, and methods for their preparation. The linker compound has a structure represented by the following General Formula (1) (wherein a, b, d and e are each independently an integer of 0 or more and 6 or less). The said X has the structure which is a multi-branched structural site which has an aromatic amino group at the terminal, and contains three or more chains of the hydrocarbon derivative chain which may have a carbon-nitrogen bond in the main chain. In the ligand conjugate, a sugar molecule is introduced into the linker compound.

Description

LINKER COMPOUND, LIGAND COMPLEX AND PROCESS FOR PRODUCING THEM}

The present invention relates to a linker compound capable of immobilizing a sugar chain such as oligosaccharide on a support for protein analysis such as a surface plasmon resonance sensor chip, and a ligand conjugate, a ligand carrier, and a method for producing the linker compound. It is about.

The various sugar chains in living organisms play an important role in the activity of living things and the mechanisms for sustaining life. In order to elaborate the functions of such sugar chains, it is necessary to interpret their functions based on the complex structure of sugar chains. In the functional analysis of sugar chains, a technique of partially reproducing the sugar chains using oligosaccharides of which the structure is elucidated, thereby revealing the relationship between the structure and the function of the entire sugar chain is used.

As the functional analysis technique of the sugar chain, for example, a surface plasmon resonance (hereinafter referred to as SPR) method is known. That is, a ligand conjugate comprising oligosaccharides that simulates a part of sugar chains is immobilized on the surface of the sensor chip, whereby a protein that specifically interacts with the oligosaccharides using a sensor chip in which the oligosaccharide is immobilized is used. Specify the substance. Thereby, the correct evaluation of the biological activity based on oligosaccharide structure can be performed.

However, since the oligosaccharide is not very high in activity alone, it is necessary to aggregate the oligosaccharide chain on the sensor chip when evaluating the biological activity of the oligosaccharide. In other words, by analyzing the interaction with the protein using the aggregated oligosaccharide chain, it becomes possible to evaluate the biological activity of the oligosaccharide chain.

Here, the inventors of the present invention have obtained a linker compound having a moiety that can be immobilized on the surface of the sensor chip and a site capable of introducing an oligosaccharide chain in a molecule, and a ligand conjugate formed by introducing one or two oligosaccharide chains into the linker compound. . And it discovered that it is possible to collect and introduce | transduce oligosaccharide chain on a sensor chip by using this ligand complex (for example, refer patent document 1, nonpatent literature 1, etc.).

[Patent Document 1] Japanese Patent Publication No. 2003-836969 (published March 19, 2003)

[Non-Patent Document 1] Japanese Chemistry Society 79th Spring Banquet-Presentation Preview II Japanese Chemistry Society, March 15, 2001, p. 1042

However, in the ligand conjugate described in Patent Document 1 and Non-Patent Document 1, it is possible to arrange oligosaccharide sugar chains two-dimensionally on the surface of the sensor chip, but technical problems remain that it is difficult to obtain the arrangement with high reproducibility.

That is, as described above, in the case of analyzing the biological activity of the oligosaccharide chain by collecting oligosaccharide chains of a plurality of molecules on the surface of the sensor chip, the interaction between the oligosaccharide chain and the protein is made equal by the same state of aggregation of the sugar chain of the oligosaccharide. Observation with high reproducibility is required. In particular, in order to observe the biological activity of the oligosaccharide chain, by evaluating the biological activity of the oligosaccharide chain with high reproducibility by collecting three or more units of the oligosaccharide chain on the surface of the sensor chip and arranging them in two dimensions with high reproducibility on these sensor chips. Becomes important.

However, in the ligand conjugate described in Non-Patent Document 1, the oligosaccharide chain included in one ligand complex consists of one unit or two units. In other words, the ligand conjugate is composed of one or two oligosaccharide chains bonded to one linker compound. Therefore, in order to observe the biological activity of the oligosaccharide chain, when the ligand conjugate is arranged on the surface of the sensor chip, the ligand complex concentration is increased to aggregate the sugar chains, which are ligands, to aggregate at least three units of the oligosaccharide chain on the surface of the sensor chip. There is a need.

When oligosaccharide chains are aggregated according to such a technique, it is difficult to obtain the arrangement of oligosaccharides with high reproducibility by controlling the sugar chains of oligosaccharides at predetermined intervals. Therefore, in the conventional ligand conjugate, it is impossible to observe the biological activity of the oligosaccharide with high reproducibility, and there is a possibility of difficulty in clarifying the sugar structure and evaluating the biological activity of the oligosaccharide.

The present invention has been made to solve the above problems, and an object thereof is to provide a novel linker compound capable of controlling the distance between sugar chains on the surface of the sensor chip and arranging oligosaccharides in two dimensions with high reproducibility, and this linker compound. There is provided a novel ligand complex, a ligand carrier, and a method for producing these, wherein sugar molecules are introduced.

In order to solve the above problems, the inventors of the present invention have a site capable of introducing more than 3 units of sugar molecules, and also analyzes proteins such as surface plasmon resonance (SPR) sensor chips and carriers of affinity chromatography. It has been found that by using a novel linker compound having a site capable of binding to a support, two or more units of sugar molecules can be two-dimensionally arranged on the support with high reproducibility.

On the other hand, the inventors of the present application, the prior application (application number: Japanese Patent Application No. 2003-190568, publication number: Japanese Patent Publication No. 2004-157108 (published: June 3, 2004), the priority date of the present application (2004 Another linker compound found for the purpose of resolving the above problem is disclosed. However, this other linker compound has a problem of non-specifically binding and interacting with the alkyl group of the linker when analyzing a protein having a very high hydrophobicity. In addition, this other linker compound has a problem that the length of the alkyl group forming the linker is not sufficient, and in the case where the immobilized oligosaccharide chain is large, metal-sulfur bonds are not formed efficiently due to steric hindrance of the oligosaccharide chains. .

Here, the inventors further introduce oligoethylene oxide groups in the linker portion to suppress nonspecific hydrophobic interactions as much as possible, and also to easily adjust the length to the disulfide groups provided for the metal bonds, thereby efficiently metal-sulfur bonds. It was found that it can be formed to complete the present invention.

That is, the linker compound which concerns on this invention is general formula (1) in order to solve the said subject.

…(1)

… (One)

(Wherein a, b, d and e are each independently an integer of 0 to 6), wherein X has an aromatic amino group at the terminal and a carbon-nitrogen bond in the main chain. It is characterized by including a structure which is a multi-branched structural site including three or more chains of hydrocarbon derivative chains which may be present.

In addition, the linker compound which concerns on this invention is general formula (2)

…(2)

… (2)

Wherein n is an integer of 1 or more and 6 or less), wherein X has an aromatic amino group at the terminal and may have a carbon-nitrogen bond in the main chain. It may be provided with the structure which is a multi-branched structure site | part including chain or more.

Here, the hydrocarbon derivative chain means that some carbon or hydrogen may be substituted with other atoms or substituents in the hydrocarbon chain composed of carbon and hydrogen. That is, the hydrocarbon derivative chain has an aromatic amino group at the terminal, and a part of the carbon-carbon bond (CC bond) which is the main chain structure of the hydrocarbon chain is a carbon-nitrogen bond (CN bond), a carbon-oxygen bond (CO bond), It points out that it may be substituted by the amide bond (CO-NH bond).

According to the said structure, the said linker compound has aromatic amino group as a site | part which can introduce | transduce a sugar molecule easily. Since the aromatic amino group is contained in each hydrocarbon derivative chain, three or more units of sugar molecules can be introduced into the linker compound. In addition, it has an S-S bond as a site that can be fixed to the support for protein analysis.

Therefore, it is possible to aggregate by introducing three or more units of sugar molecules to the support through the linker compound. In addition, since three or more units of sugar molecules are introduced into one linker compound, three or more units of sugar molecules can be arranged on the surface of the support with high reproducibility. As a result, the interaction between the sugar molecules and the protein can be observed on the surface of the support, and the biological activity of the sugar molecules can be evaluated with high reproducibility.

Further, since the linker compound has an oligoethylene oxide group in the linker portion, the linker compound can significantly reduce the possibility of causing non-specific interaction with an analyte having high hydrophobicity as compared to the case in which the linker compound has an alkyl group. In addition, since the linker portion is composed of oligoethylene oxide, the length from the disulfide group provided to the metal bond to the oligosaccharide chain bonded to the amino terminal can be easily adjusted. Thereby, a disulfide group can form a metal-sulfur bond efficiently, without being influenced by an oligosaccharide chain.

In the linker compound having the structure represented by the general formula (1) or (2), X is the general formula (3)

…(3)

… (3)

It is preferable to have a structure represented by (in formula, m <^1> , m <^2> , m <^3> , m <^4> , p <^1> , and p <^2> are integers 1-6 at each, respectively.).

In addition, in the linker compound provided with the structure represented by the said General formula (1) or (2), said X is General formula (4)

…(4)

… (4)

(Wherein q ¹ , q ² , q ³ , r ¹ , r ² , r ³ , t ¹ , t ² , t ³ , u ¹ , u ² , u ³ are each independently an integer of 0 or more and 6 or less) It is preferable to have the structure shown by ().

Since X of the linker compound has three or more of the hydrocarbon derivative chains, three or more units of sugar molecules can be introduced onto the support through the linker compound. Therefore, since the arrangement of sugar molecules can be obtained with high reproducibility by controlling the interval between three or more units of sugar molecules on the support surface, the biological activity of the sugar molecules can be evaluated with high reproducibility.

Further, the ligand conjugate of the present invention is characterized in that a sugar molecule is introduced to the aromatic amino group of any of the linker compounds described above in order to solve the above problems.

And, the ligand complex, specifically, the general formula (5)

…(5)

… (5)

(Wherein m ¹ , m ² , m ³ , m ⁴ , n, p ¹ , p ² are each independently an integer of 1 or more and 6 or less. R ′ is hydrogen (H) or a structure represented by R.) Wherein R is represented by formulas (6-1) to (6-6)

It is preferable that it is an oligosaccharide derived compound chosen from.

In addition, the ligand complex is specifically, Formula (7)

…(7)

… (7)

(Wherein a, b, d, e, q ¹ , q ² , q ³ , r ¹ , r ² , r ³ , t ¹ , t ² , t ³ , u ¹ , u ² , u ³ are each independent Is an integer equal to or greater than 0 and equal to 6, except that t ¹ , t ^2, and t ³ are not 0 when b is 0, and b is not O when t ¹ , t ^2, and t ³ are O. Has a structure represented by hydrogen (H) or R.), and R is preferably an oligosaccharide selected from formulas (6-1) to (6-6).

By using the ligand complex, a sugar molecule of three or more units or four or more units (when a ligand complex having a structure represented by the formula (5) or (7) is used) is collected on the surface of the support for protein analysis. Can be immobilized. Thus, since one ligand complex has three or more units of sugar molecules, three or more units of sugar molecules can be aggregated by using one ligand complex without aggregation of the ligand complexes on the surface of the support. Therefore, the biological activity of sugar molecules can be measured with high reproducibility. In addition, a plurality of sugar molecules can be arranged on the surface of the support in two dimensions with high reproducibility. Therefore, it is possible to evaluate the biological activity of sugar molecules with high reproducibility by using the protein analysis support to which the ligand conjugate of the present invention is immobilized.

In addition, the method for producing a linker compound of the present invention, in order to solve the above problems, the step of performing a condensation reaction of thio compounds and amine compounds having at least three branched chains in which the aromatic amino group terminal is protected by a protecting group; And deprotecting the protecting group at the terminal of the aromatic amino group.

According to the above method, the linker compound of the present invention having an S-S bond as a site that can be immobilized on the support for protein analysis and an aromatic amino group as a site to which sugar molecules can be easily introduced can be obtained.

Moreover, the manufacturing method of the ligand conjugate of this invention is characterized by performing a reduction amination reaction using the said linker compound and a sugar molecule, in order to solve the said subject.

According to the above method, the sugar complex can be introduced into the linker compound by a reduction amination reaction to obtain the ligand conjugate of the present invention.

In addition, as said sugar molecule, all kinds of sugar molecules which have a reducing terminal can be used.

Specifically as said sugar molecule, General formula (8)

…(8)

… (8)

It is preferable to use the sulfated oligosaccharide having a heparin partial disaccharide structure represented by.

Moreover, as said sugar molecule specifically, group (9)

…(9)

… (9)

It is preferable to use at least one oligosaccharide selected from.

In addition, the method for introducing a sugar molecule of the present invention is characterized in that the solution containing the ligand conjugate is brought into contact with a metal on the surface of the support to solve the above problems.

According to the above method, the S-S bond of the linker compound included in the ligand complex may be converted into a bond with a metal on the surface of the support, and the sugar chain, which is a ligand, may be immobilized on the surface of the support. Thus, the sugar molecules bound to the linker compound can be arranged on the surface of the support by a simple method of contacting the support with a solution comprising the ligand conjugate.

In addition, the ligand carrier of the present invention is characterized in that the ligand conjugate is immobilized on a support having a metal on its surface in order to solve the above problems.

According to the above configuration, the ligand conjugate can be firmly fixed to the surface of the support through the metal-sulfur bond, thereby providing a ligand carrier formed by arranging a plurality of sugar molecules on the surface of the support with high reproducibility. Therefore, the ligand carrier enables the quantitative evaluation of the biological activity of sugar molecules because the interaction between sugar molecules contained in the ligand complex and substances such as proteins interacting with the sugar molecules can be observed with high reproducibility. .

Still other objects, features, and advantages of the present invention can be fully understood by the following description. In addition, the advantages of the present invention can be seen from the following description with reference to the accompanying drawings.

1 is a schematic diagram showing an example of a synthetic route of a linker compound (Compound 15) according to the present invention.

Figure 2 is a schematic diagram showing an example of the synthetic route of the ligand conjugate (Compound 17) according to the present invention.

3 is a graph showing the binding behavior of bFGF to the chip immobilized with Mono-GlcNS6S-IdoA2S-Glc in the presence of heparin.

4 is a graph showing the inhibition rate of heparin on the binding interaction of bFGF to the chip to which Mono-GlcNS6S-IdoA2S-Glc, Tri-GlcNS6S-IdoA2S-Glc, and Tetra-GlcNS6S-IdoA2S-Glc were immobilized, respectively.

FIG. 5A is a graph showing the total reflection infrared absorption spectrum of Tri-GlcNS6S-IdoA2S-Glc in which the mixing ratio in the solution is changed.

(B) is a graph which shows the total reflection infrared absorption spectrum of Tetra-GlcNS6S-IdoA2S-Glc which changed the mixing ratio in a solution.

FIG. 6A is a graph showing the relative strength of sulfuric acid groups on a chip with respect to the mixing ratio of Tri-GlcNS6S-IdoA2S-Glc in the solution.

FIG. 6B is a graph showing the relative strength of sulfuric acid groups on a chip with respect to the mixing ratio of Tetra-GlcNS6S-IdoA2S-Glc in the solution.

FIG. 7A is a graph showing the results of observing the binding interaction of h-vWF when the mixing ratio of Mono-GlcNS6S-IdoA2S-Glc and Mono-Glc is 100/0 according to the SPR method.

FIG. 7B is a graph showing the results of observing the binding interaction of h-vWF when the mixing ratio of Tri-GlcNS6S-IdoA2S-Glc and Mono-Glc is 100/0 according to the SPR method.

FIG. 7C is a graph showing the results of observing the binding interaction of h-vWF when the mixing ratio of Tetra-GlcNS6S-IdoA2S-Glc and Mono-Glc is 100/0 according to the SPR method.

8 (a) is a graph showing the results of observing the binding interaction of h-vWF when the mixing ratio of Mono-GlcNS6S-IdoA2S-Glc and Mono-Glc is 20/80 according to the SPR method.

8B is a graph showing the results of observing the binding interaction of h-vWF when the mixing ratio of Tri-GlcNS6S-IdoA2S-Glc and Mono-Glc is 20/80 according to the SPR method.

8C is a graph showing the results of observing the binding interaction of h-vWF when the mixing ratio of Tetra-GlcNS6S-IdoA2S-Glc and Mono-Glc is 20/80 according to the SPR method.

FIG. 9A is a graph in which the binding amounts obtained from the results shown in FIGS. 7A and 8A are plotted for each concentration of h-vWF.

FIG. 9B is a graph in which the binding amounts obtained from the results shown in FIGS. 7B and 8B are plotted for each concentration of h-vWF.

FIG. 9C is a graph in which the binding amounts obtained from the results shown in FIGS. 7C and 8C are plotted for each concentration of h-vWF.

FIG. 10 (a) shows the result of measuring the interaction between rhvWF-A1 and a chip in which Mono-GlcNS6S-IdoA2S-Glc is immobilized when Mono-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0. It is a graph.

(B) of FIG. 10 shows the result of measuring the interaction between rhvWF-A1 and a chip in which Mono-GlcNS6S-IdoA2S-Glc is immobilized when Mono-GlcNS6S-IdoA2S-Glc / Mono-Glc = 50/50. It is a graph.

11 (a) shows the result of measuring the interaction between rhvWF-A1 and a chip in which Tri-GlcNS6S-IdoA2S-Glc is immobilized when Tri-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0. It is a graph.

(B) of FIG. 11 shows the result of measuring the interaction between rhvWF-A1 and a chip in which Tri-GlcNS6S-IdoA2S-Glc is immobilized when Tri-GlcNS6S-IdoA2S-Glc / Mono-Glc = 50/50. It is a graph.

FIG. 12 (a) shows the result of measuring the interaction between rhvWF-A1 and a chip in which Tetra-GlcNS6S-IdoA2S-Glc is immobilized when Tetra-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0 It is a graph.

12 (b) shows the result of measuring the interaction between rhvWF-A1 and a chip in which Tetra-GlcNS6S-IdoA2S-Glc is immobilized when Tetra-GlcNS6S-IdoA2S-Glc / Mono-Glc = 50/50 Is a graph.

Fig. 13 is a schematic diagram showing one example of the synthetic route of the linker compound (Compound 26) according to the present invention.

14 is a schematic view showing an example of the synthetic route of the ligand conjugate (Compound 27) according to the present invention.

15 is a schematic diagram showing an example of a synthetic route of a linker compound (Compound 32) according to the present invention.

FIG. 16 is a schematic diagram illustrating an example of a synthesis route of H ₂ N-TEG-NHBoc (Compound 30). FIG.

17 is a schematic diagram showing an example of the synthetic route of the ligand conjugate (Compound 34) according to the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The linker compound of the present invention contains a sugar molecule on the support via a protein analysis support such as a surface plasmon resonance (SPR) sensor chip or an affinity chromatography carrier and a sugar such as oligosaccharide (hereinafter referred to as a sugar molecule). Used to immobilize. Therefore, it is necessary for the linker compound to have a moiety that can be immobilized to the support and a moiety to which sugar molecules can be easily introduced.

In addition, the SPR or affinity chromatography aims to identify or isolate substances such as proteins that specifically interact with sugar molecules. Therefore, the linker compound should be one that does not have nonspecific interaction with a substance such as a protein.

Therefore, the linker compound of this invention has a disulfide bond (S-S bond) as shown to the said General formula (1) or (2) as a site | part which can be fixed to the said support body. The sulfur (S) of the disulfide bond can form a metal-sulfur bond with a metal such as gold (Au) coated on the surface of the support for protein analysis, for example, and can be firmly bound to the support.

In addition, the linker compound has a plurality of amino groups as a site to which sugar molecules can be easily introduced in order to arrange a plurality of sugar molecules two-dimensionally on the surface of the protein analysis support and to control the distance between sugar chains of individual sugar molecules. Has a multi-branch site, including. That is, the multi-branched moiety of the linker compound of the present invention is a moiety having a structure represented by X in General Formula (1) or (2), and as described above, X is aromatic at the terminal. It has a structure which has an amino group and contains three or more hydrocarbon derivative chains which may have a carbon-nitrogen bond or an amide bond in a principal chain.

The amino group (-NH ₂ group) of the said aromatic amino group becomes a reactor for introduce | transducing a sugar molecule into the said linker compound by reduction amination reaction with sugar molecules, such as an oligosaccharide. That is, an aldehyde group (-CHO group) or a ketone group (-CRO group, R is a hydrocarbon group) generated by the equilibrium in a sugar molecule reacts with an amino group of the linker compound. The sugar molecules can be easily introduced into the aromatic amino group by continuously reducing the Schiff's base formed by this reaction.

Therefore, X of the said General formula (1) or (2) has the structure which is a multi-branched site | part which has two or more aromatic amino groups which can introduce a sugar molecule by including three or more chains of the above-mentioned hydrocarbon derivative chain. Since sugar molecules such as oligosaccharides are introduced into each of the aromatic amino groups included in the multi-branched moiety, 2 is added to the surface of the support for protein analysis through a linker compound having a structure represented by the general formula (1) or (2). It is possible to arrange a plurality of sugar molecules with high reproducibility in dimensions.

Moreover, the linker compound of this invention has an oligoethylene oxide between a disulfide group and an aromatic amino group, as shown to the said General formula (1) or (2). Thereby, metal-sulfur bonds can be efficiently formed by suppressing non-specific hydrophobic interactions as much as possible and also making it possible to easily adjust the length to the disulfide groups provided for the metal bonds. In General Formula (1), a, b, d, and e may each be an integer of 0 or more and 6 or less. However, when b is 0, it is necessary to have oligoethylene oxide inside X. In the said General formula (2), if n is an integer of 1 or more and 6 or less, it will not be limited.

Specifically, as shown by the general formula (3), X has two two-branched structures in which two hydrocarbon derivative chains are bonded to one nitrogen (N) at a terminal opposite to the aromatic amino group. You may be provided with the structure which is. In this case, the nitrogen of two bi-branched structures is bonded to one nitrogen (N) via, for example, -CO-CH ₂ -to form a branched structure. As a result, X has a structure that is a multi-branched moiety having four hydrocarbon derivative chains. In addition, in said general formula (3), m <^1> , m <^2> , m <^3> , m <^4> is not limited as long as it is an integer of 1 or more and 6 or less, It may be different integers, and some or all may be the same integer. Among these, for convenience in the preparation of the compound having the multi-branched moiety, m ¹ to m ⁴ are preferably the same integers, and particularly preferably 2. Further, p ¹ and p ² are not particularly limited as long as they are integers of 1 to 6, and may be different integers or the same integers. Among these, for convenience of manufacture, p ¹ and p ² are preferably the same integers, and particularly preferably 1.

On the other hand, in X provided with four hydrocarbon derivative chains shown in the said General formula (3), it is also possible to set it as the structure which has oligoethylene oxide in each hydrocarbon derivative chain. For example, as shown in the formula (4), up to between CH ₂ and NH in each hydrocarbon derivative chain can be a structure having ethylene oxide.

In addition, said X has the structure which has a tribranched structure couple | bonded with one carbon (C) in the terminal on the opposite side to an aromatic amino group as the three-chain hydrocarbon derivative chain is represented by the said General formula (4). You may be provided. In this case, the carbon of the three branched structure forms a branched structure by, for example, bonding to one nitrogen (N) via -C-N-. As a result, X has a structure that is a multi-branched moiety having three hydrocarbon derivative chains.

In the formula (4), q ¹ , q ² and q ³ are not limited as long as they are integers of 0 to 6, and may be different integers or some or all of the same integers. Among these, for the convenience of preparing the compound having the multi-branched moiety, the q ¹ to q ³ are preferably the same integer, and particularly preferably 2. In addition, r ^1, r ^2, r ³ are not limited is an integer of 0 to 6, and is also different integers also are integers such that some or all. Among these, for convenience of manufacture, it is preferable that the said r <^1> -r <^3> is the same integer mutually, It is preferable that it is especially 1. In addition, u ^1, u ^2, u ³ are not limited is an integer of 0 to 6, and is also different integers also are integers such that some or all. Among these, for convenience of manufacture, it is preferable that the above u ¹ to u ³ are the same integers, and particularly preferably 1. Further, t ¹ , t ² , and t ³ are not limited as long as they are integers of 0 to 6, and may be different integers or some or all of the same integers. However, the in the general formula (1) when X is the art the general formula (4), wherein the case is 0 in the formula (1) b t ^1, t ^2, t ³ is preferably an integer of 1 to 6. Do. For convenience of production, t ¹ to t ³ are preferably the same integers, and particularly preferably 4.

As described above, X has a structure which is a multi-branched moiety which forms a branched structure by combining a plurality of hydrocarbon derivative chains with an atom such as carbon or nitrogen. On the other hand, it is preferable that all of the plurality of hydrocarbon derivative chains included in X are the same, but may have different structures as long as they have an aromatic amino group at the terminal.

As mentioned above, the linker compound which has a structure represented by General formula (1) or (2) has the SS bond couple | bonded with the support for protein analysis, and the amino group couple | bonded with sugar molecules, such as an oligosaccharide chain. . Thus, for example, the linker compound is immobilized on the support for protein analysis by metal-sulfur bonds such as Au-S bonds, so that the sugar molecules can be firmly and simply attached onto the support through the linker compound.

In addition, the linker compound has a multi-branched moiety and has an aromatic amino group at each terminal of the multi-branched moiety. Therefore, by using a ligand conjugate (described later) in which sugar molecules are introduced into the linker compound, sugar molecules can be efficiently collected on the surface of the support. In addition, since it has a multi-branched moiety, when a ligand conjugate comprising a linker compound is bound to the surface of a support, a plurality of sugar molecules can be arranged in two dimensions with high reproducibility.

In addition, the linker compound can almost ignore the effects of nonspecific interactions with the protein. Therefore, by using the linker compound of this invention, it becomes possible to evaluate the bioactivity of a sugar molecule with high reproducibility.

In addition, the linker compound has an oligoethylene oxide between the disulfide group and the aromatic amino group, as shown in the general formula (1) or (2). Thereby, metal-sulfur bonds can be efficiently formed by suppressing non-specific hydrophobic interactions as much as possible and also making it possible to easily adjust the length to the disulfide groups provided for the metal bonds.

The said linker compound is manufactured by the manufacturing method shown below. That is, the linker compound performs a condensation reaction of chioctic acid with an amine compound comprising a multi-branched structure having three or more branched chains in which an aromatic amino group terminal is protected by a protecting group, thereby removing the protecting group at the terminal of the aromatic amino group. It is manufactured by protecting.

The chioctic acid is the following general formula (10)

…(10)

… 10

It has a structure represented by.

In addition, the said amine compound will not be specifically limited if it contains the branched chain which has the terminal terminal of the aromatic amino group protected by the protecting group, What is necessary is just to include the structure corresponded to the multi-branched site | part of said linker compound.

Therefore, the branched chain may have a structure included in the hydrocarbon derivative chain except that the branched chain has an aromatic amino group terminal protected by a protecting group instead of the aromatic amino group included in the hydrocarbon derivative chain. That is, the said branched chain is a hydrocarbon chain which consists of carbon and hydrogen, and some carbon and hydrogen may be substituted by the other atom or substituent. More specifically, the branched chain has an aromatic amino group terminal protected by a protecting group, and a part of the carbon-carbon bond (CC bond), which is the main chain structure of the hydrocarbon chain, is a carbon-nitrogen bond (CN bond), or carbon- It may be substituted by the oxygen bond (CO bond).

In addition, the said protecting group is a substituent introduce | transduced so that the amino group of an aromatic amino group may not react by the said condensation reaction. Such protecting groups are not particularly limited as long as they are not affected when the protecting group of the secondary amino group is deprotected. Examples of the protecting group include t-butoxycarbonyl group (-COOC (CH ₃ ) ₃ group; described as Boc group), benzyl group, aryl carbamate group (-COOCH ₂ CH = CH ₂ , Alloc group) etc. are mentioned.

As said amine compound, For example, the following general formula (11)

…(11)

… (11)

The compound provided with the structure shown by is mentioned. In addition, n, m <^1> -m <^4> , p <^1> , p <^2> in the said General formula (11) is an integer of 1 or more and 6 or less each, respectively. The synthesis | combining method of these amine compounds is explained in full detail in the Example mentioned later.

By the condensation reaction of the chioctic acid and the amine compound, the carboxyl group (-COOH group) of the chioctic acid and the amino group (-NH ₂ group) of the amine compound are condensed to form an amide bond. Then, the above-mentioned linker compound can be obtained by deprotecting a protecting group at the terminal of an aromatic amino group, removing a protecting group, and making it an aromatic amino group.

On the other hand, since the linker compound has a structure in which the linker portion is provided with oligoethylene oxide as described above, it is preferable to use a material containing an oligoethylene oxide structure as a raw material in the production method. As this raw material, for example, bis [2- (2-hydroxyethoxy) ethyl] ether (Compound 1 of Example), commercially available polyethylene glycol (Mw: 200, 300, 400, 600, 1000) (manufactured by Sigma Co., Ltd.) and the like, and bis [2- (2-hydroxyethoxy) ethyl], among others, because of the fact that the degree of polymerization is completely controlled, that is, the length is controlled. Preference is given to using ethers (compound 1 in the examples).

Next, the ligand conjugate of the present invention will be described. Here, "ligand complex" shall mean that the sugar molecule is introduce | transduced into the aromatic amino group of the said linker compound. In the ligand conjugate of the present invention, a sugar group can be introduced to an aromatic amino group by reacting an amino group of a linker compound with an aldehyde group or a ketone group generated by equilibrium in a sugar molecule and subsequently reducing the seed base formed by this reaction. That is, the linker compound and sugar molecules are bonded by this reduction amination reaction.

As the sugar molecule contained in the ligand conjugate of the present invention, any kind of sugar molecule can be used without particular limitation as long as it is a reducing sugar having a reducing terminal. Specific examples of the sugar molecules include, for example, monosaccharides such as glucose, galactose, and mannose, maltose, lactose, and sulfated oligosaccharides described below, wherein the number of sugars bound is 2 to 10 sugars. Polysaccharides, such as heparin, chondroitin sulfuric acid, and heparan sulfuric acid in which oligosaccharide, monosaccharide, and oligosaccharide are combined, and sugar number is 11 or more, are mentioned.

Further, as the oligosaccharide, the following general formula (8) in sulfated polysaccharide heparin known to have anticoagulant activity

…(8)

… (8)

Sulfated oligosaccharides having a specific partial disaccharide structure (referred to as "GlcNS6S-IdoA2S") represented by the following formula (12) formed by introducing glucose into a hydroxyl group which is the reducing end of this sulfated oligosaccharide.

…(12)

… (12)

Oligosaccharides having a structure represented by the above can be mentioned.

The oligosaccharide or polysaccharide may be a single oligosaccharide or a single polysaccharide composed of the same monosaccharide molecules, a complex sugar composed of various monosaccharide molecules or derivatives thereof, a complex comprising various monosaccharide molecules or derivatives thereof, and oligosaccharides. Polysaccharides may be sufficient. Moreover, all the said sugar molecules may be various natural sugars obtained by isolating and refining | refining from a natural system, and the sugar synthesize | combined artificially may be sufficient.

Specifically, the ligand conjugate of the present invention has a structure represented by the general formula (5). The ligand conjugate having the structure represented by the general formula (5) is represented by the above general formula (2), and a sugar molecule is introduced into the linker compound having the structure represented by the above general formula (3). It is done. The sugar molecule is not limited as long as it is a reducing sugar having a reducing end, but is preferably selected from the general formula group (9) and general formula (12). Since X represented by the general formula (3) has a structure having four hydrocarbon derivative chains, the ligand conjugate having the structure represented by the general formula (5) is 4 units or more to the linker compound. The sugar molecule is combined. In the other hand, the above-mentioned formula ^{(5), m 1 ~ m} 4 are of the general formula (3) as in the m ¹ ~ m ^4, not only is an integer of 1 to 6, different integers also be some or all May be the same integer. In addition, if n is an integer of 1 or more and 6 or less, it will not specifically limit. R 'may be hydrogen (H) or an oligosaccharide-derived compound.

Moreover, the ligand conjugate of this invention is equipped with the structure shown by the said General formula (7). Ligand complexes having the structure represented by the general formula (7) are represented by the general formula (1), wherein a sugar molecule is introduced into the linker compound having the structure represented by the general formula (4). It is done. The sugar molecule is not limited as long as it is a reducing sugar having a reducing end, but is preferably selected from the general formula group (9) and general formula (12). Since X represented by the general formula (7) has a structure having three hydrocarbon derivative chains, the ligand conjugate having the structure represented by the general formula (7) has a sugar molecule of at least 3 units in the linker compound. Will combine.

Since all of the ligand conjugates include a linker compound and a sugar molecule, the metal and the metal-sulfur (s) bond, eg, gold-sulfur (A-S), on the surface of the support for protein analysis at the SS bond in the linker compound. ) Can be combined by bonding. Thereby, the ligand carrier which aggregates and immobilizes 3 or more units of sugar molecules on the support surface through this Au-S bond can be provided. Therefore, by using the ligand conjugate, a ligand carrier is obtained by arranging a plurality of sugar molecules with high reproducibility two-dimensionally on the surface of a support for protein analysis, for example, and by using the ligand carrier, biological activity of the sugar molecule can be improved. It becomes possible to evaluate by high reproducibility. On the other hand, as the metal on the support surface, metals such as Cu, Ag, and Pt can be used in addition to Au, and it is particularly preferable to use Au.

Moreover, the said ligand conjugate has oligoethylene oxide in a linker part. Thereby, metal-sulfur bonds can be efficiently formed by suppressing non-specific hydrophobic interactions as much as possible and also making it possible to easily adjust the length to the disulfide groups provided for the metal bonds.

As described above, the ligand carrier formed by immobilizing the ligand conjugate of the present invention on the surface of the support via a metal-sulfur bond is also included in the present invention. This ligand carrier is not limited to protein analysis, and may be used for the analysis of substances other than protein to investigate the interaction with sugar molecules.

The ligand carrier is formed by contacting a solution containing the ligand conjugate with a support having a metal film on the surface, so that each S atom of the SS bond of the ligand conjugate is bound by a metal-sulfur bond with a metal on the support surface. Ligand complex is introduced. Specifically, SS of the linker compound included in the ligand complex by immersing the support for protein analysis in the ligand complex solution for a predetermined time or injecting the ligand complex solution into the support (flowing the ligand complex solution on the support surface). The ligand conjugate may be fixed to the surface of the support by converting a bond into an Au-S bond with gold or the like on the surface of the support.

Although it does not specifically limit as a solvent used for a ligand complex solution, For example, methanol, water, dimethylacetamide (DMAc), these mixed solvents, etc. are mentioned. In addition, immersion time should just be about 0.5 hour-about 12 hours, and injection density should just be about 1 micrometer-about 1 mM.

As described above, since the ligand conjugate of the present invention has an S-S bond, the ligand conjugate of the present invention can be easily immobilized on the surface of a protein analysis support, and a sugar molecule can be simply introduced onto the support.

On the other hand, the method of introducing a sugar molecule into the support as described above is also included in the present invention.

The ligand carrier of the present invention can be used for analysis of the interaction of sugar molecules with other substances such as proteins. Specifically, the ligand carrier can be applied to SPR measurement, affinity chromatography and the like.

For example, in order to perform SPR measurement as a protein analysis, it is good to carry out as follows. That is, the ligand carrier and the protein are contacted with a ligand carrier formed by immobilizing the ligand conjugate of the present invention on a support on which a metal thin film such as a gold thin film is deposited, and using a surface plasmon resonance apparatus according to a conventional method. By measuring the resonance angle, the binding behavior between the ligand carrier and the protein can be observed. On the other hand, as said support body (sensor chip) used for SPR measurement, glass, plastics, etc. can be used, for example, glass is used preferably. The ligand carrier may be brought into contact with the protein, for example, by introducing a solution in which the protein is dissolved in a running buffer to the surface of the ligand carrier. As this running buffer, a phosphate buffer solution etc. are mentioned, for example.

Since the ligand carrier of the present invention has the ligand conjugate, a plurality of sugar molecules are arranged on the surface of the support with high reproducibility in two dimensions. Therefore, the biological activity of sugar molecules can be observed with high reproducibility, and the structure of sugar molecules and the quantitative evaluation of the biological activity of sugar molecules can be performed.

Moreover, the sensor chip which immobilized the sugar chain as a ligand carrier of this invention can be used, for example for the following SPR measurements. That is, a 1st sensor chip | tip is used using the 1st sensor chip | tip by which the 1st sugar molecule is immobilized on the support surface, and the 2nd sensor chip | tip where the 2nd sugar molecule from which the terminal structure differs from the said 1st sugar molecule is immobilized on the support surface. The difference between the detection result of the SPR measurement obtained using the chip and the detection result of the SPR measurement obtained using the second sensor chip can be detected to observe the interaction of the sugar molecules. These sensor chips may use ligand conjugates having different sugar molecules to be immobilized. Examples of sugar molecules to be compared include lactose and glucose, maltose and glucose, kojibiose and glucose. Although two sensor chips are used here, more than this number of sensor chips with different kinds of sugar molecules introduced may be used. In addition, the terminal of a sugar molecule means the one which is not fixed to a sensor chip.

In the SPR measurement, using a protein or the like specifically acting on the first sugar molecule, the measurement conditions are made constant and acted on the two sensor chips to observe the resonance angles of the two. By detecting the difference in the resonance angles of the two, it can be measured as a specific interaction between sugar molecules and proteins.

In addition, the substance which observes interaction with a sugar molecule is not limited to a protein.

Although two types of sensor chips were simultaneously measured in the above, it is not limited to this, Two or more types of sensor chips may be measured, and it is not necessary to measure simultaneously. Moreover, you may use the thing which did not introduce a sugar molecule into at least 1 sensor chip. For example, what immobilized only the linker compound may be used.

When the SPR measurement as described above is carried out, at least two sensor chips immobilizing ligand conjugates having the same structure except for sugar molecules can be measured, and therefore, the difference in the magnitude of the interaction measured by the at least two sensor chips is different. It is observed as due to. Therefore, by using the measurement method, it is possible to observe the specific interaction between sugar molecules and other substances by reducing the non-specific interactions between portions other than sugar molecules and other substances.

<Example>

Hereinafter, the synthesis of the linker compound and the ligand conjugate of the present invention will be described in more detail. In this example, an experiment was also conducted in which the characteristics were compared using the synthesized ligand conjugate and another ligand conjugate. This will also be described.

Example 1 Synthesis of Linker Compound (Compound 15)

In one of the linker compounds according to the present invention, that is, in general formula (2), n is 4, X is represented by the general formula (3), p ¹ , p ² is 1, m ¹ , m ² A linker compound (Compound 15) having a structure of 2, m ³ and m ⁴ was synthesized in the following order. 1 shows a process of synthesizing this linker compound (Compound 15). In addition, in description of this Example 1, the number added to each compound corresponds to the number described in FIG.

As shown in Fig. 1, first, bis [2- (2-hydroxyethoxy) ethyl] ether (Compound 1) was used as a raw material, and ethyl diazoacetate (compound was present in dichloromethane in the presence of BF ₃ · Et ₂ O. 2) was reacted to synthesize an ester (compound 3) in a yield of 40%. Subsequently, compound 3 was reacted with p-toluenesulfonylchloride in the presence of DMAP and pyridine in dichloromethane to give tosyl compound (Compound 4) in a yield of 78%. Compound 4 was reacted with sodium azide in N, N-dimethylformamide to obtain an azide compound (compound 5) in a yield of 90%.

This was hydrolyzed with 1N NaOH in methanol to give carboxylic acid derivative (Compound 6) in yield 98%. Compound 6 and Compound 7 were condensed in dichloromethane using HOBt and EDC.HCl to obtain a diester derivative (Compound 8) in a yield of 80%. The compound 8 was hydrolyzed with 0.6 N NaOH in methanol to give a dicarboxylic acid derivative (Compound 9) in 93% yield. Compound 9 and the diamine derivative (Compound 10) were condensed using FDPP and DIPEA to obtain Compound 11 in a yield of 40%. Catalytic hydrogen reduction of the azide group of Compound 11 was conducted to an amine compound (Compound 12) in a yield of 80%.

Thereafter, the compound was condensed with chioctic acid (compound 13) to obtain compound 14 in a yield of 59%. Finally, by reacting TFA with this compound 14, the Boc group was deprotected to obtain a linker compound (Compound 15) having 4 units of the desired aromatic amino group in 91% yield.

Hereinafter, the synthesis | combining method of each compound obtained in the synthesis process mentioned above is shown more concretely, The result of having measured the ¹ H-NMR spectrum and the mass spectrometry about each synthesize | combined compound is shown. In addition, the relative concentration on the chip | tip of the sugar chain which is a ligand was calculated | required by measuring total reflection FT-IR (ATR-FT-IR). Each was performed in the following order.

[ ¹ H-NMR spectrum, mass spectrometry, ATR-FT-IR measurement, reagents, etc.]

JEOL-JNM-Lambda-500 NMR spectrometer, JEOL JNM-GSX400 NMR spectrometer and JEOL EX-270 NMR spectrometer were used for the measurement of the ¹ H-NMR spectrum. Chemical shifts are expressed as δ values for CDCl ₃ with tetramethylsilane as reference material. CD ₃ OD and DMSO-d ₆ are shown as δ values based on the chemical shift of the protons of the remaining solvent. Mass spectrometry was measured using AppliedBiosystems, Mariner ^™ . For ATR-FT-IR measurement, the one equipped with a one-time reflective ATR accessory (MIRacle Ge prism) was used in Shimadzu, IRPrestige-21. The sensor chip for ATR-FT-IR measurement was the same as the SPR measurement. Medium pressure column silica gel chromatography used Silica gel 60 No. 9385 (Merck) was used and thin silica gel chromatography was used for Silica gel 60 F254 (Merck). Dichloromethane anhydrous was used by distillation under nitrogen stream using calcium hydride as a drying agent. The other dehydration solvent purchased the product of Kanto Chemical Corporation. Reagents and solvents other than the above were basically used as an express.

(1) Synthesis of Compound 3

Bis [2- (2-hydroxyethoxy) ethyl] ether (Compound 1) (14.57 mL, 80 mmol) and BF ₃ Et ₂ 0 (252 mL, ₂ mmol) were dissolved in 50 mL of anhydrous dichloromethane, and 0 ° C. Ethyl diazo acetate (Compound 2) (1.8 ml, 17.35 mmol) was added dropwise, followed by stirring at room temperature for 70 minutes. 20 ml of saturated ammonium chloride aqueous solution was added to the reaction solution, the mixture was extracted with dichloromethane and dried over anhydrous magnesium sulfate. The drying agent was filtered off, concentrated under reduced pressure, and the concentrated residue was purified by medium pressure preparative chromatography (600 g, hexane: ethyl acetate = 1: 3) to obtain compound 3 (2.26 g, yield: 47%) as a colorless liquid. .

As a result of the ¹ H-NMR (400 MHz, CDCl ₃ ) measurement for the compound 3, δ 4.22 (2H, q, J = 7.0, 14.2 Hz, CO ₂ C H ₂ ), 4.14 (2H, s, OC H ₂ CO), 3.75-3.62 (14H, m, C H ₂ CH ₂ O × 3, HOCH ₂ C H ₂ ), 3.61 (2H, t, J = 4.4 Hz, HOC H ₂ ), 1.84 (lH, bs, 0 H ), 1.28 (3H, t, J = 7.3 Hz, CH ₂ C H ₃ ). Moreover, the ESI-MS (positive) measurement of the said compound 3 was carried out, and it was m / z 303.27 [(M + Na) ⁺ ]. This confirmed the structure of compound 3. On the other hand, the molecular mass of this compound 3 is C ₁₂ H ₂₄ O ₇ : 280.15.

(2) Synthesis of Compound 4

The ethyl compound 3 (2.15 g, 7.66 mmol) and DMAP (41.7 mg, 337 mmol) were dissolved in 8 ml of anhydrous pyridine. The solution which melt | dissolved p-toluenesulfonic acid chloride (1.75 g, 9.19 mmol) in 8 ml of anhydrous dichloromethane was dripped at this solution at 0 degreeC, and it stirred at room temperature for 3 hours. Dichloromethane and ice water were added to the reaction solution, followed by extraction with dichloromethane. The organic layer was washed once with saturated aqueous sodium hydrogen carbonate solution, water and brine, and dried over anhydrous magnesium sulfate. The drying agent was filtered off, concentrated under reduced pressure, and the concentrated residue was purified by medium pressure preparative chromatography (100 g, chloroform: acetone = 4: 1) to obtain compound 4 (2.59 g, yield: 78%) as a yellow liquid.

As a result of the ¹ H-NMR (400 MHz, CDCl ₃ ) measurement for the compound 4, δ 7.80 (2H, d, J = 8.4 Hz, aromatic), 7.35 (2H, d, J = 8.4 Hz, aromatic), 4.21 (2H, q, CO ₂ C H ₂ ), 4.16 (2H, t, J = 4.8 Hz, TsOC H ₂ ), 4.14 (2H, s, OC H ₂ CO), 3.76-3.59 (14H, C H ₂ C H ₂ O × 3, TsOCH ₂ C H ₂ ), 2.45 (3H, s, C H ₃ Ar), 1.28 (3H, t, J = 7.0 Hz, CH ₂ C H ₃ ). Moreover, the ESI-MS (positive) measurement of the said compound 4 was carried out, and it was m / z 457.16 [(M + Na) ⁺ ]. This confirmed the structure of compound 4. On the other hand, the molecular mass of this compound 4 is C ₁₉ H ₃₀ 0 ₉ S: 434.16.

(3) Synthesis of Compound 5

The tosyl compound 4 (1.01 g, 2.31 mmol) and sodium azide (1.53 g, 2.31 mmol) were dissolved in 50 ml of anhydrous dimethylformamide, light-shielded, and stirred for 10 hours in a 120 degreeC nitrogen atmosphere. The reaction solution was extracted with chloroform, and the organic layer was washed once with water and brine, and dried over anhydrous magnesium sulfate. The drying agent was filtered off and concentrated under reduced pressure, and the concentrated residue was purified by medium pressure preparative chromatography (10 g, chloroform: acetone = 2: 1) to obtain compound 5 (638 mg, yield: 90%) as a yellow liquid.

As a result of the ¹ H-NMR (400 MHz, CDCl ₃ ) measurement for the compound 5, δ 4.22 (2H, q, J = 7.3 Hz, CO ₂ C H ₂ ), 4.15 (2H, s, OC H ₂ CH ₂ Et), 3.75-3.63 (12H, m, OC H ₂ C H ₂ O), 3.69 (2H, m, N ₃ CH ₂ C H ₂ ), 3.39 (2H, t, J = 5.1 Hz, N ₃ C H ₂ ), 1.29 (3H, t, J = 7.3 Hz, CO ₂ CH ₂ C H ₃ ). In addition, ESI-MS (positive) measurement of the compound 5 was carried out, and the result was m / z 328.14 [(M + Na) ⁺ ]. This confirmed the structure of compound 5. On the other hand, the molecular mass of this compound 5 is C ₁₂ H ₂₃ N ₃ O ₆ : 305.16.

(4) Synthesis of Compound 6

The azide compound 5 (614 mg, 2.01 mmol) was dissolved in 24 ml of methanol, and 4.3 ml of 1N NaOH was added at 0 ° C under light shielding, followed by stirring at room temperature for 21 hours. The reaction solution was concentrated under reduced pressure, chloroform was added to the concentrated residue, and 1N HCl was added until pH = 2, followed by extraction with chloroform. The organic layer was washed once with saturated brine and dried over anhydrous magnesium sulfate. The drying agent was filtered off and concentrated under reduced pressure to obtain compound 6 (549 mg, yield: 90%) as a colorless liquid.

The compound 6 was subjected to ¹ H-NMR (400 MHz, CDCl ₃ ) measurement. As a result, δ6.19 (1H, bs, CO ₂ H ), 4.16 (2H, s, OC H ₂ CO ₂ H), 3.75-3.64 (12H, m, OC H ₂ C H ₂ 0), 3.68 (2H, m, N ₃ CH ₂ C H ₂ ), 3.41 (2H, t, J = 5.1 Hz, N ₃ C H ₂ ). Moreover, it was m / z 328.14 [(M + Na) ⁺ ] when the ESI-MS (negative) measurement of the said compound 6 was performed. This confirmed the structure of Compound 6. Meanwhile, the molecular mass of the Compound 6 is _{C 10 H 19 N 3 O 6} : 277.13 a.

(5) Synthesis of Compound 7

Imino diacetic acid (10.0 g, 75.1 mmol) and BF ₃ · OEt ₂ (22 mL, 173 mmol) were dissolved in anhydrous methanol (50 mL), refluxed under an argon atmosphere for 5 hours, and then saturated sodium bicarbonate aqueous solution was added thereto. Neutralize and extract with chloroform. Triethylamine was added to the aqueous layer until pH reached 9, extracted with chloroform again, dried using anhydrous sodium sulfate as a drying agent, and the drying agent was filtered off and concentrated under reduced pressure, compound 7 (9.61 g, yield: 79 %) Was obtained as a yellow oil.

As a result of performing ¹ H-NMR (400 MHz, CDCl ₃ ) measurement with respect to this compound 7, δ3.74 (6H, s, OMe), 3.48 (4H, s, CH ₂ N), 2.00 (1H, s, NH) It was. In addition, ESI-MS (positive) measurement of the compound 7 was carried out, and the result was m / z 162.1 [(M + H) ⁺ ]. This confirmed the structure of Compound 7. Meanwhile, the molecular mass of the Compound 7 is C ₆ H ₁₁ NO _2: 161.07 a.

(6) Synthesis of Compound 8

Compound 6 (0.35 g, 1.26 mmol), EDC.HCl (0.27 g, 1.39 mmol), and HOBt (0.19 g, 1.39 mmol) were dissolved in 2 ml of anhydrous dichloromethane, and shaded under argon atmosphere for 80 minutes at 0 ° C. After stirring, a solution obtained by dissolving Compound 7 (1.42 g, 6.83 mmol) in 1 mL of anhydrous dichloromethane was added, followed by stirring at room temperature for 17 hours. The reaction solution was extracted with chloroform, and the organic layer was washed once with 10% citric acid and saturated aqueous sodium hydrogen carbonate solution. After drying using anhydrous sodium sulfate as a drying agent, the drying agent was filtered off and concentrated under reduced pressure. The concentrated residue was purified by preparative silica gel chromatography (50 g, chloroform: acetone = 10: 1) to give compound 8 (0.42 g, yield: 80%) as a white solid.

The compound 8 was subjected to ¹ H-NMR (400 MHz, CDCl ₃ ) measurement. As a result, δ 4.23, 4.11 (4H, s, s, CONC H ₂ ), 4.18 (2H, s, OC H ₂ CON), 3.69 , 3.66 (4H, s, s, CO ₂ C H ₃ ), 3.69-3.56 (12H, m, OC H ₂ C H ₂ O), 3.61 (2H, t, J = 5.1 Hz, N ₃ CH ₂ C H ₂ ), 3.32 (3H, t, J = 5.0 Hz, N ₃ C H ₂ ). Moreover, it was m / z 443.17 [(M + Na) +] when the ESI-MS (positive) measurement of the said compound 8 was performed. This confirmed the structure of Compound 8. On the other hand, the molecular mass of this compound 8 is C ₁₆ H ₂₈ N ₄ O ₉ : 420.19.

(7) Synthesis of Compound 9

The compound 8 (398 mg, 947 μmol) was dissolved in methanol (5 mL), 2N NaOH (2.1 mL) was added, the mixture was stirred at 0 ° C. for 2.5 hours, and then Dowex 50WX-8 (H ⁺ form) was added to pH = 2. The mixture was added until neutralized, and the Dowex 50WX-8 was filtered off and concentrated under reduced pressure. Water was added to the concentrated residue obtained by vacuum concentration, the insolubles were filtered off, and the residue was concentrated under reduced pressure and freeze-dried to obtain compound 9 (346 mg, yield: 93%) as a white solid.

The compound 9 was subjected to ¹ H-NMR (400 MHz, CDCl ₃ ) measurement. As a result, δ5.66 (2H, bs, CO ₂ H x2), 4.26 (2H, s, OC H ₂ CON), 4.24, 4.18 ( 4H, s, s, CONC H ₂ ), 3.71-3.63 (12H, m, OC H ₂ C H ₂ 0), 3.67 (2H, m, J = 5.1 Hz, N ₃ CH ₂ C H ₂ ), 3.40 ( 3H, t, J = 4.9 Hz, N ₃ C H ₂ ). Moreover, the ESI-MS (negative) measurement of the said compound 9 was carried out, and it was m / z 391.15 [(MH)]. This confirmed the structure of compound 9. On the other hand, the molecular mass of this compound 9 is C ₁₄ H ₂₄ N ₄ O ₉ : 392.15.

(8) Synthesis of Compound 10

N-Boc amino benzoic acid derivative (3.33 g, 14.0 mmol) and HOBt (1.93 g, 14.3 mmol) were suspended in anhydrous dichloromethane (60 mL), and stirred for 15 minutes at 0 ° C. under an argon atmosphere, followed by EDC HCl ( 2.87 g, 15.0 mmol) was dissolved in anhydrous dichloromethane (30 mL), and stirred for 50 minutes. Diethylenetriamine (0.79 mL, 7.00 mmol) was added to this solution, and the light was stirred overnight at room temperature under light shielding to obtain white crystals. The white crystals were collected by filtration and then recrystallized from methanol to give compound 10 (3.53 g, yield: 92.9%) as white crystals.

The compound 10 was subjected to ¹ H-NMR (400 MHz, CDCl ₃ ) measurement. As a result, δ 7.77-7.74 (4H, d, J = 8.67 Hz, aromatic), 7.50-7.48 (4H, d, J = 8.57 Hz , aromatic), 3.70-3.66 (4H, m, J = 5.19 Hz CONHC H ₂ ), 3.34-3.28 (4H, m, J = 5.61 Hz C H ₂ CH ₂ ONH), 1.53 (18H, s, C H ₃ Was. Moreover, the ESI-MS (positive) measurement of the said compound 10 was carried out, and it was m / z 542.4 [(M + H) ⁺ ]. This confirmed the structure of compound 10. On the other hand, the molecular mass of this compound 10 is C ₂₈ H ₃₉ N ₅ O ₆ : 541.29.

(9) Synthesis of Compound 11

Compound 9 (333 mg, 847 μmol), diisopropylethylamine (435 mL, 2.54 mmol), and FDPP (1.00 g, 2.60 mmol) were dissolved in anhydrous dimethylformamide (5 mL) and shielded to light at 0 ° C. under argon atmosphere. After stirring for 30 minutes, the compound 10 (1.15 g, 2.11 mmol) was dissolved in anhydrous dimethylformamide (11 mL) and added, followed by stirring at room temperature for 20 hours. The concentrated residue obtained by concentrating the reaction solution under reduced pressure was extracted with chloroform, and the water and organic layers were washed once with 10% citric acid and saturated aqueous sodium hydrogen carbonate solution, dried over anhydrous magnesium sulfate as a drying agent, and then the drying agent was filtered. It removed and concentrated under reduced pressure. The concentrated residue obtained by concentration under reduced pressure was purified by preparative silica gel chromatography (80 g, chloroform: methanol = 10: 1) to give compound 11 (125 mg, yield: 59%) as a white solid.

The compound 11 was subjected to ¹ H-NMR (400 MHz, CDCl ₃ ) measurement. As a result, δ 8.18 (lH, bs, N H COPh), 7.86 (2H, d, J = 8.4 Hz, aromatic), 7.80 (lH , bs, PhN H CO), 7.75-7.68 (8H, m, N H COPh, aromatic, PhN H CO), 7.54 (1H, bs, PhN H CO), 7.48 (2H, d, J = 8.4 Hz, N H COPh, aromatic), 7.42 (5H, m, aromatic, N H COPh), 7.34 (2H, d, J = 8.8 Hz, aromatic), 7.28 (1H, bs, PhN H CO), 3.84 (4H, bs, CONC H ₂ ), 3.62-3.48 (20H, m, OC H ₂ C H ₂ 0, NCH ₂ C H ₂ NH), 3.56 (2H, t, J = 5.1 Hz, N ₃ CH ₂ C H ₂ ), 3.43 (2H, bs, OCH ₂ CON), 3.35-3.30 (4H, m, NC H ₂ CH ₂ NH), 3.26 (2H, t, J = 5.1 Hz, N3CH ₂ ), 3.13, 2.98 (4H, bs, bs , NCH ₂ CH ₂ NH), 1.52, 1.50, 1.49 (36H, s, s, s, t-butyl). In addition, ESI-MS (positive) measurement of the compound 11 was carried out, and the result was m / z 1461.72 [(M + Na) ⁺ ]. This confirmed the structure of Compound 11. On the other hand, the molecular mass of this compound 11 is C ₇₀ H ₉₈ N ₁₄ O ₁₉ : 1438.71.

(10) Synthesis of Compound 12

The compound 11 (165 mg, 114 μmol) was dissolved in methanol (12 mL), 5% Pd / C (55 mg) was added, the mixture was stirred at room temperature under hydrogen atmosphere for 5 hours, and the Pd / C was filtered off to reduce pressure. The resulting concentrated residue was concentrated and purified by silica gel chromatography (10 g, chloroform: methanol = 7: 1) to give compound 12 (128 mg, yield: 79%) as a white solid.

The compound 12 was subjected to ¹ H-NMR (400 MHz, CDCl ₃ ) measurement. As a result, δ7.78-7.68 (8H, m, aromatic), 7.48 (8H, m, aromatic), 4.21, 4.10 (4H, bs , bs, CONC H ₂ ), 3.85 (2H, bs, OC H ₂ CON), 3.62-3.44 (26H, m, OC H ₂ C H ₂ 0, NCH ₂ C H ₂ NH, NC H ₂ CH ₂ NH) , 3.50 (2H, t, J = 5.1 Hz, H ₂ NCH ₂ C H ₂ ), 2.76 (2H, t, J = 5.1 Hz, H ₂ NC H ₂ CH ₂ ), 1.50 (36H, s, t-butyl Was. Furthermore, the ESI-MS (positive) measurement of the said compound 12 was carried out, and it was m / z 1413.74 [(M + H) ⁺ ]. Meanwhile, the molecular mass of the Compound 12 is _{C 70 H 100 N 12 O 19} : a 1412.72.

The compound 12 is the following formula and n is 4 in the (11), p ^1, and p ² is 1, the amine compound is ^{m 1, m 2, m 3} , m 4 having a second structure.

(11) Synthesis of Compound 14

Compound 13 (chioctic acid) (3.4 mg, 16.6 imol) and HOBt (1.6 mg, 16.6 μmol) EDCHCl (3.2 mg, 1.66 μmol) were dissolved in anhydrous dimethylformamide (2 mL), and the mixture was heated under argon atmosphere. It light-shielded at stirring and stirred. Subsequently, Compound 12 (23.5 mg, 16.6 µmol) was dissolved in anhydrous dimethylformamide (2 ml) and added, followed by stirring at room temperature for 22 hours. The concentrated residue obtained by concentrating this reaction solution under reduced pressure was extracted with dichloromethane, and the organic layer was washed once with 10% citric acid and saturated aqueous sodium hydrogen carbonate solution. After drying using anhydrous sodium sulfate as a drying agent, the drying agent was filtered off and concentrated under reduced pressure. The concentrated residue was purified by preparative silica gel chromatography (7 g, chloroform: methanol = 10: 1) to give compound 14 (15.7 mg, yield: 59%) as a white solid.

Performing a measuring ^{1 H-NMR (400MHz, CDCl} 3) relative to the compound 14 results, δ8.20, 8.00 (4H, bs , bs, N H COPh), 7.86 (2H, d, J = 8.8Hz, aromatic) , 7.77-7. 72 (7H, m, COPhN H , aromatic), 7.53 (lH, bs, N H COPh), 7.50-7.36 (10H, m, aromatic, J = 8.8 Hz, COPhN H ), 7.27 (2H, bs, COPhN H , CON H CH ₂ ), 3.89 (4H, bs, CONC H ₂ CO), 3.64-3.37 (26H, m, NCH ₂ C H ₂ NH, NC H ₂ CH ₂ NH, OC H ₂ C H ₂ 0, CONHC H ₂ , CONHCH ₂ C H ₂ ), 3.53 (lH, m, SSC H ), 3.48 (2H, m, NC H ₂ CH ₂ NH), 3.32 (4H, m, OC H ₂ CON, NC H ₂ CH ₂ NH), 3.18, 2.85 (4H, bs, bs, NC H ₂ CH ₂ NH), 3.17-3.04 (2H, m, C H ₂ SSCH), 2.44-2.36 (lH, m, C H ₂ CH ₂ SS) , 2.16 (2H, m, CH ₂ CH ₂ C H ₂ CONH), 1.89-1.81 (lH, m, C H ₂ CH ₂ SS), 1.69-1.56 (4H, m, CH ₂ C H ₂ CH ₂ CONH, C H ₂ CH ₂ CH ₂ CH ₂ CONH), 1.51, 1.50 (36H, s, s, t-butyl), 1.42-1.34 (2H, m, C H ₂ CH ₂ CH ₂ CONH). Moreover, the ESI-MS (positive) measurement of the said compound 14 was carried out, and it was m / z 1601.81 [(M + H) ⁺ ]. This confirmed the structure of Compound 14. On the other hand, the molecular mass of this compound 14 is C ₇₈ H ₁₁₂ N ₁₂ O ₂₀ S ₂ : 1600.76.

(12) Synthesis of Linker Compound (Compound 15)

The compound 14 (60.3 mg, 31.2 mol) was dissolved in dichloromethane (1 ml), TFA (3 ml) was added thereto, and the mixture was stirred at 0 ° C under light shielding for 1 hour, followed by concentration under reduced pressure, and the resulting residue was dissolved in methanol. Was injected into a column (1.0 cmφ × 3.0 cm) filled with Dowex Marathon A (OH ^- form) to carry out ion exchange. The eluate was concentrated under reduced pressure to give compound 15 (41.2 mg, yield: 91%) as a white solid.

For this compound 15, ¹ H-NMR (400 MHz, DMSO-d ₃ ) measurement was performed. As a result, δ 8.19, 8.05 (4H, m, m, N H COPh), 7.82 (1H, bt, CON H CH ₂ ), 7.53 (8H, m, aromatic), 6.51 (8H, dd, J = 8.4, 1.5 Hz, aromatic), 5.61-5.55 (8H, m, N H ₂ ), 4.24, 4.11 (4H, s, s, CONC H ₂ CO), 3.93 (2H, bs, OC H ₂ CON), 3.60-3.37 (31H, m, NCH ₂ C H ₂ NH, NC H ₂ CH ₂ NH, OC H ₂ C H ₂ O, CONHC H ₂ , CONHCH ₂ C H ₂ , SSCH), 3.19-3.06 (4H, m, CONHC H ₂ CH ₂ , C H ₂ SSCH), 2.42-2.32 (lH, m, C H ₂ CH ₂ SS), 2.04 (2H , m, CH ₂ CH ₂ C H ₂ CONH), 1.87-1.78 (lH, m, C H ₂ CH ₂ SS), 1.64-1.45 (4H, m, CH ₂ C H ₂ CH ₂ CONH, C H ₂ CH ₂ CH ₂ CH ₂ CONH), 1.34-1.28 (2H, m, C H ₂ CH ₂ CH ₂ CONH). In addition, ESI-MS (positive) measurement of the compound 15 was carried out, and the result was m / z 623.27 [(M + 2Na) ²⁺ ]. On the other hand, the molecular mass of this compound 15 is C ₅₈ H ₈₀ N ₁₂ O ₁₂ S ₂ : 1200.55.

In the compound (15), n is 4 in formula (2), X is represented by formula (3), p ¹ , p ² are 1, m ¹ , m ² , m ³ , m ⁴ are It is a linker compound which has a structure of two.

Example 2: Synthesis of Ligand Complex (Compound 17)

Using the linker compound 15 obtained in Example 1, in the general formula (5), n is 4, p ¹ , p ² is 1, m ¹ , m ² , m ³ , m ⁴ is 2, and R 'Is hydrogen (H), and R is a ligand conjugate having a structure derived from oligosaccharide represented by the general formula (12) in the following order. 2, the chemical reaction formula of this synthesis is shown.

As shown in FIG. 2, the reduction amination reaction was performed using the linker compound 15 obtained in Example 1, and the compound 16 (7 equivalent) which is a sugar molecule represented by the said General formula (12). This obtained the compound 17 which is an example of the ligand conjugate of this invention in yield 22%.

Specifically, the linker compound 15 (2.0 mg, 1.67 μmol) and compound 16 (10 mg, 11.7 mmol) were dissolved in a mixed solvent of 100 mL of water, 400 mL of dimethylacetamide, and 10 mL of acetic acid, and then shaded in a sealed tube. Heated at 37 ° C. for 25 hours. NaBH ₃ CN (3.51 mg, 50.2 mmol) was dissolved in 15 ml of acetic acid, added to the reaction solution, heated at 37 ° C. for 6 days, and concentrated under reduced pressure. Sephadex G-50 (1.6 cmφ × 80 cm, 0.3 M in PBS) Purified with NaCl). The target fraction obtained by purification was concentrated under reduced pressure, and the concentrated residue was desalted using Sephadex G-25 (1.6 cmφ × 40 cm, water). The desired fraction obtained by desalting was concentrated under reduced pressure, dissolved in water and lyophilized to obtain compound 17 (1.7 mg, yield: 22%) as a white powder.

The compound 17 was subjected to ¹ H-NMR (400 MHz, D ₂ O) measurement by the method described in Example 1, whereupon δ7.65-7.58 (8H, m, aromatic), 6.78-6.67 (8H, m, aromatic), 5.37 (4H, bs, H-1 ”), 5.13 (4H, bs, J = 2.5 Hz), 4.52 (4H, bs, H-5 '), 4.29 (10H, m, H-6a”, H-3 ', CONC H ₂ CO), 4.19 (lOH, m, H-6b'',H-2', CONC H ₂ CO), 4.05 (3H, m, H-4 '), 3.99-3.92 (14H , m, H-2, H-6a, H-5 ”, OC H ₂ CON), 3.87 (8H, m, H-5, NCH ₂ C H ₂ NH), 3.83 (8H, m, H-3, NCH ₂ C H ₂ NH), 3.77-3.70 (8H, m, H-4, NCH ₂ C H ₂ NH), 3.71 (4H, t, J = 9.9 Hz, H-3 ”), 3.64-3.50 (25H , m, H-6b, NC H ₂ CH ₂ NH, OC H ₂ C H ₂ O, CONHC H ₂ , CONHCH ₂ C H ₂ , SSC H ), 3.54 (3H, s, OC H ₃ ), 3.45-3.19 (l4H, m, H-1a, H-1b, NC H ₂ CH ₂ NH, C H ₂ SS), 3.34 (4H, t, J = 9.6 Hz, H-4 ”), 3.24 (4H, dd, J = 3.4, 10.5 Hz, H-2 ”), 2.35-2.28 (1H, m, C H ₂ CH2SS), 2.27 (2H, bt, CH ₂ C H ₂ CONHCH ₂ ), 1.89-1.84 (lH, m, C H ₂ CH ₂ SS), 1.56-1.46 (2H, m, C H ₂ CH ₂ CONH), 1.35-1.14 (2H, m, C H ₂ CH ₂ (CH ₂ ) ₂ CONH). Moreover, the ESI-MS (negative) measurement of the said compound 17 was carried out, and it was m / z 1449.93 [(M-10Na + 7H) ³ ]. This confirmed the structure of Compound 17. On the other hand, the molecular mass of this compound 17 is C ₁₃₄ H ₁₉₆ N ₁₆ Na ₁₆ 0 ₁₀₈ S ₁₄ : 4572.48.

In the compound (5), n is 4, p ¹ , p ² are 1, m ¹ , m ² , m ³ , m ⁴ are 2, and R 'is hydrogen (H). , Is a ligand conjugate having a structure in which R is an oligosaccharide-derived compound represented by General Formula (6-3).

Example 3: Validation of Interaction of Ligand Sugar Chain with Protein

In the present example, in the general formula (5) obtained in Example 2, n is 4, p ¹ , p ² are 1, m ¹ , m ² , m ³ , m ⁴ are 2, and R 'is Ligand complex having a structure of hydrogen (H) and R is an oligosaccharide-derived compound represented by General Formula (6-3) (hereinafter, the ligand complex is described as 'Tetra-GlcNS6S-IdoA2S-Glc'). Were used to verify intermolecular interactions with the protein.

In the present Example, the same experiment was performed also about the other two types of ligand complexes which the inventors discovered previously for the comparison, and the interaction was compared and examined. The other two kinds of ligand conjugates are specifically ligand conjugates described in Patent Document 1, and are ligand conjugates represented by the following general formula (13). Hereinafter, this ligand conjugate is described as 'Mono-GlcNS6S-IdoA2S-Glc'.

…(13)

… (13)

The other ligand conjugate is another ligand conjugate described in Japanese Patent Laid-Open No. 2004-157108, which is a ligand conjugate represented by the following general formula (14). Hereinafter, this ligand conjugate is described as 'Tri-GlcNS6S-IdoA2S-Glc'. On the other hand, Japanese Unexamined Patent Application Publication No. 2004-157108 is unpublished at the time of this priority date.

…(14)

… (14)

Example 3-1: Identification of Specific Interactions

First, as Example 3-1, inhibition experiments were conducted to confirm specific interactions between the chip to which the disaccharide structure (GlcNS6S-IdoA2S) represented by the general formula (8) was immobilized and the heparin binding protein. In other words, an inhibitor that inhibits the binding of the heparin binding protein and the GlcNS6S-IdoA2S structure coexists and examined whether binding of the heparin binding protein to the chip was inhibited.

In this experiment, heparin (Mw = 17600 derived from pig small intestine) was used as an inhibitor, and bFGF was used as a heparin binding protein. bFGF, also called FGF-2, is known to act on vascular endothelial cells or fibroblasts to promote wound healing by exerting angiogenesis and promoting granulation formation. In vivo, it interacts with heparan sulfuric acid, a heparin-like substance on the cell surface, and expresses biological activity. It has been reported that the minimum binding unit necessary for binding to bFGF is 5-saccharide represented by the following general formula (15) (M. Maeearana, B. Casu & U. Lindahl, J. Biol. Chem. 268, page 8857, 1993).

…(15)

… (15)

The above structure did not include a structure in which the sixth position of glucosamine was sulfated. However, the association of the IdoA2S-GlcNS structure and bFGF in heparan sulfuric acid does not require a large number of non-specific 6-position sulfates, but it is confirmed that it is necessary for the formation of the active site. Therefore, bFGF was chosen as the protein to observe the interaction with the GlcNS6S-IdoA2S structure.

Subsequently, binding inhibition experiments with bFGF in the presence of heparin were performed using the chips immobilized with Mono-GlcNS6S-IdoA2S-Glc, Tri-GlcNS6S-IdoA2S-Glc, and Tetra-GlcNS6S-IdoA2S-Glc. In other words, heparin was mixed in 200 nM bFGF solution at 3, 10, 100, 300 and 1000 nM, and mixed and injected into the chip.

3 shows the binding behavior of bFGF to the chip on which Mono-GlcNS6S-IdoA2S-Glc is immobilized. From this figure, it was confirmed that binding of bFGF to the chip on which Mono-GlcNS6S-IdoA2S-Glc was immobilized was reduced depending on heparin concentration. That is, it was confirmed that binding of bFGF to the chip on which Mono-GlcNS6S-IdoA2S-Glc was immobilized was inhibited by heparin.

The inhibition rate of bFGF binding in each chip was calculated from the data of each of the three types of chips obtained by the above experiment. The result is shown in FIG. On the other hand, the inhibition rate is expressed as a percentage of the value obtained by dividing the maximum angular change amount under different concentrations of heparin by the maximum angular change amount when heparin does not coexist.

From the graph shown in Figure 4, where we define the binding inhibition rate of bFGF on the chip is 50% as IC _50. As a result, IC ₅₀ = 2.5 nM for the chip immobilized with Mono-GlcNS6S-IdoA2S-Glc, IC = 94nM for the chip immobilized with Tri-GlcNS6S-IdoA2S-Glc, and IC for the chip immobilized with Tetra-GlcNS6S-IdoA2S-Glc _It was confirmed that IC = value of the chip | tip which fixed ₅₀ = 71nM and fixed Mono-GlcNS6S-IdoA2S-Glc was 1 order lower _{than the} value of the other two types of chips, and had a strong inhibitory effect by heparin. . Since the inhibition rate of heparin varies in any chip, it can be concluded that these chips specifically recognize bFGF, a heparin binding protein.

Example 3-2: Examination of Relative Density of Sugar Chain on Chip Surface

First, a Tri-GlcNS6S-IdoA2S-Glc or Tetra-GlcNS6S-IdoA2S-Glc and a linker compound (a non-sugar chain-linker binding compound, hereinafter referred to as Mono-Glc) to which a molecule having no sugar chain is bonded in a solution Immobilization was performed and the density change of disaccharide sulfate, which is a ligand on a chip, by the mixing ratio (injection ratio) was examined using the ATR-FT-IR method. The ratio of Tri-GlcNS6S-IdoA2S-Glc or Tetra-GlcNS6S-IdoA2S-Glc in the solution was changed to 0, 25, 50, 75, 100%. The results are shown in Figs. 5A and 5B. (A) of FIG. 5 is the total reflection spectrum of Tri-GlcNS6S-IdoA2S-Glc which changed the mixing ratio in a solution, and FIG. 5 (b) is the total reflection spectrum of Tetra-GlcNS6S-IdoA2S-Glc which changed the mixing ratio in a solution.

Since S = 0 stretching vibrations derived from sulfuric acid groups were observed in the wavenumber range of 1200-1303 cm ^-1 , sulfuric acid groups were quantified by the multivariate analysis method using the absorbance curve of this region, and the ratio of the ligand conjugates in the solution was determined. The relative strength of sulfuric acid groups on the chip is plotted. The results are shown in Figs. 6A and 6B. Figure 6 (a) is a graph showing the relative strength of sulfuric acid groups on the chip to the mixing ratio of Tri-GlcNS6S-IdoA2S-Glc in the solution, 6 (b) is a chip for the mixing ratio of Tetra-GlcNS6S-IdoA2S-Glc in the solution It is a graph which shows the relative intensity of the sulfate group of a phase. Since the correlation coefficients of the primary curves shown in Figs. 6A and 6B are 0.9993 and 0.9610, respectively, the immobilization rate (density of sugar chains on the chip surface) of the disaccharide sulfate, which is a ligand, is either ligand in solution. It was shown to be proportional to the abundance of the complex.

Example 3-3 Examination of the Influence of Relative Density of Sugar Chain on Interaction with h-vWF

Subsequently, the effect of the relative density of disaccharide sulfate, a ligand on the chip surface, on the interaction with the protein was examined. Here, the interaction with vWF derived from human plasma (h-vWF hereinafter) was analyzed.

The mixing ratio of each of the three ligand conjugates (Mono-GlcNS6S-IdoA2S-Glc, Tri-GlcNS6S-IdoA2S-Glc, Tetra-GlcNS6S-IdoA2S-Glc) and Mono-Glc was 100/0, and 20/80, respectively. 6 chips were prepared and the interaction with h-vWF was observed by the SPR method. Here, the measuring procedure by the SPR method is demonstrated.

SPR670 (made by Japan Laser Electronics Co., Ltd.) was used for the measurement. In the sensor chip, UV ozone was used by depositing 2 nm of chromium on a 13 x 20 x 0.7 mm glass substrate as an adhesive layer and then depositing a 50 nm gold thin film thereon (manufactured by Japan Laser Electronics Co., Ltd.). It was put into a cleaner (NL-UV253 manufactured by Nippon Laser Electronics Co., Ltd.), irradiated with ultraviolet rays for 30 minutes, and the chip surface was washed with ozone.

Subsequently, after mounting the sensor chip in a dedicated PTFE cell (manufactured by Nippon Laser Electronics Co., Ltd.), each of the six chips was mixed with methanol / water = 1/1 (except Mono-GlcNS6S-IdoA2S-Glc / Mono-). In the case of Glc mixing, methanol solution) was dissolved (0.1 mM), 50 µl of the solution was taken into a PTFE cell, and sealed with a parafilm. The PTFE cell equipped with the chip was gently shaken overnight on Bio Dancer (New Brunswick Scientific).

This tip was washed 6 times with methanol, once with water, and then once again with methanol and water. After air drying, it was attached to the sensor chip cartridge of SPR670. After sufficiently equilibrating the chip surface with the running buffer, the laser beam was irradiated to the gold film, and the change in the surface plasmon resonance angle observed at this time was observed. As running buffer, isotonic phosphate buffer solution (PBS) at pH 7.4 was used. In addition, all SPR measurements were performed at the temperature of 25 degreeC. bFGF uses STRATHMANN BIOTEC AG (Recombiant Human FGF-basic, MW; 17000, Lot No .; 471120), and h-vWF uses CALBIOCHEM (von Willebrand Factor, Human Plasma, MW; 270000 (Monomer Unit conversion), Lot No .; B41632).

When performing this SPR measurement, when the concentration of h-vWF was injected into each chip while changing the concentration to 10, 20, 40, 80, and 160 nM, binding interaction was observed, and the appearance of h-vWF immobilized on the chip was observed. Was observed. At this time, since no dissociating agent which completely dissociated h-vWF from the chip without denatured disaccharide sulfate was found, the calculation of the dissociation constant (K _D ) was obtained from the binding amount of h-vWF on the chip. The amount of h-vWF binding was based on the state in which the ligand conjugate was immobilized, and the difference in the curve of the sensorgram of the h-vWF injected at different concentrations was used.

(A) to (c) of FIG. 7, a mixture ratio of the three ligand conjugates (Mono-GlcNS6S-IdoA2S-Glc, Tri-GlcNS6S-IdoA2S-Glc, Tetra-GlcNS6S-IdoA2S-Glc) and Mono-Glc Is a binding interaction when is 100/0. (a) is for Mono-GlcNS6S-IdoA2S-Glc, (b) is for Tri-GlcNS6S-IdoA2S-Glc, and (c) is for Tetra-GlcNS6S-IdoA2S-Glc. 8A to 8C, each of the three ligand conjugates (Mono-GlcNS6S-IdoA2S-Glc, Tri-GlcNS6S-IdoA2S-Glc, Tetra-GlcNS6S-IdoA2S-Glc) and Mono-Glc The binding interaction when the mixing ratio of is 20/80 is shown. (a) is for Mono-GlcNS6S-IdoA2S-Glc, (b) is for Tri-GlcNS6S-IdoA2S-Glc, and (c) is for Tetra-GlcNS6S-IdoA2S-Glc.

Plotting the amount of binding obtained from this result for each concentration of h-vWF is shown in Figs. 9A to 9C. (a) is for Mono-GlcNS6S-IdoA2S-Glc, (b) is for Tri-GlcNS6S-IdoA2S-Glc, and (c) is for Tetra-GlcN S6S-IdoA2S-Glc. In this Fig. 9, there is shown also a result of calculating the dissociation constant (K _D) from curves of the graphs.

As shown in Fig. 9 (a), the sulfate is mono-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0, and Mono-GlcNS6S-IdoA2S-Glc / Mono-Glc = 20/80 as a ligand. In the case of using a chip in which disaccharide was immobilized, the dissociation constants were K _D = 35 nM and 41 nM, respectively. As shown in Fig. 9 (b), sulfidation is performed using Tri-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0, and Tri-GlcNS6S-IdoA2S-Glc / Mono-Glc = 20/80 as a ligand. In the case of using a chip in which disaccharide was immobilized, the dissociation constants were K _D = 27 nM and 24 nM, respectively. In addition, in the case of using a chip having immobilized disaccharide sulfate as a ligand using Tetra-GlcNS6S-IdoA2S-Glcto / Mono-Glc = 100/0, and Tetra-GlcNS6S-IdoA2S-Glcto / Mono-Glc = 20/80 And dissociation constants were K _D = 32 nM and 35 nM, respectively.

From these results, it was found that when h-vWF was used as the analog, even if the abundance ratio on the chip of the sugar chain as a ligand was changed, the effect on affinity was hardly found. In addition, even when using chips immobilized with ligand conjugates having different sugar chain distances, differences in sugar chain distances in interaction with h-vWF did not affect the value of dissociation constant. It is considered that since h-vWF has a multimer structure, a plurality of heparin binding domains exist, and the dissociation rate is remarkably slowed due to the influence thereof, and the difference in the distance between sugar chains is not reflected in the dissociation constant.

Example 3-4: Investigation of Influence of Relative Density of Sugar Chain on Interaction with Protein

Here, using a recombinant human vWF partial protein derived from Escherichia coli (hereinafter referred to as rhvWF-A1) having only an Al loop portion having one heparin binding domain, the interaction between the sugar chain clustered on the chip and the protein is achieved. Considering that the effect of the difference between the sugar chain distances on the interactions between the sugar chains and the sugar chain binding proteins could be examined, the following experiments were conducted. rhvWF-A1 was prepared according to M. A. Cruz, R. I. Handin & R. J. Wise, J. Biol. Chem. 268, page 21238, 1993.

Here, each of the three ligand ligands (Mono-GlcNS6S-IdoA2S-Glc, Tri-GlcNS6S-IdoA2S-Glc, Tetra-GlcNS6S-IdoA2S-Glc) described above as a ligand complex, and Mono-Glc, A chip prepared by changing the abundance ratio of each of the three ligand conjugates / Mono-Glc to 100/0 and 50/50, respectively, was used. 10 to 12 show the results of measuring the binding interaction with the chip by changing the rhvWF-A1 concentration. On the other hand, Fig. 10 (a) is a case where a chip is prepared with Mono-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0, and Fig. 10 (b) shows Mono-GlcNS6S-IdoA2S-Glc / Mono-Glc = 50/50. FIG. 11A shows a case where a chip is prepared with Tri-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0, and FIG. 11B shows Tri-GlcNS6S-IdoA2S-Glc / Mono-Glc. = 50/50. In addition, FIG. 12 (a) shows a case where a chip is prepared with Tetra-GlcNS6S-IdoA2S-Glc / Mono-Glc = 100/0, and FIG. 12 (b) shows Tetra-GlcNS6S-IdoA2S-Glc / Mono-Glc = 50/50.

Table 1 summarizes the dissociation constants, binding constants, binding rate constants, and dissociation rate constants calculated from these results. In Table 1, the dissociation constant: KD (k _d / k _a ), the coupling constant: K _A (k _a / k _d ), the binding rate constant: k _a , and the dissociation rate constant: k _d .

No. Type of ligand Composition ratio on chip K _D (μM) K _A (M ^-1 x10 ^-5 ) k _a (M ^-1S-1 x10 ³ ) k _d (S ^-1 x10 ^-3 ) One Mono-GlcNS6S-IdaA2A-Glc / Mono-Glc 100/0 2.60 3.86 8.38 21.9 2 50/50 3.79 2.64 14.6 55.2 3 Tri-GlcNS6S-IdoA2A-Glc / Mono-Glc 100/0 1.20 8.33 6.60 8.05 4 50/50 1.50 6.65 4.52 6.83 5 Tetra-GlcNS6-IdoA2A-Glc / Mono-Glc 100/0 0.99 10.1 6.50 6.44 6 50/50 1.00 9.96 5.24 5.26

As shown in Table 1, in the case of the chip in which Mono-GlcNS6S-IdoA2S-Glc was immobilized, the value of the dissociation constant became larger compared to the chip in which Tri-GlcNS6S-IdoA2S-Glc or Tetra-GlcNS6S-IdoA2S-Glc was immobilized. (K _D = 2.60 μM). In addition, when the immobilization density of sugar chains on the chip was made relatively small, the value of the dissociation constant became larger (K _D = 3.79 μM). On the other hand, in the chip immobilized with Tri-GlcNS6S-IdoA2S-Glc, when the immobilization density on the chip was relatively small, a slight increase in the value of the dissociation constant was observed (K _D = 1.20 µM → 1.50 µM). In addition, in the chip on which Tetra-GlcNS6S-IdoA2S-Glc was immobilized, the dissociation constant remained almost unchanged even if the immobilization density on the chip was changed (K _D = 0.99 μM → 1.00 μM).

In addition, as shown in Table 1, in the case of the chip immobilized with Mono-GlcNS6S-IdoA2S-Glc, it was confirmed that it had a dissociation rate constant (k _d ) that was one order higher than that of the other two ligand complexes and the immobilized chip. It became. From this, since Tri-GlcNS6S-IdoA2S-Glc and Tetra-GlcNS6S-IdoA2S-Glc have a sugar chain cluster structure in which the distance between sulfated oligosaccharide chains is controlled in a molecule, the immobilization density of sugar chains on a chip is relatively lowered. Even if it is hard to be affected.

That is, in the interaction with rhvWF-Al, in the chip | tip which immobilized Mono-GlcNS6S-IdoA2S-Glc, when the relative sugar chain immobilization density on a chip | tip was reduced, it was confirmed that the bonding force falls. On the other hand, in the chips immobilized with Tri-GlcNS6S-IdoA2S-Glc or Tetra-GlcNS6S-IdoA2S-Glc, it was found that the binding force did not change much even if the relative sugar chain immobilization density on the chip was reduced.

From the above results, in order to increase the binding force in the interaction between the sulfated oligosaccharide chain-sugar chain binding proteins, the sulfated oligosaccharide chain having the same ligand complex structure as the chip immobilized with Mono-GlcNS6S-IdoA2S-Glc was chipped. Two-dimensional clustering of phases is insufficient and supports the need for cluster structures with controlled sugar chain distances at the molecular level, such as Tri-GlcNS6S-IdoA2S-Glc or Tetra-GlcNS6S-IdoA2S-Glc.

Example 4 Synthesis of Linker Compound (Compound 26) and Ligand Complex (Compound 27)

One of the linker compounds according to the present invention, i.e., in general formula (1), a is 1, b is 4, d is 1, e is 4, X is represented by the general formula (4), q ¹ , a linker compound (compound 26) having a structure wherein q ² , q ³ is 2 and r ¹ , r ² , r ³ , t ¹ , t ² , t ³ , u ¹ , u ² , u ³ are O, and Represented by the general formula (7), a is 1, b is 4, d is 1, e is 4, q ¹ , q ² , q ³ is 2, r ¹ , r ² , r ³ , t ¹ , t ² , t ³ , u ¹ , u ² , u ³ are 0, R 'is hydrogen (H), and R is a ligand conjugate (Compound 27) having the structure of formula (6-2). Synthesized in order. 13 shows the process of synthesizing this linker compound (Compound 26). 14 shows the process of synthesizing a ligand conjugate (Compound 27) from this linker compound (Compound 26). In addition, in description of this Example 4, the number added to each compound corresponds to the number described in FIG. 13 and FIG.

[Measurement method, reagent, etc.]

^1, measurement of H-NMR spectrum, were used JOEL-Delta 600 Spectrometer. The chemical shift is represented by δ value in the case of CDCl ₃ using tetramethylsilane (0.00 ppm) as a reference substance. For D ₂ O it is shown as δ value and a D ₂ O (4.65ppm) as reference material. Mass spectrometry was measured using a PerSeptive Biosystem Mariner ^™ Biospectrometry Workstation. Medium pressure silica gel chromatography was performed using Silicagel 60 (Merck, 0.040 ~ 0.063mm). Thin layer chromatography was performed using Precoated Silicagel 60 F254 (Merck, 0.5 mm). All reagents and dehydration solvents were purchased from Kanto Chemical Co., Ltd.

(1) Synthesis of N ₃ -TEG-Trivalent- (O ^t BU) ₃ (Compound 20) (see FIG. 13)

0 ₂ N-Trivalent- (O ^t (Bu) ₃ (Compound 18) (757 mg, 1.70 mmol) and nickel chloride hexahydrate (NiCl ₂ · 6H ₂ 0) (80.8 mg, 0.340 mmol) were added to methanol (20 mL). After dissolving, sodium borohydride (322 mg, 8.50 mmol) was added in 5 equivalent portions and stirred at room temperature for 30 minutes, methanol was concentrated, and water and chloroform were added, and the filtrate was filtered with chloroform. The organic phase was dried over anhydrous sodium sulfate, and the drying agent was filtered off and concentrated under reduced pressure.The obtained concentrated residue (Compound 19) and N ₃ -TEG-COOH (Compound 6) (441 mg, 1.70 mmol) was dissolved in anhydrous dimethylformamide (10 ml), and DIEA (592 µl, 3.40 mmol), HOAt (463 mg, 3.40 mmol), and EDC-HCl (652 mg, 3.40 mmol) were added in an order of room temperature and argon atmosphere. After adding water to the reaction solution, the aqueous phase was extracted three times with ethyl acetate. After washing and drying with anhydrous magnesium sulfate, the drying agent was filtered off and concentrated under reduced pressure The obtained residue was purified by silica gel chromatography (50 g, chloroform: acetone = 50: 1) to obtain N ₃ -TEG. -Trivalent- (O ^t Bu) ₃ was obtained as a colorless oil (compound 20) (油狀物). the amount was obtained 839mg (73%).

¹ H-NMR spectrum (600 mHz, CDCl ₃ ) was measured for this compound 20. As a result, δ 3.90 (s, 2H, -0 CH ₂ CONH-), 3.70-3.67 (m, 14H, -O CH ₂ CH) ₂ O- × 3, N ₃ CH ₂ CH ₂ 0-), 3.39 (t, 2H, J = 4.8 Hz, N ₃ CH ₂ CH ₂ O-), 2.21-2.18 (m, 6H, -CH ₂ CH ₂ CO- × 3), 2.00-1.96 (m , 6H, - CH 2 CH 2 CO- × 3), was _{1.43 (s, 27H, -CH 3} × 9). ESI-MS (positive) measurement was performed and found to be m / z 697.45 [(M + Na) ⁺ ]. This confirmed the structure of Compound 20. On the other hand, the molecular mass of this compound 20 is C ₃₂ H ₅₈ N ₄ O ₁₁ : 676.41.

(2) Synthesis of TEG-Trivalent- (0 ^t BUu) ₃ (Compound 22) (see FIG. 13)

The compound 20 (N ₃ -TEG-Trivalent- (O ^t BU) ₃ ) (837 mg, 1.24 mmol) was dissolved in methanol (10 mL), and 10% Pd / C (200 mg) was added thereto under a hydrogen atmosphere at room temperature. Stir for 1.5 hours. The Pd / C was filtered off and the filtrate was concentrated under reduced pressure. The obtained concentrated residue (Compound 21) and chioctic acid (385 mg, 1.87 mmol) were dissolved in anhydrous dimethylformamide (10 mL), and DIEA (325 μl, 1.87 mmol) and HOAt (254 mg, 1.87 mmol) were added at room temperature under argon atmosphere. ) And EDC-HCl (358 mg, 1.87 mmol) were added sequentially and stirred for 13 hours. After adding saturated aqueous sodium hydrogen carbonate solution to the reaction solution, the aqueous phase was extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and then dried by filtration and concentrated under reduced pressure. The obtained concentrated residue was purified by silica gel chromatography (50g, chloroform: methanol = 30: 1) to give the, TEG-Trivaient- (O ^t Bu) ₃ was obtained (compound 22) as a colorless oil. The yield was 1.05 g (99%).

With respect to the compound 22 ^l H-NMR spectrum (600mHz, CDCl ₃₎ subjected to the measurement results, δ3.91 (s, 2H, -0 CH 2 CONH-), 3.70-3.54 (m, 13H, -O CH 2 CH ₂ 0- × 3, CH ₂ CH (CH _2- ) (S-)), 3.55 (t, 2H, J = 5.5 Hz, -CONHCH ₂ CH ₂ 0-), 3.45 (q, 2H, J = 5.5 Hz , CONH CH ₂ CH ₂ 0-), 3.20-3.16 (m, 1H, -S CH ₂ (1H)-), 3.14-3.09 (m, 1H, -S CH ₂ (1H)-), 2.49-2.43 ( m, 1H, -SCH ₂ CH ₂ (1H)-), 2.22-2.17 (m, 8H, -CH ₂ CH ₂ CO-x3, NHCO CH ₂ CH _2- ), 2.00-1.96 (m, 6H,- CH ₂ CH ₂ CO- × 3), 1.94-1.88 (m, 1H, -SCH ₂ CH ₂ (1H)-), 1.74-1.62 (m 4H, -CO CH ₂ CH ₂ CH _2- ), 1.52-1.41 (m, 2H, COCH ₂ CH ₂ CH ₂ CH _2- ) and 1.44 (s, 27H, -CH ₃ x 9). This confirmed the structure of Compound 22.

(3) Synthesis of TEG-Trivaient- (NHBoc) ₃ (Compound 25) (see FIG. 13)

The compound ^{22 (O t Bu (TEG-} Trivaient-) 3) (500mg, 0.587mmol) in dichloromethane / water (2.2㎖, 10 / l) was added TFA (2㎖) in the dissolution, and by O ℃, copper It stirred at the temperature for 1 hour. The reaction solution was concentrated and then azeotropic with toluene. The obtained concentrated residue (Compound 23) and N-Boc-phenylenediamine (Compound 24) (612 mg, 2.94 mmol) were dissolved in anhydrous dimethylformamide (1 0 mL), and DIEA (380 µL, 2.94 mmol), HOAt (400 mg, 2.94 mmol) and EDC-HCl (563 mg, 2.94 mmol) were added in this order and stirred for 19 hours. After adding saturated aqueous sodium hydrogen carbonate solution to the reaction solution, the aqueous phase was extracted three times with AcOEt. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and then the drying agent was filtered off and concentrated under reduced pressure. The obtained concentrated residue was purified by silica gel chromatography (50 g, chloroform: acetone = 3: 1) to obtain TEG-Trivalent- (NHBoc) ₃ (Compound 25) as a pale yellow oil. The yield was 230 mg (31%).

¹ H-NMR spectrum relative to the compound 25 (600mHz, CDCl ₃₎ result of the measurement, δ8.72 (bs, 3H, - NH CO-), 7.56 (bs, 3H, aromatic), 7.22-7.10 (m, 6H, aromatic), 6.91 (bs , 3H, - NH CO-), 3.85 (s, 2H, -0 CH 2 CONH-), 3.67-3.57 (m, 9H, ethylene glycol chain, CH 2 CH (CH 2 - ) (S-)), 3.55-3.47 (m, 6H, ethylene glycol chain, CONHCH ₂ CH ₂ 0-), 3.38 (q, 2H, J = 5.2 Hz, -CONH CH ₂ CH ₂ 0-), 3.14 ( ddd, 1H, J = 6.9, 6.9, 12.4 Hz, -S CH ₂ (1H)-), 3.08 (ddd, 1H, J = 6.9, 6.9, 12.4 Hz, -S CH ₂ (1H)-), 2.43- 2.35 (m, 7H, -CH ₂ CH ₂ CO-x3, -SCH ₂ CH ₂ (lH)-), 2.08 (t, 2H, J = 6.9 Hz, -NHCO CH ₂ CH _2- ), 2.17-2.12 (m, 6H, -CH ₂ CH ₂ CO- × 3), 1.88-1.83 (m, 1H, -SCH ₂ CH ₂ (1H)-), 1.65-1.50 (m 4H, -COCH ₂ CH ₂ CH ₂ CH _2- ), 1.50 (s, 27H, -CH ₃ x 9), and 1.46-1.29 (m, 2H, -COCH ₂ CH ₂ CH ₂ CH _2- ). This confirmed the structure of Compound 25.

(4) Synthesis of Ligand Complex TEG-Trivalent- (Mal) ₃ (Compound 27) (see FIGS. 13 and 14)

The compound ^{25 (O t Bu (TEG-} Trivalent-) 3) (500mg, 0.587mmool) in dichloromethane / water (4.4㎖, 10 / l) and dissolved, at 0 ℃ was added TFA (2㎖) in the same temperature Stirred for 1.5 h. The reaction solution was concentrated and then azeotropic with toluene. The obtained residue (Compound 26) was used for the next reduction amination reaction without purification. The amount obtained was 252 mg.

A description with reference to FIG. 14 is as follows. The obtained residue (Compound 26) (12.1 mg, 8.88 µmol) and maltose (9.60 mg, 26.7 µmol) were dissolved in dimethylacetamide / water (1: 1, 600 µl) and left to stand at 37 ° C for 7 hours. Acetic acid (30 µl) and sodium cyanoborohydride (5.58 mg, 88.8 µmol) were added to the reaction solution, and the mixture was left to stand at 37 ° C for 70 hours. After the reaction solution was lyophilized, the obtained residue was purified by preparative high performance liquid chromatography (ODS column, methanol / water = 50/50). Ligand complex TEG-Trivalent- (Mal) ₃ (Compound 27) was obtained as a white solid.

¹ H-NMR spectrum (600 mHz, D ₂ 0) was measured for this compound 27. As a result, δ 7.02 (dd, 3H, J = 7.6, 8.2 Hz, aromatic), 6.72 (s, 3H, aromatic), 6.60 (dd, 3H, J = 1.4, 7.6 Hz, aromatic), 6.44 (dd, 3H, J = 1.4, 8.2 Hz, aromatic), 4.91 (d, 3H, J = 3.4 Hz, H-1 ′ × 3), 3.82-3.73 (m, 8H, H-2 × 3, H-5 × 3, -0 CH ₂ CONH-), 3.73-3.67 (m, 9H, H-3 × 3, H-5 '× 3, H -6a '× 3), 3.65 (dd, 3H, J = 2.1, 12.4 Hz, H-6b' × 3), 3.59 (dd, 3H, J = 4.8, 12.4 Hz, H-6a × 3), 3.55 ( dd, 3H, J = 5.5, 6.2 Hz, H-4 × 3) 3.55 (dd, 3H, J = 9.6, 9.6 Hz, H-3 ′ × 3), 3.50-3.36 (m, 12H, -0 CH ₂ CH ₂ O- × 3), 3.45-3.40 (m, 3H, H-6b × 3), 3.42-3.38 (m, 1H, CH ₂ CH (CH _2- ) (S-)), 3.38 (dd, 3H , J = 3.4, 10.3 Hz, H-2 '× 3), 3.40 (t, 2H, J = 5.5 Hz, -CONHCH ₂ CH ₂ 0-), 3.25 (dd, 3H, J = 9.6, 9.6 Hz, H -4 '× 3), 3.15-3.10 (m, 5H, -CONH CH ₂ CH ₂ 0-, H-1a × 3), 3.02 (dd, J = 8.2, 13.7 Hz, H-1b × 3), 3.01 -2.97 (m, 1H, -S CH ₂ (1H)-), 2.96-2.91 (m, 1H, -S CH ₂ (1H)-), 2.29-2.25 (m, 6H, CH ₂ CH ₂ CO- × 3), 2.26-2.19 (m, 1H, -SCH ₂ CH ₂ (1H)-), 2.05-1.98 (m, 6H, -CH ₂ CH ₂ CO-x3), 1.99 (t, 2H, J = 6.9 Hz, -NHCO CH ₂ CH _2- ), 1.74-1.69 (m, 1H, -SCH ₂ CH ₂ (1H)-), 1.50-1.30 (m 4H, COCH ₂ CH ₂ CH ₂ CH ₂ ), 1.16-1.10 (m, 2H, -COCH ₂ CH ₂ CH ₂ CH _2- ). ESI-MS measurement showed m / z 981.41 [(M + 2Na) ²⁺ ]. These confirmed the structure of compound 27. On the other hand, the molecular mass of this compound 27 is C ₈₂ H ₁₃₂ N ₈ O ₃₉ S ₂ : 1916.80.

Example 5 Synthesis of Linker Compound (Compound 32) and Ligand Complex (Compound 34)]

In one of the linker compounds according to the present invention, that is, in general formula (1), a is 4, b is 0, d is 0, e is 0, X is represented by the general formula (4), q ¹ , a linker compound having a structure of q ² , q ³ is 2, r ¹ , r ² , r ³ is 1, t ¹ , t ² , t ³ is 4 and u ¹ , u ² , u ³ are 1 (Compound 32) and the said General formula (7), a is 4, b is 0, d is 0, e is 0, q ¹ , q ² , q ³ is 2, r ¹ , r ² , r ³ is 1, t ¹ , t ² , t ³ is 4, u ¹ , u ² , u ³ are 1, R 'is hydrogen (H), and R is of general formula (6-2) Ligand complex having a structure (Compound 34) was synthesized in the following order. 15 shows a procedure of synthesizing this linker compound (Compound 32). FIG. 16 shows a process for synthesizing Compound 30 used in the process of synthesizing the linker compound (Compound 32). 17 shows the process of synthesizing a ligand conjugate (Compound 34) from this linker compound (Compound 32). In addition, in description of this Example 5, the number added to each compound corresponds to the number described in FIG. 15, FIG. 16, and FIG.

[Measurement method, reagent, etc.]

^1, measurement of H-NMR spectrum, were used JOEL-Delta600 Spectrometer. The chemical shift is represented by δ value in the case of CDCl ₃ using tetramethylsilane (0.00 ppm) as a reference substance. In the case of D ₂ O, DHO (4.65 ppm) was used as a reference material and expressed as δ. Mass spectrometry was measured using a PerSeptive Biosystem Mariner ^™ Biospectrometry Workstation. Medium pressure silica gel column chromatography was performed using Silicagel 60 (Merck, 0.040 to 0.063 mm). Thin layer chromatography was performed using Precoated Silicagel 60 F254 (Merck, 0.5 mm). All reagents and dehydration solvents were purchased from Kanto Chemical Co., Ltd.

(1) Synthesis of Trivalent 1 (O ^t Bu) ₃ (Compound 28) (see FIG. 15)

^{_{0 2 N-Trivalent- (O t}} Bu) 3 ( Compound 18) (757mg, 1.70mmol) and _{NiCl 2 · 6H 2 0 (80.8mg} , 0.340mmol) borohydride in a dissolved, and O ℃ in methanol (20ml) Sodium (322 mg, 8.50 mmol) was added in 5 equivalent portions each time, and the reaction solution was then stirred at room temperature for 30 minutes. Methanol was removed by concentration, and water and chloroform were added to the reaction solution. After celite filtration, the aqueous phase was extracted three times with chloroform. The organic phase was dried using anhydrous sodium sulfate, and then the drying agent was filtered off and concentrated under reduced pressure. The obtained concentrated residue (Compound 19) and chioctic acid (351 mg, 1.70 mmol) were dissolved in anhydrous dimethylformamide (10 ml), and DIEA (592 µl, 3.40 mmol) and HOAt (463 mg, 3.40 mmol) at room temperature under argon atmosphere. , EDC-HCl (652 mg, 3.40 mmol) were added sequentially, and it stirred for 16 hours. After adding water to the reaction solution, the aqueous phase was extracted three times with ethyl acetate. The organic phase was washed with saturated brine and saturated aqueous sodium hydrogen carbonate solution, dried over anhydrous magnesium sulfate, and then dried by filtration and concentrated under reduced pressure. The resulting concentrated residue was purified by silica gel column Photography (50g, hexane: ethyl acetate = 3: 1) to give, Trivalent- (O ^t Bu) ₃ was obtained (compound 28) as a light yellow oil. The amount obtained was 750 mg (73%).

^L H-NMR spectrum relative to the compound 28 (600mHz, CDCl ₃₎ subjected to the measurement results, δ5.91 (s, 1H, -CO NH -), 3.57 (ddd, 1H, J = 6.2, 6.2, 12.4Hz, CH ₂ CH (CH _2- ) (S-)), 3.18 (ddd, 1H, J = 5.5, 5.5, 12.4 Hz, -S CH ₂ (lH)-), 3.11 (ddd, 1H, J = 6.9, 7.6 , 12.4 Hz, -S CH ₂ (lH)-), 2.46 (ddd, 1H, J = 6.2, 6.2, 12.4 Hz, -SCH ₂ CH ₂ (lH)-), 2.22 (t, 8H, J = 7.6 Hz , -CH ₂ CH ₂ CO-x3), 2.11 (dd, 2H, J = 6.9, 7.6 Hz, -CO CH ₂ CH ₂ CH ₂ CH _2- ), 1.97 (t, 6H, J = 7.6 Hz,- CH ₂ CH ₂ CO- × 3), 1.91 (ddd, 1H, J = 6.9, 6.9, 12.4 Hz -SCH ₂ CH ₂ (lH)-), 1.74-1.57 (m 4H, -COCH ₂ CH ₂ CH ₂ CH _2- ), 1.51-1.38 (m, 2H, -COCH ₂ CH ₂ CH ₂ CH _2- ), 1.43 (s, 27H, -CH ₃ x 9). In addition, ¹³ C-NMR (150 mHz, CDCl ₃ ) was measured and found to be δ 172.9, 172.1, 80.7, 57.3, 56.3, 40.2, 38.5, 37.2, 34.6, 30.0, 29.8, 28.9, 28.1, and 25.3. These confirmed the structure of the compound 28.

(2) Synthesis of N ₃ -TEG-NHBoc (Compound 33) (see FIG. 16)

N ₃ -TEG-CO ₂ Et (Compound 5) (500 mg, 1.64 mmol) was dissolved in 1,4-dioxane (6 mL), and aqueous sodium hydroxide solution (1 mL, 150 mg / mL) was added to the reaction solution at O ° C. It stirred at the same temperature for 3 hours. After concentration of 1,4-dioxane was concentrated, 5% aqueous potassium hydrogen sulfate solution and chloroform were added thereto. The aqueous phase was extracted three times with chloroform, and the organic phase was dried using anhydrous sodium sulfate, and then the drying agent was filtered off and concentrated under reduced pressure. The obtained residue (Compound 6) was used for subsequent coupling reaction without purification. The amount obtained was 435 mg (96%). The obtained concentrated residue and N-Boc-phenylenediamine (Compound 24) (327 mg, 1.57 mmol) were dissolved in anhydrous dimethylformamide, and dried under an argon atmosphere at room temperature with DlEA (410 µL, 2.35 mmol) and HOAt (320 mg, 2.35). mmol) and EDC.HCl (451 mg, 2.35 mmol) were added in this order and stirred for 14 hours. After adding water to the reaction solution, the aqueous phase was extracted three times with ethyl acetate. The organic phase was washed with saturated brine and saturated aqueous sodium hydrogen carbonate solution, dried over anhydrous magnesium sulfate, and then dried by filtration and concentrated under reduced pressure. The obtained concentrated residue was purified by silica gel column chromatography (50 g, toluene: ethyl acetate = 1: 1) to obtain N ₃ -TEG-NHBoc (Compound 33) as a pale yellow oil. The amount obtained was 597 mg (81%).

^L H-NMR spectrum relative to the compound 33 (600MHz, CDCl ₃₎ result of the measurement, δ8.81 (bs, 1H, - NH CO-), 7.61 (s, 1H, aromatic), 7.35 (d, 1H, J = 6.9Hz, aromatic), 7.26-7.20 (m, 2H, aromatic), 6.71 (bs, 1H, - NH CO-), 4.10 (s, 2H, -0 CH 2 CONH-), 3.78-3.70 (m , 8H, ethyleneglycol chain), 3.67-3.62 (m, 6H, ethylene glycol chain, -CONHCH ₂ CH ₂ 0-), 3.35 (t, 2H, J = 5.5 Hz, -CONH CH ₂ CH ₂ 0-). In addition, ¹³ C-NMR (150 MHz, CDCl ₃ ) was measured, resulting in δ 68.3, 152.6, 139.0, 138.0, 129.5, 114.5, 114.3, 109.8, 80.5, 71.2, 70.6, 70.6, 70.6, 70.5, 70.4, 70.2 , 70.0, 50.6, 28.3. These confirmed the structure of compound 33.

(3) Synthesis of H ₂ N-TEG-NHBoc (Compound 30) (see FIG. 16)

Compound 33 (N ₃ -TEG-NHBoc) (200 mg, 0.425 mmol) was dissolved in methanol (4 mL), 10% Pd / C (200 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1.5 hours. The Pd / C was filtered off and concentrated under reduced pressure. The obtained residue (Compound 30) was used in the subsequent reaction without purification. The amount obtained was 174 mg (93%).

(4) Synthesis of Trivalent- (TEG-NHBoc) ₃ (Compound 31) (see FIG. 15)

The compound ^{28 (O t Bu (Trivalent-)} 3) (64.2mg, 0.106mmol) in dichloromethane / water (2.2ml, 10 / l) was added TFA (2㎖) from melting, and the O ℃, this temperature Stirred for 1 hour. The reaction solution was concentrated and then azeotropic with toluene. The obtained concentrated residue (Compound 29) and H ₂ N-TEG-NHBoc (Compound 30) (174 mg, 0.425 mmol) were dissolved in anhydrous dimethylformamide (3 mL), and DIEA (92.6 µl, 0.532) was added at room temperature under argon atmosphere. mmol), HOAt (72.3 mg, 0.532 mmol) and EDC-HCl (102 mg, 0.532 mmol) were added in this order and stirred for 14 hours. After adding water to the reaction solution, the aqueous phase was extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and the drying agent was filtered off and concentrated under reduced pressure. The obtained concentrated residue was purified by silica gel column chromatography (50 g, chloroform: methanol = 30: 1) to give Trivalent- (TEG-NHBoc) ₃ (Compound 31) as a pale yellow oil. The yield was 64.7 mg (36%).

^L H-NMR spectrum relative to the compound 31 (600MHz, CDCl ₃₎ subjected to the measurement results, δ8.88 (bs, 3H, - NH CO- × 3), 7.67 (bs, 3H, aromatic), 7.42 (bs, 3H, -NHCO- × 3), 7.31 (d, 3H, J = 7.7 Hz, aromatic), 7.27 (d, 3H, J = 8.2 Hz, aromatic), 7.22 (dd, 3H, J = 7.7, 8.2 Hz, aromatic), 6.63 (bt, 3H , J = 4.8Hz, - NH CO- × 3), 4.11 (s, 6H, -O CH 2 CONH- × 3), 3.78-3.58 (m, 36H, ethylene glycol chain) , 3.57-3.49 (m, 1H, CH ₂ CH (CH2-) (S-)), 3.50 (t, 6H, J = 5.5 Hz, -CONHCH ₂ CH ₂ 0- × 3), 3.36 (q, 6H, J = 5.2 Hz, -CONH CH ₂ CH ₂ 0- × 3), 3.15 (ddd, ¹ H, J = 5.5, 6.9, 11.0 Hz, -S CH ₂ (1H)-), 3.09 (ddd, 1H, J = 6.9, 6.9, 11.0 Hz, -S CH ₂ (1H)-), 2.42 (ddd, 1H, J = 6.9, 6.9, 12.4 Hz, -SCH ₂ CH ₂ (lH)-), 2.12-2.06 (m, 8H, -CH ₂ CH ₂ CO- × 3, -NHCO CH ₂ CH ₂ CH _2- ), 1.95-1.88 (m, 6H, -CH ₂ CH ₂ CO- × 3), 1.87 (ddd, 1H, J = 6.9, 6.9, 12.4 Hz, -SCH ₂ CH ₂ (lH)-), 1.70-1.50 (m 4H, -COCH ₂ CH ₂ CH ₂ CH _2- ), 1.50 (s, 27H, CH ₃ × 9), 1.48 -1.33 (m, 2H, -COCH ₂ CH ₂ CH ₂ CH _2- ). In addition, ¹³ C-NMR (150 MHz, CDCl ₃ ) was measured and found to be δ 173.3, 172.8. 168.4, 152.8, 139.3, 137.8, 129.3, 114.5, 114.4, 110.1, 80.2, 71.1, 70.5, 70.4, 70.4, 70.3, 70.1, 70.1, 69.7, 57.3, 56.4, 40.1, 39.2, 38.3, 37.0, 34.5, 31.1, 30.5, 28.8, 28.3, and 25.4. Moreover, it was m / z 875.41 [(M + 2Na) ²⁺ ] as a result of ESI-MS measurement. From these, the compound 31 could be confirmed. On the other hand, the molecular mass of this compound 31 is C ₈₁ H ₁₂₈ N ₁₀ 0 ₂₈ S ₂ : 1704.85.

(5) Synthesis of Ligand Complex Trivalent- (TEG-Mal) ₃ (Compound 34) (see FIGS. 16 and 17)

Compound 31 (Trivalent- (TEG-NHBoc) ₃ ) (64.7 mg, 37.9 μmol) was dissolved in dichloromethane / water (2.2 ml, 10 / l), TFA (2 ml was added at O ° C. and 2.5 at the same temperature). The reaction mixture was concentrated and then azeotropic with toluene The obtained residue (Compound 32) was used for subsequent reduction amination reaction without purification.

A description with reference to FIG. 17 is as follows. The obtained concentrated residue (Compound 32) (containing 6.95 mg, 3.77 µmol) and maltose (4.07 mg, 11.3 µmol) were dissolved in dimethylacetamide / water (1: 1, 400 µl) and left to stand at 37 ° C for 13 hours. Thereafter, acetic acid (20 µl) and sodium cyanoborohydride (2.24 mg, 35.6 µmol) were added to the reaction solution, and the mixture was left at 37 ° C for 59 hours. After freeze-drying the reaction solution, the obtained residue was purified by preparative high performance liquid chromatography (ODS column, methanol / water = 50/50)) to give Trivalent- (TEG-Mal) ₃ (compound 34) as a white solid. . The amount obtained was 4.46 mg (50%).

^L H-NMR spectrum of a compound represented by 34 (600MHz, D ₂ 0) subjected to the measurement results, δ7.05 (dd, 3H, J = 7.6, 8.2Hz, aromatic), 6.77 (s, 3H, aromatic), 6.63 ( dd, 3H, J = 1.4, 7.6 Hz, aromatic), 6.47 (dd, 3H, J = 1.4, 8.2 Hz, aromatic), 4.92 (d, 3H, J = 3.4 Hz, H-l '× 3), 4.01 (s, 6H, -0 CH ₂ CONH- × 3), 3.81 (ddd, 3H, J = 2.1, 4.8, 7.6 Hz, H-2 × 3), 3.71 (ddd, 3H, J = 4.1, 7.6 Hz, H-5 × 3), 3.74-3.68 (m, 9H, H-3 × 3, H-5 '× 3, H-6a' × 3), 3.65 (dd, 3H, J = 2.1, 12.4 Hz, H -6b '× 3), 3.64-3.60 (m, 3H, H-6a × 3), 3.64-3.42 (m, 36H, -0 CH ₂ CH ₂ 0- × 9), 3.56-3.52 (m, 6H, H-4 × 3, H-3 '× 3), 3.47-3.43 (m, 3H, H-6b × 3), 3.42-3.39 (m, 1H, CH ₂ CH (CH _2- ) (S-)) , 3.38 (dd, 3H, J = 3.4, 9.6 Hz, H-2 '× 3), 3.37 (t, 6H, J = 4.8 Hz, CONHCH ₂ CH ₂ 0- × 3), 3.26 (dd, 3H, J = 9.6, 9.6 Hz, H-4 '× 3), 3.15 (dd, 3H, J = 4.8, 13.7 Hz, H-la × 3), 3.14 (t, 6H, -CONH CH ₂ CH ₂ 0- × 3 ), 3.06 (dd, J = 7.6, 13.7 Hz, H-lb × 3), 3.01 (ddd, 1H, J = 6.2, 6.2, 11.0 Hz, -S CH2 (lH)-), 2.95 (ddd, 1H, J = 6.9, 6.9, 11.0 Hz, -S CH ₂ (lH)-), 2.24 (ddd, 1H, J = 6.2, 6.2, 12.4 Hz, SCH ₂ CH ₂ (1H)-), 2. 00 (t, 2H, J = 6.9 Hz, -NHCO CH ₂ CH _2- ), 1.96-1.92 (m, 6H, CH ₂ CH ₂ CO- × 3), 1.77-1.69 (m, 7H, -CH ₂ CH ₂ CO- × 3, -SCH ₂ CH ₂ (1H)-), 1.52-1.32 (m 4H, -COCH ₂ CH ₂ CH ₂ CH _2- ), 1.20-1.14 (m, 2H, -COCH ₂ CH ₂ CH ₂ CH _2- ). Moreover, it was m / z 1214.57 [(M + 2Na) ²⁺ ] as a result of ESI-MS measurement. These confirm the structure of Compound 34. On the other hand, the molecular mass of this compound 34 is C ₁₀₂ H ₁₇₀ N ₁₀ O ₄₉ S ₂ : 2283.06.

On the other hand, specific embodiments or examples made in the terms of the best mode for carrying out the invention are intended to clarify the technical contents of the present invention to the last, and should not be construed in consultation with only such specific examples. Various modifications can be made within the spirit and scope of the claims set forth below.

As mentioned above, the linker compound of this invention has an aromatic amino group terminal as a site | part which can introduce | transduce 3 or more units of sugar molecules. Moreover, it has S-S binding as a site | part which can couple | bond with the support for protein analysis, such as a surface plasmon resonance (SPR) sensor chip and a carrier of affinity chromatography. Furthermore, an oligoethylene oxide is provided between the disulfide group and the aromatic amino group so that nonspecific hydrophobic interaction can be suppressed as much as possible, and the length to the disulfide group provided to a metal bond can be adjusted easily.

Therefore, the use of the linker compound has an effect that two or more units of sugar molecules can be two-dimensionally arranged on the support surface with high reproducibility. In addition, since the linker compound can almost ignore the influence of nonspecific interaction with the protein, it is possible to evaluate the biological activity of the sugar molecule with high reproducibility when observing the interaction between the sugar molecule and the protein. It is also possible to form metal-sulfur bonds efficiently.

The ligand conjugate of the present invention is obtained by introducing a sugar molecule into the linker compound.

Therefore, by introducing the ligand conjugate on the surface of the protein analysis support, it is possible to arrange a plurality of sugar molecules with high reproducibility two-dimensionally, and thus, it is possible to evaluate the biological activity of the sugar molecules with high reproducibility. Moreover, it exhibits the effect which can form a metal-sulfur bond efficiently.

ADVANTAGE OF THE INVENTION According to this invention, the linker compound which can control the distance between sugar chains on a sensor chip surface, and can arrange oligosaccharide two-dimensionally with high reproducibility, and the ligand complex which introduce | transduces a sugar molecule into this linker compound can be obtained. This linker compound and ligand complex are very useful for practical use and generalization of oligosaccharide chain chips.

If the chips immobilized with oligosaccharide chains are developed as tools for analyzing the functions of sugar chains and proteins, they will not only contribute to the clarification of life phenomena involving oligosaccharide chains, but are also expected to be important technologies for drug development. Therefore, the usefulness of the present invention is considered to be high.

Claims (14)

As a linker compound used for arranging sugar molecules on the surface of a support, General formula (1)

… (One) (Wherein a, b, d, and e are each independently an integer of 0 or more and 6 or less), and X is represented by General Formula (3).

… (3) In the formula, m ¹ , m ² , m ³ , m ⁴ , p ¹ , p ² are each independently an integer of 1 to 6, or a structure represented by the general formula (4)

… (4) (Wherein q ¹ , q ² , q ³ , r ¹ , r ² , r ³ , t ¹ , t ² , t ³ , u ¹ , u ² , u ³ are each independently an integer of 0 to 6) Is represented by and when b is 0, X has a structure represented by the general formula (4), and at least one of t ¹ , t ² and t ³ is not 0. The method of claim 1, General formula (2)

… (2) (Wherein n is an integer of 1 or more and 6 or less) and has a structure shown by the linker compound characterized by the above-mentioned. delete delete The ligand conjugate which consists of introducing a sugar molecule into the aromatic amino group of the linker compound of Claim 1 or 2. Ligand complex used to arrange the sugar molecules on the surface of the support, General formula (5)

… (5) (Wherein m ¹ , m ² , m ³ , m ⁴ , n, p ¹ , p ² are each independently an integer of 1 or more and 6 or less. R ′ is hydrogen (H) or a structure represented by R.) And R is formula (6-1) to formula (6-6)

Ligand complex, characterized in that the oligosaccharide-derived compound selected from. Ligand complex used to arrange the sugar molecules on the surface of the support, General formula (7)

… (7) (Wherein a, b, d, e, q ¹ , q ² , q ³ , r ¹ , r ² , r ³ , t ¹ , t ² , t ³ , u ¹ , u ² , u ³ are each independent , the integer of 0 to 6, except when b is the time 0 days t ^1, t ² and t ³ are not 0, the t ^1, t ^2, and t ³ 0 il b is not 0. Further, R 'has a structure represented by hydrogen (H) or R.), R is formula (6-1) to formula (6-6)

Ligand complex, characterized in that the oligosaccharide-derived compound selected from. As a manufacturing method of the linker compound of Claim 1 or 2, Performing a condensation reaction of thioctic acid with an amine compound having at least three branched chains in which an aromatic amino group end is protected by a protecting group; Deprotecting the protecting group at the end of the aromatic amino group, The protected branched chain is appropriately selected to react with the chioctic acid to form the structure of X according to claim 1 or 2. A method for producing a ligand conjugate, which comprises a reduction amination reaction using a linker compound according to claim 1 or 2 and a sugar molecule. The method of claim 9, As said sugar molecule, General formula (8)

… (8) A method for producing a ligand conjugate, characterized by using a sulfated oligosaccharide having a heparin partial disaccharide structure. The method of claim 9, As said sugar molecule, group (9)

… (9) Method for producing a ligand conjugate, characterized in that at least one of the oligosaccharides selected from. As a method of introducing sugar molecules in which sugar molecules are arranged on the surface of a support, A method for introducing a sugar molecule comprising contacting a solution containing a ligand conjugate according to claim 5 with a support having a metal on the surface thereof. A ligand carrier comprising the immobilization of the ligand conjugate according to claim 5 on a support having a metal on its surface. The surface of the plasmon resonance sensor chip, which is obtained by immobilizing the ligand conjugate according to claim 5 on a surface thereof.

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