JP2006022191A - Three-branched sugar chain asparagine derivative, the sugar chain asparagine, the sugar chain and their preparation methods - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a three-branched sugar chain asparagine derivative wherein an amino nitrogen in asparagine is modified with a lipophilic protective group, a biotin group or an FITC group, and its preparation method. <P>SOLUTION: The three-branched sugar chain asparagine derivative wherein the amino nitrogen in asparagine is modified with a lipophilic protective group, a biotin group or an FITC group is represented by formula (1) (wherein R<SP>1</SP>and R<SP>2</SP>are different from each other and are each a hydrogen atom or galactose; and Q is a lipophilic protective group, a biotin group or an FITC group). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a 3-branched sugar chain asparagine derivative, a 3-branched sugar chain asparagine, a 3-branch sugar chain, and a method for producing them.

In recent years, sugar chain molecules have attracted attention as a third chain life molecule following nucleic acids (DNA) and proteins. The human body is a large cell society consisting of about 60 trillion cells, and all cell surfaces are covered with sugar chain molecules. For example, the ABO blood group is determined by the difference in sugar chains on the cell surface.
Sugar chains have a role in recognition and interaction between cells, and are essential to establish a cellular society. Disruption of the cellular society leads to cancer, chronic diseases, infectious diseases, aging and so on.
For example, it is known that the structural change of sugar chains occurs when cells become cancerous. In addition, it is known that Vibrio cholerae and influenza virus invade and infect cells by recognizing and binding to a specific sugar chain.

  Sugar chains are very complex structures compared to nucleic acid and protein structures due to the diversity of monosaccharide sequences, binding modes / sites, chain lengths / branching patterns, overall higher order structures, and the like. Therefore, biological information derived from the structure is more diverse than nucleic acids and proteins. While the importance of research is recognized for sugar chains, the progress of research is delayed compared to nucleic acids and proteins due to the complexity and diversity of their structures.

  E. Meinjohanns (J. Chem. Soc. Perkin Trans1, 1998, p549-560) et al. Synthesizes a bibranched sugar chain asparagine from Bovine Fetuin (bovine-derived glycoprotein). The hydrazine decomposition reaction is used to obtain the first branched raw sugar chain. This hydrazine is highly toxic, and there is a problem in terms of safety when the obtained sugar chain derivative is applied to a pharmaceutical because there is a possibility that a small amount of hydrazine is mixed.

An object of the present invention is to provide a 3-branched sugar chain asparagine derivative in which the amino group nitrogen of asparagine is modified with a lipophilic protecting group, biotin group or FITC group, and a method for producing the same.
Another object of the present invention is to provide a 3-branched sugar chain asparagine, a 3-branched sugar chain, and a method for producing them.

The present invention relates to the following inventions.
1. A three-branched sugar chain asparagine derivative in which the amino group nitrogen of the asparagine represented by the formula (1) is modified with a fat-soluble protective group, biotin group or FITC group.

〔式中、Ｒ^１およびＲ^２は異なって、水素原子あるいはガラクトースを示す。Ｑは脂溶性の保護基、ビオチン基またはＦＩＴＣ基を示す。〕

[Wherein, R ¹ and R ² are different and each represents a hydrogen atom or galactose. Q represents a fat-soluble protective group, biotin group or FITC group. ]

2. A tribranched sugar chain from which an asparagine residue of the three-branched sugar chain asparagine of formula (2) is removed.

〔式中、Ｒ^１およびＲ^２は異なって、水素原子あるいはガラクトースを示す。〕

[Wherein, R ¹ and R ² are different and each represents a hydrogen atom or galactose. ]

3. A microplate to which a biotinylated 3-branched sugar chain asparagine derivative is bound.
4). An affinity column to which a biotinylated tri-branched sugar chain asparagine derivative is bound.

  The inventor of the present invention can obtain various isolated three-branched sugar chain asparagine derivatives from Bovine Fetuin (bovine-derived glycoprotein) easily, in large quantities and with high purity, A method for producing tri-branched sugar chains has been developed.

According to the present invention, an isolated three-branched sugar chain asparagine derivative useful in the field of drug development or the like can be obtained very easily and in a large amount as compared with the conventional one. Further, according to the present invention, isolated three-branched sugar chain asparagine and three-branched sugar chains useful together with the three-branched sugar chain asparagine derivative can be obtained very easily and in a large amount, respectively, as compared with the prior art.
Furthermore, according to the present invention, a sugar chain microchip can be easily produced by simply reacting a plurality of biotinylated sugar chains on an avidinized microplate using the binding specificity of biotin / avidin. . Thereby, a protein having a binding ability with a specific sugar chain can be elucidated.
In addition, for the purpose of separating and purifying a specific protein, a specific biotinylated sugar chain is bound and immobilized on an avidinized affinity column, and a protein having a specific binding ability with the biotinylated sugar chain is contained therein. Only the protein of interest can be isolated by passing through the mixture.

  The method for producing a three-branched sugar chain asparagine derivative of the present invention includes, for example, the three-branched sugar chain asparagine derived from a natural glycoprotein, preferably a sugar chain asparagine mixture obtained from an asparagine-linked sugar chain. One major feature is that a lipid-soluble protective group is introduced (bonded) to a sugar chain asparagine to obtain a mixture of three-branched sugar chain asparagine derivatives, and then the mixture is separated into each three-branched sugar chain asparagine derivative. To do. In the present specification, “sugar chain asparagine” refers to a sugar chain in which asparagine is bound. An “asparagine-linked sugar chain” is a group of sugar chains in which N-acetylglucosamine present at the reducing end is bonded to an acid amino group of asparagine (Asn) in a protein polypeptide by N-glycoside, (Β1-4) GlcNac (β1-4) A group of sugar chains having GlcNac as a mother nucleus. The “sugar chain asparagine derivative” refers to a sugar chain asparagine in a state where a lipophilic protecting group is bonded to an asparagine residue. In the structural formulas of the compounds, “AcHN” represents an acetamide group.

  As described above, a sugar chain derived from a natural glycoprotein is a mixture of sugar chains in which sugar residues at the non-reducing terminal are randomly deleted. The present inventors unexpectedly introduced a fat-soluble protective group into a sugar chain derived from a natural glycoprotein, specifically, the three-branched sugar chain asparagine contained in a mixture of sugar chain asparagine, It has been found that a mixture of three-branched sugar chain asparagine derivatives introduced with the protecting group can be easily separated into individual three-branched sugar chain asparagine derivatives using a known chromatographic technique. As a result, it was possible to prepare a large amount of three-branched sugar chain asparagine derivatives having the structure of the present invention.

  As described above, by introducing a fat-soluble protective group into the three-branched sugar chain asparagine and derivatizing it, it was possible to separate individual three-branch sugar chain asparagine derivatives. By introducing a group, the overall lipophilicity of the three-branched sugar chain asparagine derivative is increased, and for example, the interaction with a suitably used reversed-phase column is greatly improved, and as a result, the sugar chain is more sensitive. This is considered to be due to the separation of individual three-branched sugar chain asparagine derivatives reflecting the difference in structure. For example, the Fmoc group, which is a fat-soluble protective group preferably used in the present invention, has a very high fat solubility. That is, the fluorenyl skeleton of the Fmoc group has a highly lipophilic structure in which two benzene rings are bonded to the central 5-membered ring. For example, the fluorenyl skeleton of the ODS column, which is one of the reversed-phase columns, is used. It is considered that a very strong interaction with the octadecyl group was made possible, and it was possible to separate a tri-branched sugar chain asparagine derivative having a similar structure.

  Furthermore, according to the present invention, as described later, various three-branched sugar chain asparagines can be obtained by removing the protecting group of the obtained three-branched sugar chain asparagine derivative, and the obtained three-branched sugar chain asparagine can also be obtained. By removing the asparagine residue of asparagine, various three-branched sugar chains can be obtained easily and in large quantities.

Specifically, the method for producing a tri-branched sugar chain asparagine derivative modified with a fat-soluble protecting group of the present invention includes:
(A) A three-branched sugar chain asparagine derivative mixture is obtained by introducing a fat-soluble protective group into the three-branched sugar chain asparagine contained in a mixture containing one or more kinds of three-branched sugar chain asparagines. And (b) the mixture of the three-branched sugar chain asparagine derivative or the mixture obtained by hydrolyzing the three-branched sugar chain asparagine derivative contained in the three-branched sugar chain asparagine derivative mixture is subjected to chromatography. Separating each of the three-branched sugar chain asparagine derivatives.

  The mixture containing one or two or more types of three-branched sugar chain asparagine used in step (a) is particularly limited as long as it is a mixture containing one or more sugar chains in a state where asparagine is bound. It is not something. For example, it may be a mixture containing one or more sugar chains in which one or more asparagines are bonded. Among these, from the viewpoint of easy availability, a mixture containing one or more sugar chains having asparagine bonded to the reducing end is preferable. In the present specification, “sugar chain” refers to a combination of two or more arbitrary monosaccharides.

  Such a mixture of three-branched sugar chain asparagine is obtained by a known method, preferably a mixture of glycoproteins and / or glycopeptides from natural raw materials, for example, bovine-derived futuin, and, for example, a proteolytic enzyme, For example, by adding an enzyme such as pronase (manufactured by Wako Pure Chemical Industries), actinase-E (manufactured by Kaken Pharmaceutical Co., Ltd.), general carboxypeptidase, aminopeptidase, etc. Cleave and use components other than sugar chain asparagine as a reaction solution after the reaction or a known method such as gel chromatography column, ion exchange column, etc., or high performance liquid chromatography (HPLC It can be obtained by removing according to the purification method using).

  Using the mixture containing the three-branched sugar chain asparagine obtained as described above, a lipophilic protecting group is introduced into the three-branched sugar chain asparagine contained therein. The protective group is not particularly limited, and examples thereof include carbonate-type or amide-type such as Fmoc group, t-butyloxycarbonyl (Boc) group, benzyl group, allyl group, allyloxycarbonate group, and acetyl group. A protecting group or the like can be used. From the viewpoint that the obtained three-branched sugar chain asparagine derivative can be used immediately for the synthesis of a desired glycopeptide, the protective group is preferably an Fmoc group or a Boc group, and more preferably an Fmoc group. The Fmoc group is particularly effective when sugars such as sialic acid that are unstable to relatively acidic conditions are present in the sugar chain. In addition, the protective group may be introduced according to a known method (see, for example, Protection groups in Organic Chemistry, John Wiley & Sons Inc., New York 1991, ISBN 0-471-63031-6).

For example, when an Fmoc group is used, water and DMF are added in an appropriate amount to a mixture containing a 3-branched sugar chain asparagine, and then 9-fluorenylmethyl-N-succinimidyl carbonate and sodium bicarbonate are added. The Fmoc group can be introduced into the asparagine residue of the three-branched sugar chain asparagine by dissolving and performing a Fmoc group binding reaction to the asparagine residue at 25 ° C.
By the above operation, a mixture of tri-branched sugar chain asparagine derivatives into which a fat-soluble protecting group has been introduced is obtained.

  Subsequently, the three-branched sugar chain asparagine derivative mixture into which the fat-soluble protecting group has been introduced is subjected to known chromatography, particularly preparative chromatography, to separate each three-branched sugar chain asparagine derivative. Separation of each tri-branched sugar chain asparagine derivative by chromatography can be carried out by appropriately using known chromatography alone or in combination.

  For example, the obtained three-branched sugar chain asparagine derivative mixture is purified by gel filtration column chromatography and then purified using HPLC. As a column that can be used in HPLC, a reverse phase column is suitable, for example, an ODS, Phenyl type, nitrile type, anion exchange type column, specifically, for example, a mono Q column manufactured by Pharmacia, an eartron A company-made Iatro beads column etc. can be used. The separation conditions and the like may be appropriately adjusted with reference to known conditions. Through the above operation, each desired three-branched sugar chain asparagine derivative can be obtained from the three-branched sugar chain asparagine derivative mixture.

  In addition, by transferring various sugars (for example, sialic acid) to the various three-branched sugar chain asparagine derivatives obtained above with a glycosyltransferase, the three-branched sugar chain asparagine derivatives having a desired sugar chain structure are transferred. Can be obtained efficiently. For example, by transferring sialic acid with a glycosyltransferase, a three-branched sugar chain asparagine derivative having a desired sugar chain structure containing sialic acid can be obtained. In addition, depending on the glycosyltransferase used, a three-branched sugar chain asparagine derivative having a desired sugar chain structure with a different binding mode can be obtained.

As sialic acid, commercially available sialic acid or chemically synthesized sialic acid can be used.
As the sialyltransferase, commercially available ones, naturally-derived ones, and ones produced by gene recombination can be used, and can be appropriately selected depending on the type of sialic acid to be transferred. Specific examples include those derived from Rat Recombinant, which is an α2,3 transferase, and those derived from Rat Liver, which is an α2,6 transferase. Alternatively, the sialic acid may be transferred by shifting the equilibrium by adjusting pH using a sialic acid hydrolase.

In addition, the present invention provides a method for producing a tribranched sugar chain asparagine that can obtain various isolated three-branched sugar chain asparagines in large quantities. The method includes a step of removing a protecting group from the obtained 3-branched sugar chain asparagine derivative, following the step of producing a 3-branched sugar chain asparagine derivative according to the method for producing a 3-branched sugar chain asparagine derivative. It is a waste.
Removal of the protecting group from the tri-branched sugar chain asparagine derivative can be performed according to a known method (for example, Protection groups in Organic Chemistry, John Wiley & Sons Inc., New York 1991, ISBN 0-471-301- 6). For example, when the protecting group is an Fmoc group, the Fmoc group can be removed by adding morpholine to a three-branched sugar chain asparagine derivative in N, N-dimethylformamide (DMF). The Boc group can be removed by reacting a weak acid. After removal of the protective group, a three-branched sugar chain asparagine is obtained by purifying by a known method, for example, various chromatography using a gel filtration column, an ion exchange column, etc., or separation by HPLC as appropriate. May be.

  When the protecting group is a benzyl group, the removal of the benzyl group can be performed according to a known method (for example, Protection groups in Organic Chemistry, John Wiley & Sons Inc., New York 1991, ISBN 0-471). 62301-6).

In the present invention, there is provided a sugar chain asparagine obtained by biotinylation or FITC conversion of the amino group nitrogen of the three-branched sugar chain asparagine.
The biotinylation is to generate an amide bond by a reaction between the amino group of the tri-branched sugar chain asparagine and the carboxyl group of biotin (vitamin H).
The FITC conversion is to form an isothiamide bond by a reaction between the amino group of the three-branched sugar chain asparagine and the isothiocyanate group of fluorescein isothiocyanate (FITC).

In addition, the present invention provides a method for producing a sugar chain asparagine in which the amino group nitrogen of the three-branched sugar chain asparagine is biotinylated or FITC-converted.
The biotinylated or FITC-modified tri-branched sugar chain asparagine production method of the present invention can be obtained, for example, by biotinylating or FITC-converting the amino group nitrogen of the asparagine of the isolated three-branched sugar chain asparagine described above. be able to.
Biotinylation can be performed according to a known method. For example, a three-branched sugar chain asparagine is dissolved in water, sodium bicarbonate is added thereto, dimethylformamide in which D-(+)-biotinyl succinimide is dissolved is added, and the mixture is reacted at room temperature for 20 minutes and purified by a gel filtration column or the like. Thus, biotinylated tri-branched sugar chain asparagine can be obtained.

The microplate to which the biotinylated three-branched sugar chain asparagine of the present invention is bound is produced by reacting a biotinylated three-branched sugar chain asparagine with a commercially available avidinized microplate (for example, manufactured by Pierce). can do.
The affinity column to which the biotinylated tri-branched sugar chain asparagine of the present invention is bound can be produced by reacting a biotinylated tri-branched sugar chain asparagine with a commercially available avidinized affinity column.
The FITC conversion of the present invention can be performed according to a known method. For example, three-branched sugar chain asparagine is dissolved in water, acetone and sodium bicarbonate are added thereto, fluorescein isothiocyanate is added thereto, reacted at room temperature for 2 hours, purified with a gel filtration column, etc. Type sugar chain asparagine can be obtained.

The FITC tribranched sugar chain asparagine obtained in the present invention is useful for, for example, the study of receptors that recognize saccharides in living tissues and the study of the sugar binding specificity of lectins.
Furthermore, the removal of asparagine residues from the three-branched sugar chain asparagine can be performed according to a known method. For example, a tribranched sugar chain can be obtained by reacting tribranched sugar chain asparagine with anhydrous hydrazine and then acetylating to remove the asparagine residue. In addition, the asparagine residue can be removed by heating and refluxing the tribranched sugar chain asparagine with a basic aqueous solution, followed by acetylation to obtain a three-branched sugar chain. After removing the asparagine residue, it may be purified by a known method, for example, various chromatography using a gel filtration column, an ion exchange column or the like, or a method of separation by HPLC, if desired.
Thus, according to the present invention, a three-branched sugar chain asparagine derivative, a three-branched sugar chain asparagine, and a three-branched sugar chain (hereinafter sometimes referred to as three sugar chains) are inexpensive and efficient. Can be manufactured in large quantities.

  Such sugar chains are very useful in fields such as drug development. For example, an application example in drug development is, for example, cancer vaccine synthesis. It is known that when cells become cancerous, sugar chains that were not found in the body are expressed. It is also known that when the sugar chain is chemically synthesized and administered to an individual as a vaccine, cancer growth is suppressed. Therefore, if sugar chains can be produced according to the present invention, it is possible to synthesize a vaccine effective for cancer treatment. In addition, it is also possible to synthesize sugar chains obtained by the present invention by combining a chemical reaction and a reaction with glycosyltransferase to derivatize a new sugar residue and synthesize a new vaccine. is there.

  In addition, for example, erythropoietin (EPO), which is a glycoprotein, has been used as a therapeutic agent for anemia due to its ability to proliferate red blood cells, but it has been found that this EPO is not active unless a sugar chain is bound. . As described above, some proteins express physiological activity by binding to a sugar chain. For example, a large amount of protein is prepared using an E. coli expression system that cannot bind a sugar chain, and then a desired sugar chain structure is prepared. Introducing a three-branch type sugar chain produced according to the present invention, which imparts the expression of physiological activity by introducing a sugar chain produced according to the present invention, or has various sugar chain structures in any protein By doing so, it is also possible to synthesize a novel glycoprotein having a new physiological activity.

  It is also possible to impart new physiological activity by substituting the sugar chain present in the natural glycoprotein with the three-branched sugar chain produced according to the present invention. As a technique for replacing the sugar chain of a glycoprotein with the three-branched sugar chain obtained by the present invention, for example, P.I. Sears and C.I. H. The method described in Wong, Science, 2001, vol291, p2344-2350 can be mentioned. That is, the glycoprotein is treated with β-N-acetylglucosaminidase (Endo-H) so that only one N-acetylglucosamine residue is bound to the asparagine residue on the protein surface. Next, there is a method in which the desired three-branched sugar chain obtained by the present invention is bound to the N-acetylglucosamine residue using β-N-acetylglucosaminidase (Endo-M). In addition, after combining N-acetylglucosamine with tRNA and synthesizing a glycoprotein having an N-acetylglucosamine residue using an expression system such as Escherichia coli, the desired three-branched sugar obtained by the present invention is used. It is also possible to introduce the strand using Endo-M.

  Further, as a problem when using glycoprotein as a therapeutic agent at present, the metabolic rate of administered glycoprotein is high. This is because the glycoprotein is metabolized by the liver as soon as the sialic acid present at the end of the sugar chain of the glycoprotein is removed in vivo. Therefore, it is necessary to administer a certain amount of glycoprotein. Therefore, by producing a sugar chain in which sialic acid that is difficult to remove at the end of the sugar chain is newly incorporated according to the present invention and introducing the sugar chain into the target protein using Endo-M, the glycoprotein in vivo It is possible to control the metabolic rate of the protein, and to reduce the amount of glycoprotein to be administered.

  Examples will be described below, but the present invention is not limited to these examples.

Example 1
After FETUIN (manufactured by SIGMA, 1 g) is dissolved in a phosphate buffer (pH = 7.0, 20 ml), NaN ₃ (10 mg) is added. To this, orientase ONS (manufactured by HBI, 100 mg) is added and allowed to stand at 50 ° C. for about 24 hours. After confirming the completion of the reaction by TLC, the reaction solution is filtered through Celite. The filtrate is reduced by concentration and purified by gel filtration column chromatography (Sephadex G-25, 2.5 × 100 cm, H ₂ O). Fractions containing the desired sugar are collected and concentrated, followed by lyophilization. To the obtained residue (about 400 mg), Tris-hydrochloric acid / calcium chloride buffer solution (pH = 7.5, 20 ml) and NaN ₃ (20 mg) are added and dissolved. To this, actinase E (40 mg) is added and allowed to stand for 24 hours while checking the pH every 12 hours. Here, actinase E (20 mg) is further added and allowed to stand for 48 hours while checking the pH every 12 hours. After confirming the completion of the reaction by TLC, celite filtration is performed, the filtrate is concentrated and reduced, and purified by gel filtration column chromatography (Sephadex G-25, 2.5 × 100 cm, H ₂ O). Fractions containing the desired sugar are collected and concentrated, followed by lyophilization.

To the obtained residue (about 250 mg), water (1.25 ml) and sodium bicarbonate (66 mg) are added and dissolved. The reaction mixture is ice-cooled, and DMF (1.75 ml) is added dropwise. Furthermore, Fmoc-OSu (130 mg) was added and reacted at room temperature for 2 hours. After confirming disappearance of the raw material by TLC (isopropanol: 1M aqueous ammonium acetate solution = 3: 2), the reaction solution is slowly dropped into acetone (about 60 ml). The precipitated crystals are filtered off and dried. The crystals are dissolved in water and then purified once with an ODS column (Cosmo Seal 75C-OPN, nacalai quest). After the fractions containing the target sugar chain group are confirmed by HPLC, they are collected and concentrated. This solution was purified with an HPLC preparative column (YMC-Pack R & D ODS, D-ODS-5-A, 20 × 250 mm, AN / 25 mM AcONH ₄ buffer = 18/82, 7.5 ml / min., Wave length; 274 nm). The glycan group (1) coming out in the vicinity of 15 minutes and the glycan group (2) coming out in the vicinity of 20 minutes are collected, concentrated, and then desalted with an ODS column. After separation, this is desalted with an ODS column (Cosmo Seal 75C-OPN, nacalai tesque), concentrated and freeze-dried.

Each sugar chain group is dissolved to a concentration of 2% with 40 mM HCl solution, and reacted at 50 ° C. for about 34 hours while monitoring the reaction by HPLC. After confirming that the raw materials almost disappeared, each was neutralized with a saturated aqueous sodium bicarbonate solution under ice cooling. The reaction solution is filtered through a membrane filter and then concentrated. Each solution was purified with a HPLC preparative column (YMC-Pack R & D ODS, D-ODS-5-A, 20 × 250 mm, AN / 25 mM AcONH ₄ buffer = 18/82, 7.5 ml / min., wave length; 274 nm).
From the sugar chain group (1), a peak obtained after 37 minutes and from the sugar chain group (2), a peak obtained after 37 minutes were collected and concentrated, respectively, and then an ODS column (Cosmosil 75C-OPN, When the salt is desalted by nasalai tesque), and concentrated and freeze-dried, compound 1 is about 3.8 mg from sugar chain group (1), and compound 8.3 is about 8.3 mg from sugar chain group (2). Obtained.

The ¹ H-NMR data of the obtained compound is as follows.
¹ H NMR (400 MHz, D ₂ O, 30 ° C., HOD = 4.81)
7.96, 7.76 (each d, each 2H, Fmoc), 7.55, 7.48 (each t, each 2H, Fmoc), 5.17 (s, 1H, Man-H1), 5.05 (D, 1H, GlcNAc-H1), 4.98 (s, 1H, Man-H1), 4.66-4.58 (m, 4H, GlcNAc-H1 × 4), 4.52 (bd, 3H, Gal-H1 × 3), 4.37 (bt, 1H, Fmoc), 4.26 (bs, 2H, Man-H2 × 2), 4.16 (d, 1H, Man-H2), 2.78, 2.59 (each bdd, each 1H, Asn-CH ₂ ), 2.13, 2.10 (each, each 6 H, Ac × 4), 1.94 (bs, 3H, Ac)

The ¹ H-NMR data of the obtained compound is as follows.
¹ H NMR (400 MHz, D ₂ O, 30 ° C., HOD = 4.81)
7.98, 7.77 (each d, each 2H, Fmoc), 7.57, 7.49 (each t, each 2H, Fmoc), 5.17 (s, 1H, Man-H1), 5.06 (D, 1H, GlcNAc-H1), 4.98 (s, 1H, Man-H1), 4.67-4.50 (m, 4H, GlcNAc-H1 × 4), 4.54, 4.52, 4.50 (each d, each 1H, Gal-H1 × 3), 4.40 (bt, 1H, Fmoc), 4.27 (bs, 2H, Man-H2 × 2), 4.17 (d, 1H , Man-H2), 2.78,2.59 ( each bdd, each 1H, Asn-CH 2), 2.13,2.11 (each s, each 6H, Ac × 4), 1.95 (bs , 3H, Ac)

Example 2 (Biotinylation)
240 μL of N, N-dimethylformamide and 160 μl of morpholine were added to 1 μmol of the sugar chain asparagine Fmoc obtained in Example 1, and reacted at room temperature in an argon atmosphere. After confirming the completion of the reaction by TLC (using 1M ammonium acetate: isopropanol = 8: 5 as a developing solvent), it was cooled with ice water. Diethyl ether was added in an amount 10 times that of the reaction solution and stirred for 15 minutes, and then the deposited precipitate was filtered off. The resulting residue was dissolved in water and evaporated at 35 ° C. Further, the operation of adding 3 ml of toluene and evaporating was repeated three times. The residue was purified by gel column chromatography (Sephadex G-25, H ₂ O) to obtain a sugar chain asparagine from which the Fmoc group had been removed.
The compound obtained above (6 mg, 2.58 μmol) was dissolved in water (300 μl) and sodium bicarbonate (2.1 mg, 24.9 μmol) was added. To this was added dimethylformamide (300 μl) in which D-(+)-biotinylsuccinimide (4.2 mg, 12.3 μmol) was dissolved, and the mixture was reacted at room temperature for 20 minutes. After confirming the disappearance of the raw materials by TLC (isopropanol: 1M aqueous ammonium acetate solution = 3: 2), the mixture was concentrated using an evaporator. The residue was purified by gel column chromatography (Sephadex G-25, H ₂ O) to obtain the following biotinylated tri-branched sugar chain asparagine derivative (6.2 mg, 94%).

Example 3 (Biotinylation)
Biotinylation was performed in the same manner as in Example 2 except that Compound 1 was changed to Compound 2.

Example 4 (FITC conversion)
In the same manner as in Example 2, the sugar chain asparagine from which the Fmoc group was removed was obtained.
The compound obtained above (1.4 mg, 0.60 μmol) was dissolved in 70 μl of purified water. Acetone 70 μl and NaHCO ₃ (0.76 mg, 9 μmol) were added to this, and the mixture was stirred at room temperature, and then fluorescein isothiocyanate (FITC, 0.95 mg, 2.4 mmol, manufactured by SIGMA) was added and stirred for about 2 hours. After confirming the completion of the reaction by TLC after 2 hours, acetone was distilled off under reduced pressure, and the remaining aqueous solution was purified by gel column chromatography (Sephadex G-25, H ₂ O) to contain the desired product. Collected fractions. After the collected fractions were concentrated, HPLC (YMC-Pack ODS-AM, SH-343-5AM, 20 × 250 mm, AN / 25 mM AcONH ₄ buffer = 10/90, 7.5 ml / min., Wave length; 274 nm) After separation, the solution was concentrated and then desalted by gel column chromatography (Sephadex G-25, H ₂ O). Fractions containing the desired product are collected, concentrated and lyophilized to obtain the following fluorescently labeled (FITC-modified) 3-branched sugar chain asparagine derivative (1.2 mg, 73.5% yield). It was.

Example 5 (FITC conversion)
The FITC was formed in the same manner as in Example 4 except that Compound 1 was changed to Compound 2.

Example 6 (Production of microplate)
To 96-well BD biocoat streptavidin (BD Biosciences: bioassay, binding ability: 5 ng / well) 100 μg of biotinylated sugar asparagine of Example 2 (equivalent to about 10 times the biotin binding ability) Dissolved in distilled water to 1000 μl. This adjustment solution was added to each well so that it might become 10 microliters per hole, and it wash | cleaned 3 times with distilled water, and manufactured the microplate. The binding yield of each biotinylated sugar chain asparagine was 95% or more. The confirmation of the immobilization rate was calculated from the remaining unimmobilized sugar chain remaining.

Example 7 (Production of microplate)
A microplate was produced in the same manner as in Example 6 except that the compound obtained in Example 2 was changed to the compound obtained in Example 3.

Example 8 (Production of affinity column)
10 ml of avidin-coated beads (Hitachi Software Engineering Co., Ltd., xMAP LumAvidin Development Microspheres 1 ml) and 30 mg of the biotinylated sugar chain asparagine of Example 2 were stirred in a slurry state, and the beads were filtered and washed. Washing was performed on the filter paper three times using distilled water twice the volume of the beads. The confirmation of immobilization was confirmed by the remaining amount of biotinylated sugar chain asparagine recovered by washing. Next, 10 ml of the above-mentioned biotinylated sugar chain asparagine-fixed beads were packed in a glass open chromatographic column (φ20 mm, length 300 mm) in a slurry state with 30 ml of distilled water to produce an affinity column.

Example 9 (Production of affinity column)
An affinity column was produced in the same manner as in Example 8, except that the compound obtained in Example 2 was changed to the compound obtained in Example 3.

Claims (11)

A three-branched sugar chain asparagine derivative in which the amino group nitrogen of the asparagine represented by the formula (1) is modified with a fat-soluble protective group, biotin group or FITC group.

[Wherein, R ¹ and R ² are different and each represents a hydrogen atom or galactose. Q represents a fat-soluble protective group, biotin group or FITC group. ] The three-branched sugar chain sparagin derivative according to claim 1, wherein the fat-soluble protecting group is an Fmoc group. A three-branched sugar chain asparagine represented by the formula (2).

[Wherein, R ¹ and R ² are different and each represents a hydrogen atom or galactose. ] A tribranched sugar chain from which an asparagine residue of the three-branched sugar chain asparagine of formula (2) is removed. (A) A three-branched sugar chain asparagine derivative mixture is obtained by introducing a fat-soluble protective group into the three-branched sugar chain asparagine contained in a mixture containing one or more kinds of three-branched sugar chain asparagines. And (b) the mixture of the three-branched sugar chain asparagine derivative or the mixture obtained by hydrolyzing the three-branched sugar chain asparagine derivative contained in the three-branched sugar chain asparagine derivative mixture is subjected to chromatography. 3. The three-branched sugar chain asparagine derivative according to claim 1, wherein the three-branched sugar chain asparagine derivative is introduced, wherein a fat-soluble protecting group is introduced (Q is a fat-soluble protecting group). ) Manufacturing method. A method for producing a 3-branched sugar chain asparagine derivative modified with a biotin group, wherein the 3-branched sugar chain asparagine of formula (2) is biotinylated. A method for producing a 3-branched sugar chain asparagine derivative modified with a FITC group, wherein the 3-branched sugar chain asparagine of formula (2) is converted to FITC. A method for producing a three-branched sugar chain asparagine, comprising removing a lipophilic protecting group, biotin group or FITC group of the three-branch sugar chain asparagine derivative according to claim 1. A method for producing a 3-branched sugar chain, comprising removing an asparagine residue of the 3-branched sugar chain asparagine of formula (2). A microplate to which the biotinylated tri-branched sugar chain asparagine derivative according to claim 1 is bound. An affinity column to which the biotinylated tri-branched sugar chain asparagine derivative according to claim 1 is bound.