JP2009155353A - Method for producing sugar chain compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an N-bonded type sugar chain compound having a free hydroxy group at its reducing terminal by using safer hydrazine hydrate as compared with that of anhydrous hydrazine. <P>SOLUTION: This sugar chain compound of formula (2) is obtained by effecting the hydrazine hydrate on a sugar chain asparagine compound of formula (1) [wherein, R<SP>1</SP>to R<SP>3</SP>are each H or a sugar residue; R<SP>4</SP>is H or a fucose residue; R<SP>5</SP>is H, a lipid-soluble protecting group or the like; R<SP>6</SP>is carboxy or a group expressed by -CONHR<SP>7</SP>]. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a sugar chain compound having a free hydroxyl group at the reducing end by cleaving the reducing terminal CN bond of the sugar chain asparagine compound.

  A molecule in which a sugar chain is covalently bonded to a protein is called a glycoprotein. Sugar chains in glycoproteins have functions such as maintaining the three-dimensional structure of proteins, controlling solubility, and adding protease resistance. Recently, it has been revealed that sugar chains in glycoproteins are involved in life phenomena such as fertilization, differentiation, signal transduction, canceration, intracellular transport of proteins and regulation of physiological activity. Thus, sugar chains bound to proteins play an important role in various physiological functions. However, since the structures of these sugar chains are diverse and the types are enormous, it is extremely difficult to identify which structure of the sugar chain is involved in the life phenomenon. In order to elucidate these functions, it is indispensable to synthesize glycoproteins and glycopeptides having a sugar chain with a single structure.

  Glycoproteins can be divided into two groups based on the difference in binding mode between sugar and protein. One is an asparagine-linked sugar chain (N-linked type) in which the side chain amino group of asparagine (Asn) and a sugar chain are bonded. The other is a mucin-linked sugar chain (O-linked type) in which a sugar chain is bonded to the hydroxyl group of serine (Ser) or threonine (Thr).

  The present inventors established a method (Patent Document 1) for preparing a large amount of biantennary complex-type sugar chains from chicken eggs by combining an enzyme method and a chemical method, and a complex-type sugar having a free hydroxyl group at the sugar chain reducing end. It was shown that an aminated complex-type sugar chain derivative was produced from a chain, and the obtained derivative could be selectively introduced into a thiol group of a peptide (Patent Document 2).

Preparation of an N-linked sugar chain having a free hydroxyl group at the sugar chain reducing end includes a method of cleaving a sugar chain from a sugar chain asparagine and its derivatives and glycoproteins by an enzyme and a method of chemically cleaving it. Usually, as a method of chemically cleaving, a hydrazine decomposition method is used. When hydrazine is used in the presence of hydrazine hydrate or water, anhydrous hydrazine is used because the sugar on the sugar chain reducing terminal side is eliminated (β elimination).
WO 03/008431 Publication WO 2004/011036

  However, anhydrous hydrazine is a reagent that requires careful operation due to its toxicity and ignitability, and is unsuitable for a large amount of processing. Therefore, a safe and effective method for cleaving N-linked sugar chains is required. Yes.

  An object of the present invention is to provide a method for producing an N-linked sugar chain compound having a free hydroxyl group at the reducing end, using hydrazine hydrate that is safer than anhydrous hydrazine.

The present invention relates to the following inventions.
A method for producing a sugar chain compound represented by formula (2), wherein hydrazine hydrate is allowed to act on a sugar chain asparagine compound represented by formula (1).

[式中、Ｒ^１、Ｒ^２及びＲ^３は同一又は異なって水素原子、糖残基を示す。Ｒ^４は水素原子又はフコース残基を示す。Ａｃはアセチル基を示す。Ｒ^５は水素原子、脂溶性の保護基、アミノ酸残基、又はペプチド残基を示し、Ｒ^６はカルボキシル基又は基−ＣＯＮＨＲ^７を示す。Ｒ^７は、アミノ酸残基又はペプチド残基を示す。]

[Wherein, R ¹ , R ² and R ³ are the same or different and each represents a hydrogen atom or a sugar residue. R ⁴ represents a hydrogen atom or a fucose residue. Ac represents an acetyl group. R ⁵ represents a hydrogen atom, a fat-soluble protecting group, an amino acid residue, or a peptide residue, and R ⁶ represents a carboxyl group or a group —CONHR ⁷ . R ⁷ represents an amino acid residue or a peptide residue. ]

[式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４及びＡｃは前記に同じ。]

[Wherein, R ¹ , R ² , R ³ , R ⁴ and Ac are the same as above. ]

  The present inventors have used hydrazine hydrate that could not be used because of β-elimination so far, decomposed with hydrazine, subsequently substituted with benzylamine compound, purified, and then released to the reducing end by hydrolysis treatment. It has been found that an N-linked sugar chain compound having a hydroxyl group can be produced.

  According to the method of the present invention, an N-linked sugar chain compound having a free hydroxyl group at the reducing end can be produced using hydrazine hydrate that is safe for hydrazine decomposition reaction.

The sugar chain asparagine compound represented by the formula (1) of the present invention includes glycoproteins, glycopeptides or sugar chain asparagines in which the sugar chain binds to asparagine, and derivatives thereof.
The sugar chain compound represented by the formula (2) of the present invention is an N-linked sugar chain compound having a free hydroxyl group at the reducing end.

[式中、Ｒ^１、Ｒ^２及びＲ^３は同一又は異なって水素原子、糖残基を示す。Ｒ^４は水素原子又はフコース残基を示す。Ａｃはアセチル基を示す。Ｒ^５は水素原子、脂溶性の保護基、アミノ酸残基、又はペプチド残基を示し、Ｒ^６はカルボキシル基又は基−ＣＯＮＨＲ^７を示す。Ｒ^７は、アミノ酸残基又はペプチド残基を示す。]

[Wherein, R ¹ , R ² and R ³ are the same or different and each represents a hydrogen atom or a sugar residue. R ⁴ represents a hydrogen atom or a fucose residue. Ac represents an acetyl group. R ⁵ represents a hydrogen atom, a fat-soluble protecting group, an amino acid residue, or a peptide residue, and R ⁶ represents a carboxyl group or a group —CONHR ⁷ . R ⁷ represents an amino acid residue or a peptide residue. ]

[式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４及びＡｃは前記に同じ。]

[Wherein, R ¹ , R ² , R ³ , R ⁴ and Ac are the same as above. ]

  The sugar residue may be a monosaccharide such as mannose, N-acetylglucosamine, galactose, or fucose, which may be protected with a hydroxyl group and substituted with a halogen atom such as fluorine. Two or more glycoside bonds may form a sugar chain. Further, it may be a sugar chain containing sialic acid which may be substituted by halogen such as fluorine or whose carboxyl group may be protected.

  That is, the sugar chain asparagine compound represented by the formula (1) may be a conventionally known or unknown sugar chain asparagine, a high mannose type sugar chain asparagine compound, a complex type sugar chain asparagine compound, or a hybrid sugar chain asparagine compound. It may be. In addition, the sugar chain compound represented by the formula (2) may be a conventionally known or unknown sugar chain compound, and may be a high mannose type sugar chain compound, a complex type sugar chain compound, or a hybrid sugar chain compound. .

  The fat-soluble protective group is not particularly limited, and includes, for example, carbonates such as 9-fluorenylmethoxycarbonyl (Fmoc) group, t-butyloxycarbonyl (Boc) group, and allyloxycarbonate (Alloc) group. Groups, acyl groups such as acetyl (Ac) group, protecting groups such as allyl group and benzyl group. The introduction of the protecting group may be performed according to a known method such as Protecting groups in Organic chemistry (John Wiley & Sons INC., New York 1991, ISBN 0-471-62301-6).

The amino acid residue or peptide residue in R ⁵ is an amino acid or peptide in which the amino group and carboxyl group of asparagine are amide-bonded, and is not particularly limited.
The amino acid residue or peptide residue in R ⁷ is an amino acid or peptide in which the carboxyl group and amino group of asparagine are amide-bonded, and is not particularly limited.

  The hydrazine hydrate used in this step can be used as long as it is conventionally known, and hydrazine monohydrate can be used as it is or diluted with water. The concentration of hydrazine monohydrate is 20 ˜100 wt%, particularly preferably about 40 to 100 wt%.

  The amount of hydrazine hydrate used is not particularly limited and is 0.8 equivalents or more, preferably 1.0 equivalents or more, based on 1 equivalent of the sugar chain asparagine compound represented by the formula (1). Since the solvent is used for a long time, it is preferable to use a large excess amount, and it is 1 to 10000 parts by weight, preferably 100 to 5000 parts by weight with respect to 1 part by weight of the sugar chain asparagine compound represented by the formula (1). Is preferred.

  The reaction in this step is carried out under heating and at the reflux temperature. In this reaction, it is considered that the sugar chain asparagine compound represented by the formula (1) reacts with hydrazine to form a hydrazino compound represented by the formula (3). According to the study by the present inventors, although the reason is not clear, the β elimination reaction is a hydrazino in which most or all of the sugar chain asparagine compound represented by the formula (1) is represented by the formula (3). It was found to occur after conversion to a sugar chain compound. In other words, the β elimination reaction does not occur until most or all of the sugar chain asparagine compound represented by the formula (1) is converted into the hydrazino sugar chain compound represented by the formula (3). .

  Therefore, this reaction may be performed until all of the sugar chain asparagine compound represented by the formula (1) is consumed, but it is preferable to terminate the reaction before the consumption. Alternatively, the reaction is preferably terminated before the production of the hydrazino sugar chain compound represented by the formula (4) generated by the β elimination reaction occurs. The reaction is preferably carried out by following TLC or mass spectrum. The reaction can be completed by stopping heating under reflux.

[式中、Ｒ^１、Ｒ^２、Ｒ^３及びＲ^４は前記に同じ。]

[Wherein, R ¹ , R ² , R ³ and R ⁴ are the same as above. ]

[式中、Ｒ^１、Ｒ^２及びＲ^３は前記に同じ。]

[Wherein, R ¹ , R ² and R ³ are the same as above. ]

  In the reaction of this step, since the sugar chain asparagine compound represented by the formula (1) has an amide-bonded protecting group such as an acetyl group, the protecting group is eliminated by an excess of hydrazine to become an amino group. Therefore, it is necessary to N-acetylate by acting an acetylating agent.

As the acetylating agent, conventionally known acetylating agents that can be used for the N-acetylation reaction can be used, and examples thereof include acetyl halides such as acetyl chloride and acetyl bromide, and acetic anhydride. Can be suitably used. The amount of the acetylating agent used may be 1 to 20 equivalents, preferably about 1.5 to 10 equivalents, per 1 equivalent of amino group.
For the N-acetylation reaction using an acetylating agent, a conventionally known method can be applied. For example, after the excess hydrazine is distilled off from the reaction solution under reduced pressure, the acetylating agent is allowed to act in the presence of a base. Made.

  As the base, conventionally known ones can be used, and examples thereof include alkali metal carbonates such as sodium carbonate, potassium carbonate and sodium bicarbonate, and organic bases such as triethylamine and pyridine. Sodium bicarbonate and pyridine are particularly preferred. preferable. The amount of the base used is not particularly limited and can be used in an equal amount or more with respect to the acetylating agent, but is preferably used in a large excess, for example, in the case of an alkali metal carbonate such as sodium bicarbonate. As a saturated aqueous solution, 1 to 100 parts by weight can be used with respect to 1 part by weight of an acetylating agent.

  This reaction is carried out in a solvent, and examples of the solvent include water, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), tetrahydrofuran (THF) and the like. These may be used alone or in combination of two or more. A mixture may be used, and water can be preferably used. Although there is no restriction | limiting in particular as the usage-amount of a solvent, What is necessary is just to be about 10-2000 weight part normally with respect to 1 weight part of sugar_chain | carbohydrate compounds represented by Formula (3), Preferably it may be about 100-1000 weight part.

The reaction is carried out at −10 to 100 ° C., preferably 0 to 50 ° C., and is usually completed in about 0.1 to 24 hours.
The compound after the acetylation reaction is represented by the following formula (5).

［Ｒ^１〜Ｒ^４及びＡｃは上記に同じ。］

[R ^{1 to} R ⁴ and Ac are the same as above. ]

  The product containing the hydrazino sugar chain compound represented by the formula (5) obtained as described above is treated with gel filtration column chromatography to remove cleavage fragments such as asparagine residues and β-eliminated sugar residues. Can be removed.

  By treating the obtained hydrazino sugar chain compound represented by the formula (5) with an acid, a product containing the sugar chain compound represented by the formula (1) is obtained.

Examples of the acid to be used include mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid, carboxylic acids such as formic acid, acetic acid and trifluoroacetic acid, and sulfonic acids such as methanesulfonic acid and ethanesulfonic acid, among which acetic acid is safe. From the viewpoints of performance and ease of use.
The amount of the acid used is not particularly limited as long as it is 1 equivalent or more with respect to 1 equivalent of the hydrazino sugar chain compound represented by the formula (5), and 1 to 5 equivalents are preferable. Usually, it is preferable to add an acid to such an extent that the aqueous solution of the compound of formula (5) is sufficiently acidic.

The reaction may be performed at about 0 to 50 ° C., preferably about 10 to 40 ° C., and is usually completed for about 1 to 15 hours, preferably about 2 to 10 hours, but the reaction is traced by TLC or mass spectrum, It is preferable to confirm the completion.
As described above, a product containing the sugar chain compound represented by the formula (2) can be produced. However, in the above hydrazine decomposition, when the reaction is terminated at a stage where β elimination does not occur. As a raw material, the sugar chain asparagine compound represented by the formula (1) remains, and when the reaction proceeds somewhat, β-eliminated N- of the hydrazino sugar chain compound represented by the formula (4) Acetyl (6) will be mixed. Moreover, the hydrazino sugar chain represented by the formula (3) is unstable and causes β-elimination over time. Therefore, the sugar chain compound represented by the formula (2) obtained at this stage is mixed with other compounds such as the compounds (1), (4), (6).
The compound of the above formula (6) is shown below.

［Ｒ^１〜Ｒ^３及びＡｃは上記に同じ。］

[R ^{1 to} R ³ and Ac are the same as above. ]

  Next, an amine compound is allowed to act in a solvent on the mixture of the sugar chain compound represented by the formula (2) obtained above and another compound.

Examples of the amine compound include carbon atoms such as monoalkylamine having 1 to 4 carbon atoms such as methylamine, ethylamine, and isopropylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, cycloheptylamine, and cyclooctylamine. Examples thereof include 3 to 8 cycloalkylamines and benzylamines which may have a substituent. Examples of the substituent of benzylamine which may have a substituent include alkyls having 1 to 4 carbon atoms such as halogen atoms such as fluorine, chlorine and bromine, methyl groups, ethyl groups, propyl groups, isopropyl groups and tert-butyl groups. Groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups and other alkoxy groups, nitro groups, and the like. These substituents can be used alone or at any position on the phenyl ring. Includes those in which ˜5 are the same or differently substituted. Among these benzylamines, benzylamine, p-methoxybenzylamine and 2,4,5-trimethoxybenzyl can be preferably exemplified, and among them, p-methoxybenzylamine is particularly preferable.
The amount of the amine compound used is usually 1 to 20 equivalents, preferably 2 to 10 equivalents, relative to 1 equivalent of the sugar chain compound represented by the formula (2).

This reaction is preferably carried out in the presence of an acid such as camphorsulfonic acid.
The amount of the acid used may be 0.01 to 5 equivalents, preferably 0.05 to 1 equivalent, with respect to 1 equivalent of the sugar chain compound represented by the formula (1).

Examples of the solvent used in this reaction include water, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), tetrahydrofuran (THF) and the like. These may be used alone or in combination of two or more. May be.
The amount of the solvent used is not particularly limited, but is usually about 10 to 2000 parts by weight, preferably about 100 to 1000 parts by weight with respect to 1 part by weight of the sugar chain compound represented by the formula (2).

  This reaction is usually performed at 0 to 100 ° C., preferably about 10 to 50 ° C., and is usually completed in about 1 to 24 hours. However, when the reaction is followed by TLC or mass spectrum, the raw material disappears. What is necessary is just to complete reaction.

  By this reaction, an amino sugar chain compound represented by the formula (7) in which an amino compound is substituted at the reducing end of the sugar chain can be obtained. This compound is stable to a base and does not cause a β elimination reaction. The sugar chain structure can be maintained. In addition, the hydrazino sugar chain compound represented by the formula (6) mixed in the raw material of this reaction similarly reacts to give the corresponding amino-substituted compound.

[式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４及びＡｃは前記に同じ。Ｒ^８は炭素数１〜４のアルキル基、炭素数３〜８のシクロアルキル基、置換基を有することのあるベンジル基を示す。]

[Wherein, R ¹ , R ² , R ³ , R ⁴ and Ac are the same as above. R ⁸ represents an alkyl group having 1 to 4 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, or a benzyl group that may have a substituent. ]

Here, examples of the alkyl group having 1 to 4 carbon atoms of R ⁸ include a methyl group, an ethyl group, and an isopropyl group. Examples of the cycloalkyl group having 3 to 8 carbon atoms include a cyclopropyl group and a cyclobutyl group. , Cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, and the like. Examples of the substituent of the benzyl group which may have a substituent include halogen atoms such as fluorine, chlorine and bromine, alkyls having 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl and tert-butyl groups. Groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups and other alkoxy groups, nitro groups, and the like. These substituents can be used alone or at any position on the phenyl ring. Includes those in which ˜5 are the same or differently substituted. Among these benzyl groups, a benzyl group, a p-methoxybenzyl group and a 2,4,5-trimethoxy group can be preferably exemplified, and among them, a p-methoxybenzyl group is particularly preferable.

The amino sugar chain compound represented by the formula (7) can be isolated and purified by treating the amino sugar chain compound represented by the formula (7) and other compounds by column chromatography.
Separation by chromatography can be appropriately performed by using known chromatography alone or in combination, for example, purification by gel filtration chromatography and then purification by reverse phase column chromatography. Can do.
Examples of the reversed-phase column include ODS, Phenyl, nitrile, and anion exchange columns, and the amino group of the amino sugar chain compound represented by the formula (7) is ODS. It produces a strong interaction with the octadecyl group of the column and excels in resolution.
The separation conditions and the like may be appropriately adjusted with reference to known conditions.
The resulting amino sugar chain compound represented by the formula (7) is a novel compound.

The target sugar chain compound represented by the formula (2) can be obtained by allowing an acid to act on the amino sugar chain compound represented by the formula (7) isolated by the chromatography.
Examples of the acid to be used include mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and carboxylic acids such as formic acid, acetic acid and trifluoroacetic acid. Carboxylic acids are preferred, and among them, acetic acid is preferred because it is safe and simple to use.
The amount of the acid used is not particularly limited as long as it is 1 equivalent or more with respect to 1 equivalent of the amino sugar chain compound represented by the formula (7), and 1 to 5 equivalents are preferable. Usually, it is preferable to add an acid so that the aqueous solution of the compound of the formula (7) is sufficiently acidic.
The reaction may be performed at about 0 to 50 ° C., preferably about 10 to 40 ° C., and is usually completed for about 1 to 15 hours, preferably about 2 to 10 hours, but the reaction is traced by TLC or mass spectrum, It is preferable to confirm the completion.
The obtained sugar chain compound represented by the formula (2) can be purified by chromatography or the like.

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

Example 1
To 10 mg of the sugar chain asparagine compound (1-1), 10 ml of hydrazine hydrate (hydrazine 55%) was added and dissolved at room temperature. This was heated to reflux at 100 ° C. The reaction was followed by TLC (isopropanol: 1M ammonium acetate aqueous solution = 1: 1), and when the raw material disappeared on TLC, the reflux with heating was stopped. In the sugar chain asparagine compound (1-1), since the Fmoc group was released immediately after the addition of hydrazine hydrate, the sugar chain asparagine compound from which the Fmoc group was eliminated was handled as a raw material.

The reaction solution was concentrated to dryness under reduced pressure, and 1 ml of water was added to the resulting residue to dissolve it. To this aqueous solution was added sodium hydrogen carbonate powder until saturation, followed by acetic anhydride (0.1 mL). The progress of acetylation was followed by TLC (isopropanol: 1M aqueous ammonium acetate = 1.5: 1). Further, the disappearance of the raw materials was confirmed by TLC (isopropanol: 1M ammonium acetate aqueous solution = 1: 1), and neutralized by adding sodium bicarbonate powder so that the pH of the reaction solution was 7-8.
The reaction solution was concentrated to dryness under reduced pressure, and the resulting residue was dissolved in 1 ml of water and subjected to gel filtration chromatography (column carrier: Sephadex G-25, column size: φ16 mm × 345 mm, flow rate: 0.8 ml / min). The fraction containing the compound (5-1) was separated with a developing solvent: water and concentrated under reduced pressure.

  5 mg of the obtained concentrated residue was dissolved in 1 ml of water, and 572 μl of acetic acid was added to make an acetic acid aqueous solution. The mixture was stirred at room temperature, and the reaction was followed by TLC (isopropanol: 1M aqueous ammonium acetate = 1.5: 1). After 6.5 hours, the completion of the reaction was confirmed, neutralized with 1M aqueous sodium hydroxide solution, and lyophilized to obtain a powder.

The obtained powder is purified by gel filtration column chromatography (same as above), fractions containing the target compound (2-1) are collected, concentrated under reduced pressure, and compound (2-1). Got. However, contamination of the compound (1-2) derived from the unreacted raw material and the β-eliminated compound (8) was observed.
Yield: 8.9 mg [Compound (2-1): Compound (2-2): Compound (8) = 90: 8: 2]

Compound (2-1)
¹ H-NMR (400 MHz, 295 K, HOD = 4.81), 5.28 (bd, 1H, GlcNAc1-H-1), 5.23 (s, 1H, Man4-H-1), 5.03 ( s, 1H, Man4'-H-1), 4.86 (s, 1H, Man3-H-1), 4.70 (m, 3H, GlcNAc2, 5, 5'-H-1), 4.53 (D, 2H, Gal6, 6'-H-1), 4.34 (bs, 1H, Man3-H-2), 4.28 (bd, 1H, Man4-H-2), 4.20 (bd , 1H, Man4′-H-2), 2.76 (bdd, 2H, NeuAc7, 7′-H-3 eq), 2.17 (s, 3H, Ac), 2.16 (s, 6H, Ac × 2), 2.13 (s, 6H, Ac × 3), 1.80 (dd, 2H, NeuAc7, 7′-H-3ax).
Mass: ESI calcd for 2222, found; 1110 [(M-2) ⁻² ]
Compound (8)
Mass calcd for 2019, found; 1008.3 [(M-2) ⁻² ]

Example 2
45 mg of a mixture of the compound (2-1), the compound (1-2) and the compound (8) obtained in the same manner as in Example 1 was dissolved in 3 ml of DMSO. To this solution, 2 ml of p-methoxybenzylamine and 5 mg of camphor sulfonic acid were added, and the reaction was performed while maintaining the temperature at about 37 ° C. in a constant temperature layer.
The reaction was followed by mass spectrum (disappearance of 1110 / -2 peak and generation of 1170 / -2 peak) to confirm the completion of the reaction. 10 mM ammonia water was added to the reaction solution to dilute it twice, and gel filtration column chromatography (column carrier: Sephadex G-25, column size: φ10 mm × 900 mm, flow rate: 0.8 ml / min, developing solvent: 50 mM ammonium carbonate) Purification with an aqueous solution or aqueous ammonia (pH 9 to 10)), fractions containing the compound (7-1) were collected, concentrated under reduced pressure, and lyophilized to obtain a powder of the compound (7-1). However, mixing of the compound (1-2) and the compound (9) was observed.

The resulting powder 20mg dissolved in 10mM ammonium bicarbonate solution, ODS column (column carrier, which was completely replaced with 10mM ammonium bicarbonate solution: Cosmosil _75C 18 -OPN (manufactured by Nacalai Tesque, Inc.), column size: 0.75 × 0.75 × 10 cm). Thereafter, a 10 mM aqueous ammonium hydrogen carbonate solution was flowed in an amount 5 times that of the carrier to discharge the compound (1-2). Thereafter, 10 mM ammonium hydrogen carbonate aqueous solution: acetonitrile (= 98: 2) was allowed to flow 5 times the amount of the carrier, and after washing the carrier, 10 mM ammonium hydrogen carbonate aqueous solution: acetonitrile (= 96: 4) was caused to flow. After the outflow, the compound (7-1) was collected.
Yield: 9mg
Compound (7-1)
¹ H-NMR (400 MHz, HOD = 4.81), δ 5.11 (s, 1H, Man4-H-1), 4.93 (s, 1H, Man4′-H-1), 4.75 (s , 1H, Man3-H-1), 4.59 (m, 3H, GlcNAc2, 5,5'-H-1), 4.43 (d, 2H, Gal6, 6'-H-1), 4. 24 (bs, 1H, Man3-H-2), 4.18 (bd, 1H, Man4-H-2), 4.10 (bd, 1H, Man4′-H-2), 2.65 (bdd, 2H, NeuAc7, 7'-H-3eq), 1.71 (dd, 2H, NeuAc7, 7'-H-3ax).
Mass: ESI calc for 2341.8, found; 1169.9 [(M-2) ⁻² ]
Compound (9)
Mass: ESI calcd for 2139.8, found; 1068.4 [(M-2) ⁻² ]

Example 3
After adding 1 ml of water to 3 mg of the compound (7-1) obtained in Example 2 to make an aqueous solution (pH 10), about 20 μl of acetic acid was added. The pH at this time was about 4.
The reaction was followed by mass spectrum (disappearance of 1170 / -2 peak and generation of 1110 / -2 peak) to confirm the completion of the reaction. Sodium hydroxide aqueous solution was added to the reaction solution to adjust to pH 5-6, and gel filtration column chromatography (column carrier: Sephadex G-25, column size: φ10 mm × 900 mm, flow rate: 0.8 ml / min, developing solvent: water) The obtained fraction was concentrated under reduced pressure to obtain 2.5 mg of compound (2-1) having a purity (98%).
The NMR and mass spectral data of the obtained compound (2-1) were the same as those obtained in Example 1.

Claims (7)

A method for producing a sugar chain compound represented by formula (2), wherein hydrazine hydrate is allowed to act on a sugar chain asparagine compound represented by formula (1).

[Wherein, R ¹ , R ² and R ³ are the same or different and each represents a hydrogen atom or a sugar residue. R ⁴ represents a hydrogen atom or a fucose residue. Ac represents an acetyl group. R ⁵ represents a hydrogen atom, a fat-soluble protecting group, an amino acid residue, or a peptide residue, and R ⁶ represents a carboxyl group or a group —CONHR ⁷ . R ⁷ represents an amino acid residue or a peptide residue. ]

[Wherein, R ¹ , R ² , R ³ , R ⁴ and Ac are the same as above. ] The process according to claim 1, wherein the action of hydrazine hydrate is terminated before β-elimination of N-acetylglucosamine on the sugar chain reducing terminal side. (A) a step of allowing hydrazine hydrate to act on the sugar chain asparagine compound represented by formula (1),
(B) a step of allowing an acetylating agent to act;
(C) a step of allowing an acid to act,
(D) a step of acting at least one amine compound selected from monoalkylamines having 1 to 4 carbon atoms, cycloalkylamines having 3 to 8 carbon atoms and benzylamine which may have a substituent;
(E) a step of purification by column chromatography,
(F) a step of allowing an acid to act,
Are performed in this order, The manufacturing method of the sugar_chain | carbohydrate compound represented by Formula (2) characterized by the above-mentioned. The process according to claim 3, wherein the action of hydrazine hydrate (A) is terminated before β-elimination of N-acetylglucosamine on the sugar chain reducing terminal side. The production method according to claim 3, wherein the amine compound of (D) is p-methoxybenzylamine. The process according to claim 3, wherein the acid of (C) and (F) is acetic acid. An amino sugar chain compound represented by the formula (7):

[Wherein, R ¹ , R ² , R ³ , R ⁴ and Ac are the same as above. R ⁸ represents an alkyl group having 1 to 4 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, or a benzyl group that may have a substituent. ]