WO2007114482A1 - Process for production of sugar chain compound - Google Patents

Abstract

Disclosed is a process for production of a sugar chain compound represented by the formula (2), wherein the process comprises reacting a sugar chain asparagine compound represented by the formula (1) with hydrazine hydrate. (1) [wherein R1, R2 and R3 independently represent a hydrogen atom or a sugar residue; R4 represents a hydrogen atom or a fucose residue; Ac represents an acetyl group; R5 represents a hydrogen atom, a lipid-soluble protecting group, an amino acid residue or a peptide residue; and R6 represents a carboxyl group or a group -COR7 where R7 represents an amino acid residue or a peptide residue.] (2) [wherein R1, R2, R3, R4 and Ac are as defined above.]

Description

 Method for producing sugar chain compound

Technical field

 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 end C-covalent bond of the sugar chain asparagine compound.

 Background art

 book

 A molecule in which a sugar chain is covalently bonded to a protein is called a glycoprotein. Sugar chains in glycoproteins play a role in 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 vital phenomena such as fertilization, differentiation, signal transduction, canceration, intracellular transport of proteins and regulation of physiological activities. 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 life phenomena. In order to elucidate these functions, it is essential to synthesize glycoproteins and glycopeptides with a single-structure sugar chain.

 Glycoproteins can be divided into two groups based on the differences in the manner of binding between sugar and protein. One is an asparagine-linked sugar chain (N-linked) in which the side chain amino group of asparagine (A sn) is linked to a sugar chain. The other is a mucin-linked sugar chain (O-bonded type) in which a sugar chain is bound to the hydroxyl group of serine (Se r) or threonine (T h r).

The inventors of the present invention established a method (Patent Document 1) for preparing a large amount of biantennary complex type glycans from chicken eggs by combining an enzyme method and a chemical method. It was shown that an aminated complex-type sugar chain derivative was produced from a complex-type sugar chain possessed, and that 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 reducing end of the sugar chain includes a method of cleaving the sugar chain from a sugar chain asparagine and its derivatives and glycoproteins by an enzyme and a method of chemically cleaving it. Usually, a hydrazine decomposition method is used as a method of chemically cleaving. When hydrazine is used in the presence of hydrazine hydrate or water, saccharide on the reducing end side of the sugar chain is eliminated (eliminated), so anhydrous hydrazine is used.

 [Patent Document 1] W 0 0 3/0 0 8 4 3 1

 [Patent Document 2] W 0 2 0 0 4/0 1 1 0 3 6

 However, anhydrous hydrazine requires careful handling due to its toxicity and ignitability, and is unsuitable for large-scale 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. Disclosure of the invention

 The present invention relates to the following inventions.

 A method for producing a sugar chain compound represented by the formula (2), wherein hydrazine hydrate is allowed to act on the sugar chain asparagine compound represented by the 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 a acetyl group. R ⁵ is hydrogen atom, fat Represents a soluble protecting group, an amino acid residue, or a peptide residue, and R ⁶ represents a force lpoxyl group or a group C OR 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 present inventors used hydrazine hydrate that could not be used because of / 3 elimination until now, hydrazine decomposition, followed by substitution with benzylamine compound, purification, and hydrolysis to the reducing end. It has been found that an N-linked sugar chain compound having a free hydroxyl group can be produced.

 The sugar chain asparagine compound represented by the formula (1) of the present invention includes glycoproteins, glycopeptides, sugar chain asparagine and their derivatives, etc., in which the sugar chain binds to asparagine.

 The sugar chain compound represented by the formula (2) of the present invention is an N 1-linked sugar chain compound having a free hydroxyl group at the reducing end.

[Wherein, RR 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 a acetyl group. R ⁵ represents a hydrogen atom, a lipophilic protecting group, an amino acid residue, or a peptide residue, and R ⁶ represents a force lpoxyl group or a group —COR 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 sugar residue may be a monosaccharide such as mannose, N-acetyl darcosamine, galactose, or fucose, which may have a hydroxyl group protected, may be substituted with a halogen atom such as fluorine, Two or more of these monosaccharides may be bonded with daricoside to form a sugar chain. Further, it may be a sugar chain containing a sialic acid which may be substituted with a halogen such as fluorine, and whose strong lpoxyl 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, 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 lipophilic protecting group is not particularly limited. For example, 9-fluorenyl methoxycarbonyl (Fmo c) group, t-butyloxycarbonyl (Bo c) group, aryloxycarbonate (A 1 1 oc ) Groups such as carbonate-containing groups, acetyl groups such as acetyl (Ac) groups, protecting groups such as allyl groups and benzyl groups. 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 an amino group of asparagine and a force loxyl group 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 carboxy 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 as follows. It may be about 20 to 100% by weight, particularly preferably about 40 to 100% by weight.

 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). A large excess is preferably used because it is used as a solvent. 1 to 100 parts by weight, preferably 1 to 100 parts by weight per 1 part by weight of the sugar chain asparagine compound represented by the formula (1) It is preferably 5 0 0 parts by weight.

 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, the reason is not clear, but the / 3 elimination reaction is represented by the formula (3) 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 hydrazino sugar chain compound. In other words, S elimination reaction does not occur until most or all of the sugar chain asparagine compound represented by the formula (1) is converted to the hydrazino sugar chain compound represented by the formula (3). Become.

 Therefore, this reaction may be carried out until all of the sugar chain asparagine compound represented by the formula (1) is consumed, but it is preferable to terminate the reaction before consumption. Alternatively, the reaction is preferably terminated before formation of the hydrazino sugar chain compound represented by the formula (4) generated by the j6 elimination reaction occurs. The reaction is preferably carried out by thin-layer chromatography (TLC) or mass spectrometry. The reaction can be completed by stopping heating under reflux.

[式中、 R ¹ , R R ³及び R ⁴は前記に同じ。

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

[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 bond-type protecting group such as an acetyl group, the protecting group is eliminated by excess hydrazine to form an amino group. Become. Therefore, N-acetylation must be performed by the action of an acetylating agent.

 As the acetylating agent, a conventionally known acetylating agent that can be used in the N-acetylating reaction can be used. For example, acetylated halides such as acetyl chloride and acetyl bromide can be exemplified by acetic anhydride. Acetic anhydride can be preferably used. The amount of the acetylating agent used may be about 1 to 20 equivalents, preferably about 1.5 to 10 equivalents, relative to 1 equivalent of the 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. It is made with.

 As the base, conventionally known ones can be used. Examples thereof include alkali metal carbonates such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, and organic bases such as tritylamine, pyridine, etc. Is particularly preferred. The amount of the base used is not particularly limited and can be used in an equal amount or more than the acetylating agent, but it is preferably used in a large excess, for example, an alkali metal carbonate such as sodium hydrogen carbonate. In this case, the saturated aqueous solution may be used in an amount of 1 to;

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. You can mix Water is preferably used. The amount of solvent used is not particularly limited.

The amount is usually about 10 to 2000 parts by weight, preferably about 100 to 1000 parts by weight per 1 part by weight of the sugar chain compound represented by (3).

 The reaction is carried out at -10 to 100, preferably 0 to 50 and is usually completed in about 0.1 to 24 hours, but it is preferable to confirm the progress of the reaction by TLC or mass spectrum. .

 The compound after the acetylation reaction is represented by the following formula (5).

 (Five)

[~ 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 obtain fragments such as asparagine residues and j6 desorbed 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 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. It is preferable from the viewpoint of safety and ease of use. The amount of the acid used is not particularly limited as long as it is 1 equivalent or more per 1 equivalent of the hydrazino sugar chain compound represented by the formula (5), and 1 to 5 equivalents are preferable. Usually an expression

 It is preferable to add an acid to such an extent that the aqueous solution of the compound (5) is sufficiently acidic.

The reaction may be carried out at about 0 to 50 ° C, preferably about 10 to 40 ° C. Usually, the reaction is completed within about 1 to 15 hours, preferably about 2 to 10 hours, but the reaction can be carried out with TLC or mass spectrum. It is preferable to track and confirm the end. 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 6 elimination does not occur In this case, the sugar chain asparagine compound represented by the formula (1) as a raw material remains, and if the reaction proceeds slightly, the | N-acetyl (6) will be mixed. Also, the expression

The hydrazino sugar chain represented by (3) is unstable and causes δ elimination over time. Therefore, the sugar chain compound represented by the formula (2) obtained at this stage includes the compound (1),

Contamination with other compounds such as (4) and (6) is observed.

The compound of the above formula (6) is shown below.

 ― (6)

 1 ~! ^ And A c are the same as above. ]

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

Examples of the amine compound include monoalkylamines having 1 to 4 carbon atoms such as methylamine, ethylamine, isopropylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, cycloheptylamine, cyclooctylamine. Examples thereof include cycloalkylamines having 3 to 8 carbon atoms such as amine and benzylamines which may have a substituent. Examples of the substituent of benzylamine that may have a substituent include halogen atoms such as fluorine, chlorine and bromine, alkyl groups having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group, an isopropyl group and a tert-butyl group. , A methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an alkoxy group such as a tert-butoxy group, a nitro group, etc., and 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 However, benzylamine, P-methoxybenzylamine, and 2,4,5-trimethoxybenzyl can be preferably exemplified, and p-methoxybenzylamine is particularly preferred. The amount of the amine compound used is usually 1 to 20 equivalents, preferably 2 to 10 equivalents, per 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 equivalents, 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 (DMS O), N, N-dimethylformamide (DMF), tetrahydrofuran (TH F) and the like. These may be used alone or in combination of two or more. May be used.

 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, the reaction is followed by TLC or mass spectrum, and the raw material disappears. The reaction may be terminated at the time of performing.

[式中、 R¹, R²、 R³、 R⁴及び Acは前記に同じ。 R⁸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べンジル 基を示す。 ] 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; It does not occur and the sugar chain structure can be maintained. The hydrazino sugar chain compound represented by the formula (6) mixed in the raw material of this reaction reacts in the same manner to give the corresponding amino-substituted compound.

[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. 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, carbon numbers such as methyl group, ethyl group, propyl group, isopropyl group and tert-butyl group. An alkyl group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an alkoxy group such as a tert-butoxy group, a nitro group, and the like. These substituents can be used alone or at any position on the phenyl ring. And those in which 2 to 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 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. it can.

 Separation by chromatography can be appropriately performed by using known chromatography alone or in combination. For example, after purification by gel filtration chromatography, reverse phase column chromatography is used. It can be purified.

 Examples of the reverse phase column include ODS, Phenyl system, nitrile system, anion exchange system power ram, etc. The amino acid of the amino sugar chain compound represented by the formula (7) The base produces a strong interaction with the octyldecyl group of the ODS column, and has excellent 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 above chromatography.

 Examples of the acid 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 safe and simple to use. Is preferable.

 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 the acid to such an extent that the aqueous solution of the compound of the formula (7) is sufficiently acidic.

 The reaction may be carried out at about 0 to 50 ° C, preferably about 10 to 40 ° C. Usually, the reaction is completed within about 1 to 15 hours, preferably about 2 to 10 hours, but the reaction can be carried out using TLC or mass spectrum. It is preferable to track and confirm the end.

 The obtained sugar chain compound represented by the formula (2) can be purified by chromatography or the like. BEST MODE FOR CARRYING OUT THE INVENTION

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

Example 1

Sugar chain asparagine compound (1 1 1) 1 Omg of hydrazine hydrate (hydrazine 55%) 1 Oml 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, heating and reflux were 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 treated as a raw material.

 (1-1)

 The reaction solution was concentrated to dryness under reduced pressure, and the resulting residue was dissolved in 1 ml of water. To this aqueous solution was added sodium hydrogen carbonate powder until saturation, followed by acetic anhydride (0.1 mL). The progress of the acetylation was followed by TLC (isopropanol: 1M ammonium acetate aqueous solution = 1.5: 1). The disappearance of the raw materials was confirmed by TLC (Isopropanol: 1M ammonium acetate aqueous solution = 1: 1), and neutralized by adding sodium hydrogen carbonate powder so that the pH of the reaction solution was 7-8. The reaction solution is concentrated to dryness under reduced pressure, and the resulting residue is dissolved in 1 ml of water and subjected to gel filtration chromatography (column support: Sepha ex G-25, column size: 16 mmX 345 mm, flow rate: The fraction containing the compound (5-1) was fractionated at 0.8 ml / min, developing solvent: water, and concentrated under reduced pressure.

(5-1) 得られた濃縮残渣の 5mgを水 lmlに溶かし、 酢酸 572 ^ 1を加えて酢酸 水溶液とした。 室温で撹拌し、 反応を TLC (イソプロパノール： 1M酢酸アン モニゥム水溶液 =1. 5 ： 1) で追跡した。 6. 5時間後、 反応終了を確認し、 1 M水酸化ナトリゥム水溶液で中和し、 凍結乾燥して粉体とした。

(5-1) 5 mg of the obtained concentrated residue was dissolved in 1 ml of water, and acetic acid 572 ^ 1 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 ammonium acetate aqueous solution = 1.5: 1). 6. After 5 hours, the completion of the reaction was confirmed, neutralized with 1 M 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 the compound (2-1) is collected. ) However, contamination of unreacted raw material derived compounds (1 1 2) and / 3 desorbed compound (8) was observed.

Yield: 8.9 mg [Compound (2_ 1): Compound (2-2): Compound (8) = 9 0: 8: 2]

Compound (2-1)

^X H-NMR (40 ΟΜΗζ, 2 95 K, HOD = 4. 8 1), 5. 28 (bd 1H, G l cNAc l— H— 1), 5. 23 (s, 1 H, Man 4-H -1) 5. 03 (s, 1 H, Man 4 '-H- 1), 4.86 (s, 1 H, Man 3-H— 1), 4.70 (m, 3 H, G 1 c NAc 2, 5, 5 '-H- 1), 4. 53 (d, 2H, Ga 1 6, 6'—H— 1), 4. 34 (bs, 1 H, Man 3 -H- 2 ), 4.28 (bd, 1 H, Man 4-H- 2), 4.20 (bd, 1H, Man 4 '-H- 2), 2.76 (bdd, 2 H, N eu Ac 7 7 '-H- 3 eq), 2.17 (s, 3 H, Ac), 2.16 (s, 6 H, Ac X 2), 2. 1 3 (s, 6 H, Ac X 3), 1. 80 (dd, 2 H, NeuAc 7, 7'— H— 3 ax).

 Ma s s: ES I c a 1 c d f o r 2222, f ound; 1 1 10

[(M-2) _ ² ]

Compound (8)

Ma s s c a 1 c d f o r 20 19, f oun d; 1008. 3

[(M-2) 1 ² ]

実施例 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 DMS03m1. P-Methoxy in this solution 2 ml of sibenzylamine and 5 mg of camphor sulfonic acid were added, and the reaction was carried out while maintaining the temperature at about 37 ° C in the thermostatic layer.

 The reaction was followed by mass spectrum (disappearance of 1 110Z-2 peak and formation of 1170/2 peak) to confirm the completion of the reaction. 1 OmM aqueous ammonia is added to the reaction solution to dilute it twice and gel filtration column chromatography (column support: Sephade G-25, column size: φ 10 mmX 900 mm, flow rate: 0.8 ml / min, Developing solvent: Purified with 5 OmM ammonium carbonate aqueous solution or aqueous ammonia (pH 9-10)), fraction containing compound (7-1), fractionated, concentrated under reduced pressure, lyophilized to compound (7- 1) powder was obtained. However, contamination of compound (1-2) and compound (9) was observed.

 (7-1)

得られた粉末 2 Omgを 1 OmM炭酸水素アンモニゥム水溶液に溶かし、 10 mM炭酸水素アンモニゥム水溶液で完全に置換した ODSカラム （カラム担体： Co smo s i 1 75 C₁₈— OPN (ナカライテスク株式会社製） 、 カラム サイズ： 0.75X 0. 75X 10 cm) に充填した。 その後、 1 OmM炭酸水素 アンモニゥム水溶液を担体の 5倍量流し、 化合物 （1_2) を流出させた。 その 後、 1 OmM炭酸水素アンモニゥム水溶液：ァセトニトリル （=98 : 2) を担 体の 5倍量流し、 担体を洗浄後、 1 OmM炭酸水素アンモニゥム水溶液：ァセト 二トリル （=96 ： 4) を流して、 化合物 （9) の流出後、 化合物 （7— 1) を 分取した。 収量： 9mg

ODS column (column support: Co smo si 1 75 C ₁₈ — OPN (manufactured by Nacalai Tesque)), in which 2 Omg of the obtained powder was dissolved in 1 OmM ammonium hydrogen carbonate aqueous solution and completely replaced with 10 mM ammonium hydrogen carbonate aqueous solution, Column size: 0.75X 0.75X 10 cm). After that, 1 OmM ammonium bicarbonate aqueous solution was flowed 5 times the amount of the carrier, and the compound (1_2) was discharged. Then, 1 OmM ammonium hydrogen carbonate aqueous solution: acetonitrile (= 98: 2) was flowed 5 times the carrier, the carrier was washed, and 1 OmM ammonium hydrogen carbonate aqueous solution: acetonitrile nitrile (= 96: 4) was flowed. After the outflow of Compound (9), Compound (7-1) was collected. Yield: 9mg

Compound (7— 1)

 -NMR (400 MHz, H o D = 4. 81), 55. 11 (s, 1 H, Man 4 -H- 1), 4.93 (s, 1 H, Man4'— H— 1), 4.75 ( s, 1 H, Man 3 -H- 1),. 59 (m, 3H, G 1 cNAc 2, 5, 5'— H-1), 4.43 (d, 2 H, Ga 1 6, 6'_H -1),

4.24 (bs, 1H, Man 3 -H- 2), 4.18 (bd, 1 H, Man 4 one H-2), 4. 10 (bd, 1 H, Man 4 '-H- 2 ),

2. 65 (b d d, 2H, NeuAc 7, 7'-H-3 e Q), 1.71 (dd, 2H, NeuAc 7, 7'-H-3 ax).

 Ma s s: ES I c a 1 c d f o r 2341.8, f ound;

1169. 9 [(M - 2) - 2]

Compound (9)

 Ma s s: ES I c a l c d f o 2139.8, f ound;

1068.4 [(M - 2) - 2]

Example 3

1 mg of water was added to 3 mg of the compound (7-1) obtained in Example 2 to make an aqueous solution (pHI 2 O), and then about 201 of acetic acid was added. The pH at this time was about 4. The reaction was followed by mass spectrum (disappearance of 1170 / —2 peaks and generation of 1110 no 2 peaks) to confirm the completion of the reaction. Sodium hydroxide aqueous solution is added to the reaction solution to adjust the pH to 5-6, and gel filtration column chromatography (column support: Sephade G-25, column size: φ 1 OmmX 90 Omm, flow rate: 0.8 mlZmin, developing solvent) :water)

The obtained fraction was concentrated under reduced pressure to obtain 2.5 mg of the compound (2 1 1) of purity (98%).

 The NMR and mass spectral data of the obtained compound (2-1) were the same as those obtained in Example 1. Industrial applicability

 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.

Claims

The scope of the claims 1. A process 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 a acetyl group. R ⁵ represents a hydrogen atom, a lipophilic protecting group, an amino acid residue, or a peptide residue, and R ⁶ represents a force lpoxyl group or a group COR ⁷ . R ⁷ represents an amino acid residue or a peptide residue. ]

[Wherein, RR ² , R ³ , R ⁴ and Ac are the same as above. ]  2. The production method according to claim 1, wherein the action of hydrazine hydrate is terminated before N-acetylyldarcosamine on the sugar chain reducing end side;  3. (A) a step of allowing a hydrazine hydrate to act on the sugar chain asparagine compound represented by formula (1),  (B) a step of acting a acetylating agent,  (C) a step of reacting an acid, (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 reacting an acid, Are carried out in this order. A method for producing a sugar chain compound represented by formula (2):  4. The production method according to claim 3, wherein the action of the hydrazine hydrate of (A) is terminated before the N-acetyldarcosamine on the sugar chain reducing terminal side is eliminated.  5. The production method according to claim 3, wherein the amine compound (D) is p-methoxybenzylamine.  6. The process according to claim 3, wherein the acid of (C) and (F) is acetic acid. 7. An amino sugar chain compound represented by the formula (7).

[Wherein, RR 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 which may have a substituent. ]