TW200804429A - Process for the preparation of sugar-chain compound - Google Patents

Abstract

This invention provides a process for the preparation of sugar-chain compound represented by formula (2), characterized by reacting a sugar-chain asparagine compound represented by formula (1) with a hydrazine hydrate. [wherein, R1, R2 and R3 are the same or different, each independently representing hydrogen or sugar residue, R4 represents hydrogen or fucose residue, Ac represents acetyl group, R5 represents hydrogen, fat-soluble protecting group, amino acid residue or peptide residue, R6 represents carboxyl group or-COOR7, R7 represents amino acid residue or peptide residue] [wherein, R1, R2, R3, R4 and Ac are the same meaning as above mentioned.]

Description

200804429 IX. Description of the invention: - [Technical field to which the invention pertains] The present invention relates to a method for cutting a sugar chain aspartate compound, a spear "method. a raw sugar & a sugar chain compound having a free radical. [Prior Art] The sugar chain and the protein are covalently bonded to each other. The glycoprotein is attached to the protein. The glycoprotein is attached to the protein. The protein is maintained in a three-dimensional structure or the solubility is adjusted, and the protease resistance is added. The chain participates in life phenomena such as fertilization or differentiation of proteins in the fertilization or differentiation, signal transmission, and regulation of proteins. For this reason, the glycoside chain plays an important role in various physiological functions. , the type of the evening ~ ..., because the sugar chain is involved in the phenomenon of life is very difficult: Shape: Special: pointed out that the structure of the sugar chain with a single-structured sugar chain of sugar and egg two functions, such as. The synthesis of the shellose peptide is indispensable according to the combination of sugar and protein. The group of amines is the amine of the aspartame and the glycoprotein can be roughly divided into the side chain of the ammonia-binding sugar chain (N• knot). The base group of the tyrosine acid TM combined with the sugar chain and the other group are the serine acid ((4) silk chain (0 binding type). ... 3 'mucin binding type sugar. The inventors combined enzymes The method of chemical and chemical methods for the divergence of complex glycochains is based on the compound of the free radicals in the end of the quail eggs. It reveals that the amino chain-reduced I-chains from the sugar chains are used to make the aminated complexes. 3906074044 The thiol group of the peptide can be selectively introduced into the peptide (Patent Document 2). When the N-binding sugar chain having a free hydroxyl group at the reducing end of the sugar-coated chain is present, From the sugar chain aspartame and its derivatives or glycoproteins, the method of cutting sugar chains by enzymes or the method of chemical cutting. Generally, the chemical cutting can be used to break down the hydrazine. When hydrazine is used in the presence of hydrate or water, anhydrous sugar is used because the sugar at the end of the sugar chain is detached (no detachment). [Patent Document 1] WO 03/008431 [Patent Document 2] WO 2004/011036 Bulletin, however, anhydrous hydrazine requires operation due to its toxicity or flammability. It is a reagent which is not suitable for large-scale treatment, and therefore it is desirable to have a safe and effective method for binding sugar chain cutting. The object of the present invention is to provide safe use of hydrazine hydrate as compared with anhydrous hydrazine, and to have a free hydroxyl group at the reducing end. The present invention relates to the following invention. A method for producing a sugar chain compound represented by the formula (2), which comprises a sugar chain aspartame of the formula The amine compound acts with the hydrazine hydrate.

[wherein, R2 and R3 are the same or different and represent a nitrogen atom or a sugar 319076 6 200804429 Insult: R4 represents a hydrogen atom or a fucose residue; Ac represents an ethyl hydrazide group; and a dihydrogen atom of a dihydrogen atom , amino acid residue or peptide residue; R represents a reading group; R7 represents an amino acid residue or a peptide residue] (2) NHAc NHAc [wherein R, R, and R4 and Ac are as described above The same]. In the first month, it was found that the hydrazine hydrate which has not been used so far due to the occurrence of cold detachment is decomposed by hydrazine, and then purified by benzylamine compound, and then purified by hydrolysis. The end has an N-binding type sugar chain compound which slides on each surface. The oligosaccharide aspartame compound represented by the formula (1) of the present invention contains a sugar chain and a day; an amine-bound glycoprotein, a glycopeptide, a sugar chain aspartate, a derivative such as a pot, and the like. /, ★" The sugar chain compound represented by the formula (2) of the present invention is an N-binding type sugar chain compound having a free surface group at the reducing end.

[Formula t V is the same or different ' represents a hydrogen atom or a sugar residue; R4 represents a hydrogen atom or a fucose residue; Ac represents an ethyl hydrazide group; and R5 represents a hydrogen atom, a fat-soluble protecting group, an amino acid residue or Peptide residue; R6 is a reading group S-COR7; V represents an amino acid residue or a peptide residue] 319076 7 200804429 R1 R4 NHAc NHAc (2) [wherein R1, R2, R3 and R4 and Ac are Same as above]. The sugar residue may be a monosaccharide such as mannose, N-ethyl glucosamine, galactose or fucose which may be protected by a hydroxyl group or a halogen atom such as fluorine, or may be more than 2 of these monosaccharides. A glycoside is formed by a glycoside. Further, it may be a sugar chain substituted with a halogen such as fluorine, or a sugar chain containing a protected sialic acid of a carboxyl group. That is, the sugar chain aspartame compound represented by the formula (1) may be a conventionally known or unknown sugar chain aspartate, a high mannose type sugar chain aspartate compound, a complex sugar chain aspartate. An amine compound, a mixed sugar chain aspartate compound. The sugar chain compound represented by the formula (2) may be a conventionally known or unknown sugar chain compound, a high mannose type sugar chain compound, a complex type sugar chain compound, or a mixed type sugar chain compound. The fat-soluble protective group is not particularly limited, and examples thereof include a carbonyl group such as a 9-fluorenyloxyl group (Fmoc) or a tert-butyloxycarbonyl group (Boc) or a propyloxy group (Alloc). A protecting group such as a fluorenyl group such as an acetamino group (Ac), an allyl group or a benzoinyl group. The introduction of such protecting groups can be based on, for example

Protecting groups in Organic Chemistry (John Wiley & Sons INC., New York 1991, ISBN 0-471-62301-6) and the like are carried out. The amino acid residue or the peptide residue of R5 is an amino acid of aspartame and an amino acid or a peptide having a carboxyl group which is a guanamine, and is not particularly limited. 8 319076 200804429 The amino acid residue or the peptide residue of R7 is an amino acid of the aspartame-based amino acid or a peptide, and is not particularly limited. The hydrazine hydrate used in the method can be used in the prior art, and can be directly used as a hydrazine-hydrate or a water-diluted solution, and the arrogant can be weight%, particularly preferably 40 to 1 〇〇 weight; The amount of the hydrazine hydrate to be used is not particularly limited, and the relative::::醯: the compound 1 equivalent is preferably 0.88 equivalent or more, which is more than a large amount of excess due to the use of a solvent in advance. The sugar chain aspartame compound represented by the formula (1) is preferably 100 to 5 parts by weight and is 1 to the reaction of the method, which can be heated under the reflux temperature and the formula (1) The chain of the amine (4) amine compound material u = the thiol compound shown. According to the study by the present inventors, it has been found that the dissociation reaction is based on the conversion of most of the two or all of the conversions shown in the formula (1) to the conversion of the thiol sugars represented by the formula (3) to the second '&(1) days. Most of the winter amine compounds or reactions. () The saccharide chain compound does not occur, and the detachment does not occur. However, the glycosylation of the sugar bond as shown in the formula (1) or the (3) oxime is preferably close to complete consumption. The reaction of the thiol chain compound represented by the formula (4) before birth is preferred. And Yancha six / soil death one mass spectrometry tracking. The final reaction of the reaction:: chromatography (TLC) or J褚® is stopped by heating and refluxing. 3] 9076 9 200804429

Rx

NHNH2 (3) [wherein, ^ is ... and ... is the same as above]. R\

Nhnh2 (4) [wherein R, R2 and R3 are the same as above]. In the reaction of the present method, the sugar chain aspartate compound represented by the formula (1) has a protecting amine group such as an ethylamine group, and the protecting group is removed from the silk amine group due to excessive tillage. The m-ethyl acylating agent is necessary for N-acetoxylation. The ethylenating agent may be used in the N-acetation reaction, and the acetylating agent may be exemplified by, for example, acetamidine or acetic anhydride such as acetamidine chloride or acetamidine bromide. Preferably, acetic acid liver can be used. The amount of the acetylation agent to be used is m equivalents, preferably about 10 equivalents, per equivalent of the amine group. · The N-acetylation reaction using an acetylation agent can be applied to a conventional method, for example, after distilling off the excess hydrazine under reduced pressure in the reaction solution, in the presence of a base, The action of the agent is completed. For example, a conventional metal pyrite such as sodium carbonate or sodium hydrogen carbonate may be used, and triethylamine or hydrazine may be mentioned. The organic test is determined, and it is particularly preferred to be hydrogen carbonate. Although the amount of the test is not particularly limited, it may be used in an equal amount or more with respect to the ethylating agent, and it is preferably used in a large amount. For example, it is used for hydrogen carbonate steel, for example, for B. The thiolating agent 丨 parts by weight gold == is a saturated aqueous solution, and the phase reaction can be carried out in a solvent, weight:. (OMSO), N N--甲甲甲萨a 〇 J Niu Shui, monomethyl sulfoxide These can be I. ―Types, ^ t" early collar and more than 2 kinds of mixed use, there is no special system compared to ^ usage, relative to the use of water. The share of Luo Media is usually 丨. To about the weight. The damage is about 1 to 1000, preferably from -10 to 10 (rc is carried out, often (U i is completed in about 24 hours. It is carried out in comparison with the second enthalpy, and the general formula confirms the progress of the reaction. The compound after the acetylation reaction is represented by the following formula (7).

NHNHAc [wherein, R1 to]^4 are: . . . and Ac is the same as the above]. As described in the above manner, the product can be removed by the cutting #p of the chain compound amine residue or the like via the sheng filter tube (four) method, or the ruthenium fragment or the sugar residue decomposed by β. The mercapto sugar chaining represented by the formula (5) is a product of a saccharide chain compound of the formula (1), which is contained in the formula, and is mainly treated with an acid. The acid used can be used with formic acid, acetic acid, ... inorganic acids such as cucurbitic acid, phosphoric acid, etc., difosmetic acid, acid, and ethyl tartaric acid 319076 ] 3 200804429 ^ where, by safety or "convenience" In view of the above, it is preferable that the amount is two: it is not particularly limited with respect to the thiol chain compound represented by the formula (5), and is preferably: ! It is preferred to add an acid in a manner of 1 to 10. The liquid phase of the liquid is preferably about 10 hours, and the above method can be used to produce: 42) sure / 1 should be carried out. In the decomposition of hydrazine, when the reaction is terminated at the stage of the detachment of the formula (1) in which the 4 R non-sugar chain compound is not produced, the J VIII (4) saccharide chain is mixed. , σ Ν 乙 乙 乙 乙 ( ( ( ( ( ( ( ( 乙 。 。 。 。 。 。 。 ( ( ( ( ( ( ( ( ( ( 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 糖 。 。 Mix in. R3

The formula (4) is a handle of the formula. V^NHNHAc NHAc (6) 匕:,: two to ~c are the same as above]. The compound of the glyco bond represented by the formula (2) and the like are reacted with an amine compound in a solvent. 319076 12 200804429 The amine compound may, for example, be a carbon amine such as methylamine, ethylamine or isopropylamine. 'Cyclopropylamine, cyclobutylamine, cyclopentylamine, • cyclohexylamine, cycloheptyl. Amine, ring Xinmei face 笪 # * α - female annoying number 3 to 8 cycloalkylamine, two beautiful, methylamines. The benzylamines having a substituent include a fluorine atom, a chlorine atom, a chlorine atom, a water atom, a methyl group, a methyl group, a propyl group, a propyl group, and a tributyl group. Methyl decyl, ethoxy, propyl acrylate, isopropoxy, succinimide & j sheep L 丞 虎 虎 虎 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , each package + Dingmen t, Shisi location, for 2 to 5 identical - or different replacement. The benzylamine-one-face field ## #,,中中,车家四土 can be exemplified as benzyl methyl moon female 'p-methyl benzyl benzylamine, 2, 4, 5 _ three: terte good P-methoxybenzylamine. The amount of the soil compound, the relative amount, is usually from i to 20, and the compound 1 is preferably 2 to 1 equivalent in the field. The reaction is preferably carried out in the presence of an acid such as camphorsulfonic acid: the amount of the acid used is 0.01 to 5 equivalents, preferably 0 05 θ, relative to the formula (1). 1 in the field is 1 summer. The solvent used in the reaction, (DMSO), hydrazine, hydrazine-dimethyl ketoamine (D water, methyl sulfoxide, etc. may be used alone or in combination of two or more. (THF) or the like, the amount of the solvent to be used is not particularly limited, and the amount of the compound is usually from 1 Torr to 2 Torr = about 5,000 parts by weight in the formula (2). Preferably, the reaction is usually carried out in 0 to one row, usually about 1 to 24 hours, and the 蛀k bauxite is at 10 to 5 (Tc into °. preferably by TLC or mass spectrometry 3) 9076 13 200804429 In the process of . 及 及 及 雇 雇 雇 雇 雇 原 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ It is determined that the U-lin is not produced, and the compound is also mixed with the thiol-chaining compound represented by the formula (6) in the raw material, and the appropriate amine-substituted compound is given.

NHAc NHAc (7) [wherein, R1, R2, r3, and r4 represent C of the carbon number of 1 to 4 are the same as those of the above; benzyl group of the R8 group]. The earth-alkaline is a cycloalkyl group of 3 to 8, having a carbon number of 1 to 4 isopropyl, etc., substituted by 'R8 herein; a carbon number of 3 to 8 WA... may be exemplified by a group, an ethyl group, a cyclopentyl group, and a cyclohexyl group. : % = exemplified by cyclopropyl, cyclobutyl, and the like, the base thereof, the base, and the like. Benzene f having a substituent: a group: a fluorine atom such as fluorine, chlorine or molybdenum, a carbon number such as a methyl group, a ethyl chloride-propyl group, a third butyl group, etc., an earth ethoxy group, a propoxy group, Isopropoxy group, third butoxide oxygen to the same; St-based system - phenylbenzyl is particularly preferred. The ～4,5-dimethyl emulsion group in which the amino sugar chain compound represented by the formula (7) and the other compound are treated with " can be used to separate the amino sugar chain compound represented by the formula (7). 319076 14 200804429 Separation by chromatography - or a combination of several combinations of 佶田二, Γ separate and appropriate chromatographic methods (use the rod, / /, after using the reverse phase, total 4, for example, by colloid Purification by reversed-phase chromatography by filtration chromatography. Anion exchange system such as 0DS, 'system, nitrile system, or 10 parts of non-amino sugar chain compound of ODS column has excellent amine base separation ability. The eight-burning base produces a strong interaction, and the fraction-to-adjustment. ' can refer to suitable and well-known conditions and add: the chain compound is a new compound. The effect can be the target +() A non-amino sugar-bonding compound and a sugar chain compound represented by formula (2) are used as an acid. Examples of the acid to be used include hydrochloric acid, acetic acid, rg ^ Λ ^ force L owed, and inorganic acids such as hydrazine. It is better to find a few acids, preferably _ _ _ _ _ _ _ _ _ _ _ _ _ _ ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... Chain compound 1 is 5 equivalents. Usually, compound 2 represented by formula (7) is preferably 1 to It is preferred to add an acid in a manner of a degree. / The liquid phase is sufficiently acidic. The reaction is usually carried out at 0 to 5 Torr, preferably at 1 Torr, usually at about 1 to 15 hours, preferably at from 1 to 10 It is preferable to use a sugar chain compound of the formula (2) which is a T2 or a mass spectrometer, such as a TIX or a mass spectrometer, and the like. (Example) Purification by hydrazine chromatography or the like. (Best form for carrying out the invention) 319076 15 200804429 The following description of the examples is given, but the present invention is not limited to the examples. Example 1 In the sugar chain aspartate compound (1-1) 1 Omg, hydrazine hydrate (肼55%) 10 ml, dissolved at room temperature, and heated to reflux at 100 ° C. The reaction was followed by TLC (isopropanol: 1 M aqueous ammonium acetate = 1 : 1), and the reflux was stopped at the time when the material disappeared on the TLC. In the amine compound (1-1), after the addition of the hydrazine hydrate, the Fmoc group is immediately detached, and the oligosaccharide chain aspartate compound is taken as a raw material by the -Fmoc group.

(1-1) The reaction solution was concentrated to dryness under reduced pressure, and 1 ml of water was added to the residue to dissolve. After the sodium hydrogencarbonate powder was added to the aqueous solution to be saturated, acetic anhydride (0.1 ml) was added. The reaction was followed by TLC (isopropanol··1Μ acetic acid aqueous solution = 1.5:1) to follow the progress of acetylation. After confirming the disappearance of the raw material by TLC (isopropyl alcohol: 1 Μ aqueous ammonium acetate solution: 1), the sodium hydrogencarbonate powder was neutralized so that the pH of the reaction liquid became 7 to 8. The reaction mixture was concentrated to dryness under reduced pressure, and the obtained residue was dissolved in water (1 ml) 6 319076 200804429 to colloidal filtration chromatography (column carrier: Sephadex G-25, column size: 0 16 mm><345 mm, Flow rate: 0.8 ml/min, developing solvent: water) • A fraction containing the compound (5-1) was prepared and concentrated under reduced pressure.

5 mg of the concentrated residue thus obtained was dissolved in 1 ml of water, and acetic acid 5 72 μl was added to prepare an aqueous acetic acid solution. Stir at room temperature and the reaction was followed by TLC (isopropanol: 1 aqueous ammonium acetate = 1.5:1). After 6.5 hours, it was confirmed that the reaction was completed, and the aqueous solution was neutralized with 1 Torr of nitrogen oxide to form a powder. The obtained powder was purified by a gel column chromatography (same conditions as above) to prepare a fraction containing the objective compound (2-1), and concentrated under reduced pressure to give a compound (2-1). ). However, it was confirmed that the compound (1-2) derived from the unreacted raw material and the compound (8) desorbed by β were mixed. Yield: 8.9 mg [Compound (2-1): Compound (2-2): Compound (8) = 90: 8 : 2] Compound (2-1) 17 319076 200804429 'H-NMR (4 0 0 MHz, 2 9 5 K, HOD = 4.8l), 5.2 8 (bd, 1H, GlcNAcl - H-1), 5.23 ( s , 1 Η, M an 4 - H - 1), 5.0 3 ( s , 1 H, Man4 ' Hl), 4.8 6 ( s , 1 H, Man3 — H — 1) “4.7 0 (m, 3H, GlcNAc2, 5, 5′-H – 1), 4.5 3 (d, 2H, G a 1 6, 6 '一H-1', 4.34 (bs, 1 H,

Man3 —H—2) , 4.2 8 (bd, 1 H,M an 4 — H — 2 ), 4.20 (bd, lH, Man4, H-2), 2.76 (bdd, 2H, N eu A c 7, 7'-H-3eq), 2.17 ( s , 3 H, Ac) , 2.16 ( s , 6 H, A c X 2) , 2.13 ( s , 6H, A c X 3) , 1.80 (dd, 2H, N Eu A c 7 , 7'-H — 3ax). Mass: ESI calculated 2222, found: 1110 [(M-2)-2] Compound (8) Mass: ESI Calculated value 2019, measured value ······ (M-2)·2]

(1-2) 18 319076 200804429

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 the solution, 2 ml of p-methoxybenzylamine and 5 mg of camphorsulfonic acid were added, and the reaction was carried out at about 37 ° C in a constant temperature layer. The reaction was followed by mass spectrometry (the disappearance of the 1110/-2 peak and the formation of the 1170Λ2 peak) to confirm the end of the reaction. The reaction was diluted 2 times with 1 OmM aqueous ammonia, and colloidal filtration column chromatography (column carrier: Sephadex G-25, column size: 0 10 mm X 900 mm, flow rate: 0.8 ml/min, developing solvent: 50 mM ammonium carbonate aqueous solution) Or aqueous ammonia (pH 9 to 10)), and the fraction containing the compound (7-1) is prepared and concentrated under reduced pressure to obtain a freeze-dried compound (7-1) powder. However, it was confirmed that the compound (1-2) and the compound (9) were mixed.

19 319076 200804429

20 mg of the obtained powder was dissolved in 10 mM ammonium hydrogencarbonate water, and filled in an ODS tube branch completely replaced with a 10 mM aqueous solution of ammonium hydrogencarbonate (manufacturer carrier: Cosmosil 75C18-〇PN (manufactured by Nacalai Tesque)) Column size: 0.75 X0.75x10cm). Thereafter, a 5-fold amount of an aqueous solution of ammonium hydrogencarbonate was passed through the carrier to cause the compound (1_2) to flow out. Thereafter, a carrier of 5 times the amount of 10 mM aqueous solution of ammonium hydrogencarbonate: acetonitrile (= 98: 2) was introduced, and after washing the carrier, '10 mM aqueous solution of ammonium hydrogencarbonate: acetonitrile (=96:4) was passed, and the compound (9) was discharged. , Compound (7-1) was prepared. Yield: 9 mg of compound (7-1) 'H-NMR (400 MHz, HOD^4.8l), 6 5.11 (s, 1 Η,

Man4^Hl) , 4.93 (s, i H, Man4^H^l)\ 4.75 (s , 1 H, Ma η 3-H- 1) , 4 . 5 9 (m, 3 H, G 1 c NA c 2, 5,5'-H-l), 4.43(d,2H,Gal6,6,-H-l), 4·24 (bs, 1H,Man3—H - 2) , 4.18 (bd,1H, Man4 -H-2) , 4. 10 (bd, 1H, Ma n4, -H-2), 2·65 (bdd, 2H, NeuAc7, 7, H-3eq), 1.71 (dd, 2H, NeuAc7, 7 'H-3ax' mass: ESI calculated 2341.8, found: π69·9[(Μ-2)-2] Compound (9) 20 319076 200804429 Mass: ESI Calculated value 2139.8, found: 1068·4 [ (Μ-2)·2], Example 3, 3 ml of water (pH 10) was added to 3 mg of the compound (7-1) obtained in Example 2, and then acetic acid was added to about 20 //1 . The pH at this time is about 4. The reaction was followed by mass spectrometry (the disappearance of the 1170/-2 peak and the formation of the 1110Λ2 peak), and it was confirmed that the reaction was completed. Add sodium hydroxide aqueous solution to the reaction solution, adjust to pH 5 to 6, and colloidal filtration column chromatography (column carrier: Sephadex, G-25, column size · 0 10mm X 900mm, flow rate · · 0.8ml /min, development solvent: water), and the obtained fraction was concentrated under reduced pressure to obtain a purity (98%) of compound (2-l) 2.5 mg. The NMR and mass spectral data of the obtained compound (2-1) were the same as those obtained in the above Example 1. (Industrial Applicability) According to the method of the present invention, an N-binding type sugar chain compound having a free hydroxyl group at a reducing end can be produced by using a safe hydrazine hydrate in a hydrazine decomposition reaction. 319076

Claims (1)

200804429, Patent application range: A method for producing a sugar chain compound of the formula (2), which is characterized in that a sugar chain aspartame compound represented by the formula (1) is reacted with a hydrazine hydrate. , R5 Γ ψ 5 T? ^ 2 jj η and R are the same or different, indicating that the ruthenium atom or saccharide residue 'R is not a hydrogen atom or a fucose residue; Ac represents an acetamidine group; R5 is not a ruthenium atom , a fat-soluble protecting group, an amino acid residue or a peptide residue; V represents a carboxyl group; R7 represents an amino acid residue or a peptide residue] R4 (2) NHAc NHAc [wherein R1, P2 ^, , R, R and Ac are the same as the above one. For example, the patent application method, m. Drying ang 1 manufacturing method, wherein the action of hydrazine hydrate is terminated before sugar 4@ I. Ethyl glucosamine at the end of the chain to avoid the detachment 3. Under:);: Each: one (Α) to make the sugar chain aspartate compound and hydrazine hydrate of the formula (1, - a step of acting as a hydrazine, (C) a step of acid action, (1)) and a cycloalkyl group selected from the group consisting of a monoalkylamine having a carbon number of 4 and a cyclohexane having a carbon number of 3 to 319076 22 200804429 and having a step of at least one of the action of the substituent phenylhydrazineamine, a step of purifying the amine compound (E) by column chromatography, and (F) a step of acid action. • Affirmation of the manufacturing method of the third paragraph of the patented circumcision, the role of the 苴 苴 俜; ^ + , , (A) 肼 m m m m m 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 J. The manufacturing method of claim 3, wherein the compound is p-methoxybenzoin. 6. The method of manufacture of claim 3, wherein the acid is acetic acid.胺 (A) Amination (C) and (F) of the amino sugar chain compound of the formula (7), (7) [wherein, R], the number of turns I to 4]. R2, R3 and R4 and Ac are the same as those of the alkyl group and the carbon number of 3 to 8; R8 is a burnt group or has a substituent. 3] 9076 200804429 VII. Designated representative figure: No case in the present case, (1) The representative representative figure of this case is: the () picture. (2) A brief description of the symbol of the representative figure: 8. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: 4 319076