JPH06184183A - Production of sialyl oligosaccharide - Google Patents

Abstract

PURPOSE:To obtain the subject compound which is a raw material for a sialic acid-containing saccharide chain compound useful for efficiently synthesizing a ganglioside by carrying out a new method for treating a nest of a swallow with hydrazine and then N-acetylating a free amine. CONSTITUTION:A nest of a swallow is initially subjected to the reducing treatment with hydrazine (preferably anhydrous hydrazine) to provide a free amine, which is then N-acetylated according to a method for reacting the free amine with acetic anhydride in the presence of sodium hydrogencarbonate. The objective compound is then usually isolated from the resultant product by thin-layer chromatography or ion exchange chromatography, etc. Furthermore, the treatment with the hydrazine is preferably carried out by heating the nest and hydrazine in a sealed tube. The hydrazine can be used in an amount of, e.g. 3-10 pts.wt. based on 1 pt.wt. nest of the swallow.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing sialyl oligosaccharides. More specifically, it relates to a method for producing sialylgalactose and a sialic acid dimer that can be used as a sugar chain raw material.

[0002]

BACKGROUND OF THE INVENTION Gangliosides, also called sialic acid-containing glycolipids, are present on the surface of cell membranes and contribute to the negative charge on the cell surface. It has physiological functions that contribute to neural function and mutual discrimination of cells, differentiation, proliferation, carcinogenesis, aging, and the like. Further, it has been attracting attention as a part involved in cytotoxins such as cholera toxin, botulinum toxin, tetanus toxin, peptide hormones such as thyroid stimulating hormone, and receptor functions such as interferon.

Attempts have been made to synthesize gangliosides having such various physiological functions. For example, JP-A-61-2695, JP-A-61-282391,
JP-A-63-45293 describes various methods for synthesizing gangliosides. The mainstream of these methods is to connect a sugar chain compound and sialic acid (N-acetylneuraminic acid). Sialic acid, which is a raw material, is produced by decomposing a glycoprotein contained in a nest of swallows with an acid.

However, gangliosides are synthesized by linking a sugar chain compound and ceramide to sialic acid, and if a sialic acid compound in which a sugar chain compound is linked to sialic acid is obtained in advance, a certain ganglioside is obtained. Can be synthesized more efficiently than the conventional method using sialic acid.

[0005]

OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide a novel method for producing a sialyl oligosaccharide as a raw material for a sugar chain compound containing sialic acid.

[0006]

DESCRIPTION OF THE INVENTION The present invention comprises the steps of treating a nest of swallows with hydrazine to obtain a free amine, N-acetylating the obtained free amine, and isolating a sialyl oligosaccharide from the obtained product. The present invention relates to a method for producing a sialyl oligosaccharide, which comprises the step of:

The present invention will be described below. The bird's nest is
The same thing as that conventionally used in the method for producing sialic acid, that is, an edible swallow's nest may be used. Swallow's nest contains glycoproteins.

The swallow's nest is decomposed by reduction treatment with hydrazine, preferably anhydrous hydrazine, to give a free amine compound. Hydrazine can be usually heated
It is preferable from the viewpoint of promoting decomposition. The heating can be performed in a range of 50 to 110 ° C., for example. further,
This heating can promote decomposition more if done in a sealed tube,
And it is preferable from the viewpoint that the yield can be improved. The amount of hydrazine used in the nest of the swallow can be, for example, about 3 to 10 parts by weight with respect to 1 part by weight of the nest of the swallow. The treatment time is appropriately determined depending on the type of the nest of the swallow, the temperature, the scale of the treatment and the like. For example, it can be about 1 to 12 hours.

The free amine compound obtained by the treatment with hydrazine is then N-acetylated. N-acetylation can be carried out by a conventional method, for example, by reacting a free amine compound with acetic anhydride in the presence of sodium hydrogen carbonate.

The N-acetylation reaction using acetic anhydride in the presence of sodium hydrogen carbonate is suitably carried out by adding acetic anhydride to the reaction solution little by little in the presence of a saturated amount of sodium hydrogen carbonate. The amount of acetic anhydride used is
For example, 3 to 1 per 1 part by weight of the swallow's nest used as a raw material
It can be 0 parts by weight.

Then, the desired sialyloligosaccharide is isolated from the obtained reaction solution. For this isolation, thin layer chromatography or ion exchange column chromatography commonly used for isolating sialyl oligosaccharides can be used. The conditions are appropriately determined in consideration of the desired sialyloligosaccharide and the type of thin layer or column. In the present invention, in particular, sialylgalactose (Neu5Acα) shown below as a sialyl oligosaccharide
2 → 3Gal) and a sialic acid dimer (N
eu5Acα2 → 8Neu5Ac) can be isolated.

[0012]

[Chemical 1]

[0013]

[Chemical 2]

When the sialyl oligosaccharide is isolated from the N-acetylation reaction solution, it is preferable to remove unnecessary salts from the reaction solution in advance. Various ion exchange resins can be used to remove the salts, depending on the type of the salts to be removed.

[0015]

According to the production method of the present invention, it is possible to provide a novel method for producing a sialyloligosaccharide as a raw material for a sugar chain compound containing sialic acid.

[0016]

Utility: Sialylgalactose obtained by the production method of the present invention is useful as a raw material for the synthesis of ganglioside such as GM ₃ . Conventionally, sialylgalactose has been synthesized by chemically binding sialic acid and galactose. This synthesis required 5 to 10 steps, and the total yield was 10% or less. However, according to the method of the present invention, sialylgalactose can be directly obtained, and the synthesis of ganglioside can be remarkably efficient.

Further, the sialic acid dimer obtained by the production method of the present invention contains a sialic acid group such as GD ₃ or GQ _1b at 2
It is useful as a raw material for the synthesis of three or more gangliosides. Conventionally, a sialic acid dimer has been synthesized by chemically bonding two sialic acids (for example, JP-A-3-17092). This synthesis required about 5 steps and the yield was 10% or less. But,
According to the method of the present invention, a sialic acid dimer can be directly obtained, and the synthesis of a ganglioside having two or more sialosyl groups can be remarkably efficient.

[0018]

EXAMPLES The present invention will now be described in more detail with reference to examples. Example 1 Neu5Ac 2α → 3Gal After drying well, 5.0 g of powdered swallow nest is taken, 20 ml of anhydrous hydrazine is added, and the mixture is heated and stirred at 100 ° C. for 8 hours in a sealed tube. After stirring at room temperature for another 12 hours, the mixture is concentrated under reduced pressure.
After adding 200 ml of saturated aqueous sodium hydrogen carbonate to the residue to make a uniform solution,
While stirring, 30 ml of acetic anhydride is added dropwise little by little. After stirring for 12 hours at room temperature, it is passed through a column of Amberlite IR-120B (H type) or Dowex 50 (H type) ion exchange resin (300 ml). After washing the column with 500 ml of distilled water, the washing solution and the passing solution are combined and lyophilized. 11.24 g of crude product was obtained.

11.24 g of the crude product obtained here was dissolved in 50 ml of distilled water, and Dowex 50 (H type) (60 ml) and Dowex I were added.
Pass through a column of (HCOO type) (60 ml) ion exchange resin.
After the resin was washed with distilled water, it was eluted with 1N-formic acid from Dowex I, and the fractions containing the desired product were collected and lyophilized using thin layer chromatography to give a crude product (0.
I got 91g.

0.91 g of the crude product obtained here was added to 0.01M-
Dissolve in 20 ml of Tris hydrochloric acid buffer PH 7.6, adsorb on DEAE-Sephadex A-25 (equilibrium equilibrated with 0.01M-Tris hydrochloric acid buffer PH 7.6) column (300 ml), then add 0.25
Elute with 0.1M-Tris hydrochloric acid buffer (PH 7.6) containing% -NaCl. The eluate was freeze-dried and then Bio Gel P-3 again.
182.4 mg after desalting using the (500 ml) column
(3.6%) powder was obtained.

[Α] ²⁰ _D = 3.8 (C = 1.0 H ₂ O) ¹ HN.MR 500MHz D ₂ O (Internal mark t-BuOH) 5.278 (d, 0.3H, J = 3.7Hz, H-1a α) , 4.627 (d, 0.7H, J = 8.1H
z, H-1a β), 4.319 (dd, 0.3H, J = 2.9,10.3Hz, H-3a α), 4.6
88 (dd, 0.7Hz, J = 3.3,10.0HZ, H-3aβ), 4.005 (d, 0.3H, J = 2.
2Hz, H-4a α), 3.935 (d, 0.7H, J = 3.3Hz, H-4a β), 3.509 (d
d, 0.7H, J = 8.1,9.9Hz, h-2aβ), 2.748 (dd, 0.7H, J = 4.7,12.
5HZ, H-3beq β), 2.733 (dd, 0.3H, J = 4.8,12.5Hz-3beq
α), 2.036 (s, 0.9H, NHAc, α), 2.018 (s, 2.1H, NHAc, β), 1.
806 (t, 0.3H, J = 12.5Hz, H-3bexα), 1.787 (t, 0.7H, J = 12.1H
z, H-3bax β)

Example 2 Neu5Ac 2α → 8Neu5Ac After drying well, 1.5 g of powdered swallow's nest was taken, 15 ml of anhydrous hydrazine was added, and the mixture was heated and stirred at 100 ° C. for 7 hours in a sealed tube. After stirring at room temperature for another 12 hours, the mixture is concentrated under reduced pressure.
After adding 200 ml of saturated aqueous sodium hydrogen carbonate to the residue to make a uniform solution,
While stirring, 40 ml of acetic anhydride is added dropwise little by little. After stirring for 12 hours at room temperature, it is passed through a column of Amberlite IR-120B (H type) or Dowex 50 (H type) ion exchange resin (300 ml). After washing the column with 500 ml of distilled water, the washing solution and the passing solution are combined and lyophilized. 2.51 g of crude product was obtained.

2.51 g of the crude product obtained here is dissolved in 10 ml of distilled water and fractionated with Sephadex G-10 (520 ml). Of the fractionated fractions, the low molecular weight fractions are collected and freeze-dried. 0.46 g of crude product was obtained.

0.46 g of the crude product obtained here was added to 0.01M-Tr
is Dissolved in hydrochloric acid buffer PH 7.6, 5 ml, and adsorbed on DEAE-Sephadex A-25 (equalized with 0.01M-Tris hydrochloric acid buffer PH 7.6) column (1000 ml), then 5.0% -N
Elute with 0.01M-Tris hydrochloric acid buffer containing aCl (PH 7.6). The eluate was freeze-dried and then Sephadex G- again.
Desalting using 10 (520 ml) column, 108.3m
g (7.2%) of powder is obtained.

¹ HN.MR 500MHz D ₂ O (Internal mark t-BuOH) 2.747 (dd, 1H, J = 4.4,12.1Hz, H-3beq), 2.201 (dd, 1H, J = 4.
8,12.8Hz, H-3aeq), 2.046 (s, 3H, NHAc), 2.012 (s, 3H, NHA
c), 1.727 (t, 1H, J = 12.1Hz, H-3aax), 1.682 (t, 1H, J = 12.1H
z, H-3bax), ¹³ CN.MR 125MHz D ₂ O (internal standard dioxane) 102.9 (C-2b), 97.5 (C-2a), 76.0 (C-8a), 73.7 (C-6b), 72.9
(C-8b), 71.1 (C-6a), 69.2 (C-4b), 68.9 (C-7b), 68.4 (C-4
a), 67.8 (C-7a), 63.4 (C-9b), 61.7 (C-9a), 53.2 (C-5a), 52.
5 (C-5b), 41.9 (C-3b), 40.1 (C-3a), 23.0 (COCH _3, b), 22.9 (C
OCH _3, a)

Claims (2)

[Claims] 1. A step of treating a nest of swallows with hydrazine to obtain a free amine, a step of N-acetylating the obtained free amine, and a step of isolating a sialyl oligosaccharide from the obtained product. A method for producing a sialyl oligosaccharide, comprising: 2. The process according to claim 1, wherein at least one of sialylgalactose and sialic acid dimer is isolated from a product obtained by N-acetylating a free amine.