JP2001247990A - Method for manufacturing oligosaccharide using polymer-combined saccharide acceptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for obtaining the object oligosaccharide from above a polymer without using organic solvent or metallic catalyst. SOLUTION: A polymer-combined saccharide acceptor through an amino group is used to synthesize oligosaccharide, and then the oligosaccharide is brought out by an oxidative electrolytic reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for synthesizing an oligosaccharide, and more particularly to a method for synthesizing a sugar chain on a polymer.

[0002]

2. Description of the Related Art As a simple method for synthesizing an organic compound, a reaction on a solid phase or a polymer carrier is actively performed. In the synthesis of oligosaccharides, a solid phase reaction or a reaction on a polymer carrier is incorporated, and useful oligosaccharides are synthesized. From such a point, it is important to develop a new solid-phase reaction and a reaction on a polymer carrier for the purpose of promoting efficient reaction and easy purification (Advanced
ChemTech Catalog). Conventionally, a polyethylene glycol derivative has been known as a polymer carrier used for oligosaccharide synthesis (S. Douglas, J. Am. Chem. Soc., 11
7, 2116-2117 (1995)). However, according to the method reported so far, a large amount of an organic solvent, a metal catalyst, and the like are required when a target sugar chain is cleaved from the polymer after oligosaccharide synthesis on the polymer.

[0003] However, in the industrial production of oligosaccharides, a production method using a large amount of an organic solvent or a heavy metal catalyst has a problem.
In recent years, it has become a serious problem in terms of disposal of used solvents or catalysts.

[0004] From such a viewpoint, it has been desired to develop a method for producing an oligosaccharide without using a large amount of an organic solvent or a heavy metal catalyst.

[0005]

Accordingly, an object of the present invention is to develop a method for obtaining a target oligosaccharide from a polymer without using an organic solvent or a metal catalyst.

[0006]

The inventors of the present invention have conducted various studies to solve the above-mentioned problems. As a result, the use of a receptor having a hydrophilic polymer bonded through an amino group enables the use of an aqueous solvent (buffer solution). ) To allow the enzyme reaction to proceed efficiently, as well as the method of Fukase et al. (K. Fukas
e, Tetrahedron Lett. 31, 389-392 (1990)), an electrolysis reaction (edited by The Chemical Society of Japan, New Laboratory Chemistry Course, Basic Operation [II], Maruzen Co., 633-638 (1975)) The inventors have found that the oligosaccharides easily produced can be cut out, and have completed the present invention.

[0007]

DETAILED DESCRIPTION OF THE INVENTION A para-aminophenyl derivative obtained by reducing a para-nitrophenyl derivative of a sugar is combined with a water-soluble polymer such as a polyethylene glycol derivative. As the polyethylene glycol derivative, an activated PG derivative which is generally commercially available can be used (Shearwater Polymers, Inc. Polyethyle of NOF Corporation)
ne Glycol Derivatives catalog; Motoyuki Yamada et al., Hyundai Kagaku, 12, 62-67 (1995)). This bonding mode may be either an amide bond or a bond via an alkyl group, or a mode directly bonded to an aromatic ring (FIG. 1). The resulting polymer-bound sugar derivative can be utilized as a sugar acceptor in a conventional polymer-supported chemical glycosylation reaction or as a sugar acceptor in an enzymatic reaction. The exclusion of the oligosaccharide from the oligosaccharide-bound polymer obtained by these reactions can be efficiently excised from the polymer by an anodic oxidation electrolysis reaction. (Fig. 2)

Reference Example 1 A para-aminophenyl derivative and a polyethylene glycol derivative obtained by reducing the nitro group of para-nitrophenyl-N-acetylglucosaminide were disclosed in Japanese Patent Laid-Open Publication No. 7-319022: Oligosaccharide derivative; . & Med. Chem.
Let., 7, 255-258 (1997)) to obtain an N-acetylglucosamine-binding polymer. A glycosylation reaction was performed using the obtained N-acetylglucosamine-binding polymer (300 mg) as a sugar acceptor, an imidate derivative (20 mg) obtained from galactose in three steps as a sugar donor, and trifluoromethanesulfonic acid as an activator. (R. Schmidt, Angew. Chem. Int. Ed. Engl. 25, 2
12-235, (1986)). After removing the galactose residue from the obtained reaction mixture with sodium methoxide in methanol, the reaction residue was separated by ultrafiltration using a YM1 membrane (Amicon). The obtained polymer fraction is treated with N-acetylglucosaminidase, and then subjected to ultrafiltration (YM1).
Galβ1-6GlcNAc binding polymer was purified. FIG. 4 shows the results of measuring the compound obtained here at 500 MHz ¹ H NMR (solvent D ₂ O). The polymer fraction obtained here was used for the next electrolysis reaction. (FIG. 3) Reference Example 2 N-acetylglucosamine-binding polymer as a sugar receptor
(100 mg) and paranitrophenyl as a sugar donor
β-D-galactoside (3 mg) was added to potassium phosphate buffer (pH 60.45 ml) and dibetylformamide (0.048 ml)
Dissolve in a mixed solvent and apply β-galactosidase (Japanese Unexamined Patent Publication No. 9-313
(Adjusted according to the method of 177) (0.12 U) to carry out an enzyme reaction. After 2 hours, the reaction was stopped by heating at 100 ° C. for 5 minutes. After the reaction mixture was purified by ultrafiltration using a YM1 membrane (yield 85 mg), NMR measurement confirmed that the Galβ1-3GlcNAc-bound polymer was formed in a yield of 2%.

[0009]

EXAMPLES Example 1: Galβ1-6GlcN synthesized in Reference Example 1
Ac-bonded polymer 100mg (0.0095mmol, loading rate 50
%), 13 mmol / L sulfuric acid aqueous solution
mA current was applied for 2 hours. After neutralization with an ion exchange resin, low molecular fractions were collected with an ultrafiltration membrane (YM1), and purified with a Seppak (C18 cartridge, manufactured by Waters). The resulting solution was freeze-dried to obtain Galβ1
2 mg of -6GlcNAc was obtained (yield: quantitative).

[0010]

According to the method of the present invention, oligosaccharides constituting glycoprotein sugar chains can be easily synthesized.

[Brief description of the drawings]

FIG. 1 shows a method for bonding a glycosyl acceptor to polyethylene glycol via a para-aminophenyl group.

FIG. 2 is a schematic diagram of an oxidative electrolysis reaction.

FIG. 3 is a reaction formula of a galactose binding reaction to an N-acetylglucosamine binding polymer by a chemical synthesis technique.

FIG. 4 shows NMR of a high molecular fraction after purifying unreacted acceptors by N-acetylhexosaminidase (derived from Jack bean) by ultrafiltration after digestion.

FIG. 5 shows NMR of a polymer fraction after ultrafiltration purification.

FIG. 6 shows NMR of a low-molecular-weight fraction after purification by ultrafiltration.

Claims (1)

[Claims] 1. A method for producing an oligosaccharide, comprising synthesizing an oligosaccharide on a polymer and excising the oligosaccharide by an oxidative electrolysis reaction.