JP2001019698A - Sulfated oligosaccharide compound and its synthesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new oligosaccharide having Galβ (1-6) glycoside bond and provide a new process for producing the oligosaccharide using an enzyme β-D-galactosidase. SOLUTION: The objective new oligosaccharide having Galβ (1-6) glycoside bond, especially 6'-sulfo N-acetylisolactosamine (S6Galβ1-6GlcNA), 6'- sulfoisolactose (S6Galβ-6G1c), etc., is produced by galactosyl group transfer reaction using a β-D-galactosidase, especially a β-D-galactosidase derived from E.coli. The synthesis of oligosaccharide uses an enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for preparing Galβ (1-
6) A novel oligosaccharide having a glycosidic bond and a novel method for producing the oligosaccharide using β-D-galactosidase, specifically 4-methylumbelliferyl β
-D-galactopyranoside 6 sulfate (S6Galβ-4M
The present invention relates to the novel oligosaccharide obtained by using U) as a donor substrate and a method for producing the novel oligosaccharide.

[0002]

2. Description of the Related Art Sugar chains and carbohydrates containing a sulfate group are present on cell surfaces and the like, and function as information molecules at the time of interaction such as transmission of biological information, identification of cells, and identification phenomena.
For example, sialyl 6'-sulfo Le ^X interacts with selectins involved in cell-cell adhesion (J. Biol. Chem.
(1996) 271, 27213), and sialyl 6'-sulfolactose is present in rat and human milk and has been suggested to be important as a source of sulfate groups for infants (Pedi).
atr Res. (1985) 19,216). Recently, it has been revealed that human influenza A virus specifically binds to sulfatide (sulfated sugar chains), and its functions such as the physiological activity of sulfated sugars (sulfated sugar chains) are receiving more and more attention. I have. However, few attempts have been made to use enzymes for the synthesis of sulfated oligosaccharides. However,
It is known that in vivo synthesis of sugar chains is performed in one step by an enzyme that catalyzes the production of glycosides, a glycosyltransferase. However, glycosyltransferases are generally difficult to isolate from living organisms, require special reaction substrates, and are not common. Thus, attempts have been made to synthesize sugars based on the idea that sugar hydrolases (glycoside bond degrading enzymes) can catalyze glycosylation reactions that are the reverse of the original hydrolysis reactions. Attempts have also been made to make cells produce sugar chains having different structures using various cells.

[0003] As described above, a sugar chain having N-acetyllactosamine as a skeleton is a ligand sugar chain capable of binding to a cell adhesion molecule belonging to the selectin family and expresses an important physiological activity in vivo. I have. It is clear that the expression of these sugar chains on the cell surface is involved in specific adhesion between heterologous cells, and functional activators and inhibitors (suppression of inflammation) using these sugar chains are useful. Expected to be a drug. These sugar chains are also considered to be involved in the adhesion of microorganisms to target cells when they infect the cells, and are therefore regarded as important materials for developing drugs for protecting these diseases. As the importance of the physiological function of a sugar chain in a living body becomes clear, the present inventors have proposed that 4-methylumbelliferyl β-D-galactopyranoside 6 sulfate (S6Galβ-4MU) and N 6-Sulfo N-acetyllactosamine (S6) from acetylglucosamine (GlcNAc) using the enzyme β-D-galactosidase.
Attempts to synthesize Galβ1-4GlcNAc) or S6G
Basic research on the hydrolysis reaction of alβ-4MU using the enzyme β-D-galactosidase has been performed.

[0004]

SUMMARY OF THE INVENTION As described above, the present inventors need to synthesize new sugar chains in order to further study sugar chains present on cell surfaces and expressing various physiological activities in vivo. It was also important to know their active properties. This is the so-called sugar chain library enrichment. Therefore, an object of the present invention is to provide a novel sugar chain and a method for synthesizing the novel sugar chain.
Another object of the present invention is to find use of the obtained novel sugar chain.
In order to solve the above-mentioned problem, while further promoting the synthesis of a sugar chain by the enzyme, the present inventors have found that 4-methylumbelliferyl β-D-galactopyranoside hexasulfate (S6Galβ
-4MU) as a donor substrate, and using β-D-galactosidase from Escherichia coli as an enzyme, Galβ (1-
6) It was discovered that oligosaccharides having glycosidic bonds could be obtained. Then, utilizing the galactosyl group transfer reaction, N-acetylglucosamine (G
lcNAc), glucose, etc.
Sulfo N-acetylisolactosamine (S6Galβ1
-6GlcNAc), 6'-sulfoisolactose (S
6Galβ1-6Glc) was found to be obtained.

[0005]

A first aspect of the present invention is to use 4-methylumbelliferyl β-D-galactopyranoside hexasulfate (S6Galβ-4MU) as a donor substrate.
An oligosaccharide having a Galβ (1-6) glycoside bond obtained by a galactosyl group transfer reaction to a receptor substrate by D-galactosidase. Preferably, the oligosaccharide is characterized in that the acceptor substrate is N-acetylglucosamine, and the oligosaccharide is characterized in that the acceptor substrate is glucose. The second of the present invention is 4
-Methylumbelliferyl β-D-galactopyranoside 6-sulfate (S6Galβ-4MU) as a donor substrate, Galβ (1-6) glycoside linkage by galactosyl transfer reaction to an acceptor substrate by β-D-galactosidase This is a method for synthesizing an oligosaccharide having Preferably, the oligosaccharide is characterized in that the acceptor substrate is N-acetylglucosamine, and the oligosaccharide is characterized in that the acceptor substrate is glucose. More preferably, the method for synthesizing the oligosaccharide is characterized in that the β-D-galactosidase is derived from Escherichia coli. Still more preferably, the galactosidyl transfer reaction is carried out at 40 ° C. under alkaline conditions of pH 7 to 8. A method for synthesizing the oligosaccharide. A third aspect of the present invention is that Gal as an intermediate for the production of a drug having a bioactive function.
An oligosaccharide having a β (1-6) glycosidic bond is used. Preferably, the oligosaccharide is 6′-sulfo N-acetylisolactosamine (S6Galβ1-6GlcN
A) or 6′-sulfoisolactose (S6Galβ
1-6Glc), wherein the oligosaccharide is used. The present invention provides a method of galactosyl transfer of a specific donor substrate to an acceptor substrate to obtain a Galβ (1-6)
Β, an enzyme that produces oligosaccharides with glycosidic bonds
The present invention has solved the above-mentioned problem by discovering -D-galactosidase.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail. The synthesis method of the oligosaccharide having a novel sugar chain bond of the present invention is basically composed of: Donor substrate, 4-methylumbelliferyl β-
D-galactopyranoside hexasulfate (S6Galβ-4M
U) is to produce an oligosaccharide having a Galβ (1-6) glycosidic bond by a galactosyl transfer reaction to an acceptor substrate using an enzyme in a buffer solution. As the enzyme, β-D-galactosidase derived from Escherichia coli is particularly used. As the buffer of the buffer solution, compounds such as sodium phosphate and sodium acetate can be used. As the pH, the activity range of the enzyme, for example, neutral to alkali, can be selected. As the acceptor substrate, saccharides such as N-acetylglucosamine (GlcNAc) and glucose can be used. 2. In the synthesis reaction of oligosaccharides having a novel sugar chain bond, the donor substrate and the acceptor substrate are reacted with each other, for example, by the optimal reaction pH of an enzyme.
Then, an enzyme, for example, β-D-galactosidase is added to the solution, the reaction is adjusted to an optimal reaction temperature condition of the enzyme, and the reaction is allowed to proceed. Then, the boiling reaction is stopped. The solution after the reaction is stopped is subjected to centrifugation to remove the precipitate, and the reaction solution from which the precipitate has been removed is subjected to separation and purification of sugar chains, for example, ion exchange chromatography [Sep-PacAccel QMA column (Wat
ers Co., Ltd., φ1.0 × 4.0 cm)]. The non-adsorbed portion is removed by washing with distilled water, etc.
Elution is performed by a linear concentration gradient method such as a buffer solution or a pyridine acetate buffer. The linear concentration gradient method is, for example, 0 to 0.5 M
A pyridine acetate buffer (pH 5.4) having a linear gradient as described above is used. Thus, the target compound Galβ (1-
6) An oligosaccharide having a glycosidic bond, for example, 6'-sulfo N-acetylisolactosamine (S6Galβ1-6
GlcNAc), 6'-sulfoisolactose (S6G
alβ1-6Glc) is obtained. This reaction process is shown in FIG.

[0007] The enzyme used in the present invention may be any enzyme capable of promoting the galactosyl transfer reaction, and β-D-galactosidase derived from Escherichia coli is preferred. Β-
D-galactosidase can be obtained from Nakarai Tesque Co., Ltd., Wako Pure Chemical Industries, Ltd. or the like. The β-D-galactosidase has the following physicochemical properties. Action: The galactosyl group is transferred to the 6-position hydroxyl group of the acceptor substrate. Substrate specificity: lactose, aryl or alkyl β-
D-galactoside, etc. Optimal reaction pH: 7-8 Inhibition and activation: Inhibited by Hg ²⁺ and Ag ²⁺ . Molecular weight: 464,992 (Proc. Natl. Acad. Sci. USA (187
7) See 74,1507-1510)

The oligosaccharide having a Galβ (1-6) glycosidic bond of the present invention includes those containing a sialic acid residue. Specifically, 6′-sulfo N-acetylisolactosamine containing a sialic acid residue (S6Galβ1-6G
lcNA), 6'-sulfoisolactose (S6Gal
β1-6Glc) and the like.

[0009]

EXAMPLE 1 50 mg of 4-methylumbelliferyl β-D-galactopyranoside hexasulfate (S6Galβ-4MU) and 132 mg of N-acetylglucosamine (GlcNAc) were added in 0.1 parts.
500 μl of M acetate buffer solution (pH 6.0) and 250 μl of water
and 250 μl (76 U) of a β-D-galactosidase solution derived from Escherichia coli was added. After performing the reaction at 40 ° C. for 30 hours, the reaction was stopped by boiling for 5 minutes. After removing the precipitate by centrifugation, ion-exchange chromatography Sep-PacAccelQMA washed with 10 ml of distilled water
Purification was performed using a column (Waters, φ1.0 × 4.0 cm, flow rate 0.5 ml / ml). FIG. 2 shows the state of the separation of the reaction product by ion exchange chromatography. After washing the non-adsorbed portion with 10 ml of distilled water, the adsorbed portion of 7.0 ml of 0.1 M sodium chloride solution was eluted (fractions 9 to 2).
2). After collecting and concentrating each adsorption part, the BioLC system (D
IONEX, CarboPacPA1, φ4 × 250
mm, the flow rate was 1.0 ml / ml, and the eluate was subjected to 100 mM NaOH containing 0.4 M sodium acetate). FIG. 3 shows the reaction mixture before purification and the analysis result after purification.
The peak which seems to be a glycosyl transfer product is 10 to 13.5 min.
This fraction was fractionated and analyzed by ¹ H-NMR. FIG. 4 shows the obtained spectrum. As a result, it was revealed that the synthesized compound was 6′-sulfo N-acetylisolactosamine (S6Galβ1-6GlcNAc).

Example 2 The reaction was carried out at a temperature of 35 ° C., 40 ° C. and 48 ° C.
The temperature characteristics of the sugar transfer reaction were investigated. The result is shown in FIG. A particular temperature, about 40 ° C., was found to be the optimal temperature.

Example 3 The above reaction was carried out at pH conditions of 4, 5, 6, 7 and 8, and the pH characteristics of the sugar transfer reaction were examined. The result is shown in FIG.
Shown in pH 7-8 was found to be the optimal pH.

Example 4 50 mg of 4-methylumbelliferyl β-D-galactopyranoside hexasulfate (S6Galβ-4MU) and 132 mg of N-acetylglucosamine (GlcNAc) were added in 0.1 mg.
500 μl of M phosphate buffer solution (pH 6.0) and 250 μl of water
and 250 μl (76 U) of a β-D-galactosidase solution derived from Escherichia coli was added. After performing the reaction at 40 ° C. for 30 hours, the reaction was stopped by boiling for 5 minutes. The reaction solution from which the precipitate was removed by centrifugation was subjected to ion exchange chromatography BioLC system (DIONEX, CarboPac PA1, φ4 × 250m
m, flow rate 1.0 ml / ml, eluate contains 0.4 M sodium acetate1
(00 mM NaOH). As shown in FIG. 7, a peak considered to be a glycosyltransfer product was confirmed.
Sep-Pac Accel QMA column (Wat
ers, φ1.0 × 4.0cm, flow rate 0.5ml /
ml). After washing the non-adsorption part with distilled water,
The adsorbed part was eluted by a linear concentration gradient method using 0.5 M pyridine acetate buffer (pH 5.4). FIG. 8 shows the separation pattern. When a part of the adsorption part was analyzed by a BioLC system, the target product was detected in fractions 21 to 24 (the vertical axis in FIG. 8 indicates the color development intensity of the sugar by the phenol-sulfuric acid method), so the concentration was performed. After drying, analysis by ¹ H-NMR was performed. FIG. 9 shows the result. The results revealed that the synthesized compound was 6′-sulfo N-acetylisolactosamine (S6Galβ1-6GlcNAc).

Example 5 4-methylumbelliferyl β-D-galactopyranoside hexasulfate (S6Galβ-4MU) (50 mg) and glucose (Glc) (108 mg) were added to 0.1 phosphate buffer (500 μM).
and 250 μl of water, and 250 μl (76 U) of a β-D-galactosidase solution derived from E. coli was added. 4
After performing the reaction at 0 ° C. for 30 hours, the reaction was stopped by boiling for 5 minutes. FIG. 10 shows the synthesis reaction. The reaction solution from which the precipitate was removed by centrifugation was transferred to a BioLC system (DIONEX, Car
The analysis was performed using boPac PA1, φ4 × 250 mm, the flow rate was 1.0 ml / ml, and the eluate was 100 mM NaOH containing 0.4 M sodium acetate). The result is shown in FIG. From this result, a peak considered to be a glycosyltransfer product was confirmed, and thus the reaction solution was washed with 10 ml of distilled water using a Sep-Pac Accel QMA column (Waters, φ
1.0 × 4.0 cm, flow rate 0.5 ml / ml). After washing the non-adsorbed portion with distilled water, the adsorbed portion was eluted by a linear concentration gradient method of 0 to 0.5 M pyridine acetate buffer (pH 5.4). FIG. 12 shows the separation pattern. When a part of the adsorption part was analyzed with the BioLC system, fractions 21 to 2 were found.
Since the target product was detected in 4 and concentrated to dryness, ¹
Analysis by H-NMR was performed. As a result (FIG. 13),
The synthesized compound is 6'-sulfoisolactose (S6
Galβ1-6Glc).

By subjecting the novel sugar chain synthesized in each of the above examples to sialylation treatment, a compound having a physiologically active function such as an anti-inflammatory agent or a cancer metastasis inhibitor can be synthesized. The biological activity of sugars having residues can be expected.

[0015]

As described above, the novel method for synthesizing a sugar chain and the provision of a novel sugar chain are intended to enrich a sugar library for studying new properties of a sugar chain and to provide a drug having a new physiological function. It has an excellent effect of contributing to the development of the product.

[Brief description of the drawings]

FIG. 1. Synthesis of sulfated disaccharide (S6Galβ1-6GlcNAc) by β-D-galactosidase

Fig. 2 Separation of reaction products by ion exchange chromatography

FIG. 3 Analysis result of a reaction solution (Example 1) by a BioLC system

FIG. 4. 6′-Sulfo N-acetylisolactosamine (S6Galβ1-6GlcNAc) ¹ H- in heavy water
NMR spectrum

FIG. 5: Effect of temperature on sugar transfer reaction

FIG. 6: Effect of pH on sugar transfer reaction

FIG. 7: Analysis result of reaction solution (Example 4) by BioLC system

FIG. 8: Separation pattern of target product (Example 4) by Sep-Pac

FIG. 9: 6'-Sulfo N-acetylisolactosamine (S6Galβ1-6GlcNAc) ¹ H- in heavy water
NMR spectrum

FIG. 10. S6Ga by β-D-galactosidase
Synthesis of lβ1-6Glc

FIG. 11 shows an analysis result of a reaction solution (Example 5) by a BioLC system.

FIG. 12: Desired product by Sep-Pac (Example 5)
Isolated pattern

FIG. 13: 6'-Sulfoisolactose (S6Galβ1-6Glc) ¹ H-NMR spectrum in heavy water

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 19/26 C12P 19/26 // C12N 9/38 C12N 9/38 F-term (Reference) 4B050 CC10 DD02 LL05 4B064 AF03 AF21 CA02 CA21 CB07 CB30 CC03 CC06 CC07 CD09 CD12 DA01 4C057 AA03 BB01 BB04

Claims (11)

[Claims] 1. Use of 4-methylumbelliferyl β-D-galactopyranoside hexasulfate (S6Galβ-4MU) as a donor substrate obtained by a galactosyl group transfer reaction to an acceptor substrate by β-D-galactosidase. G
An oligosaccharide having an alβ (1-6) glycosidic bond. 2. The oligosaccharide according to claim 1, wherein the acceptor substrate is N-acetylglucosamine. 3. The oligosaccharide according to claim 1, wherein the acceptor substrate is glucose. 4. A method for preparing Galβ (4-methylumbelliferyl β-D-galactopyranoside 6-sulfate (S6Galβ-4MU) as a donor substrate by a galactosyl transfer reaction to an acceptor substrate by β-D-galactosidase. 1
-6) A method for synthesizing an oligosaccharide having a glycosidic bond. 5. The method for synthesizing an oligosaccharide according to claim 4, wherein the acceptor substrate is N-acetylglucosamine. 6. The method for synthesizing an oligosaccharide according to claim 4, wherein the acceptor substrate is glucose. 7. The method for synthesizing an oligosaccharide according to claim 4, wherein the β-D-galactosidase is derived from Escherichia coli. 8. The method for synthesizing an oligosaccharide according to claim 4, wherein the galactosyl transfer reaction is carried out under alkaline conditions at 40 ° C. and pH 7 to 8. 9. Use of an oligosaccharide having a Galβ (1-6) glycoside bond as an intermediate for the production of a drug having a physiologically active function. 10. The use of the oligosaccharide according to claim 9, wherein the oligosaccharide having a Galβ (1-6) glycoside bond is 6′-sulfoN-acetylisolactosamine (S6Galβ1-6GlcNAc). 11. An oligosaccharide having a Galβ (1-6) glycosidic bond is a 6′-sulfoisolactose (S6Ga
Use of the oligosaccharide according to claim 9, wherein the oligosaccharide is 1β1-6Glc).