JP3035748B2 - Glycolipids obtained by glycosyltransfer reaction - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel glycolipid obtained by a transglycosylation reaction.

[0002]

2. Description of the Related Art Glycolipids are a group of substances comprising sugar and lipid as components, and have both a hydrophilic part and a hydrophobic part in the molecule, and are present on the surface of biological membranes and the like. Regarding the role of glycolipids in the living body, mutual recognition, division, proliferation, differentiation, immunity, etc. of cells are known, and it is thought that they play an important role also in physiological functions. And
For research targeting them, synthesis of glycolipids having a natural sugar chain structure and novel glycolipids has been attempted. Conventionally, chemical synthesis of lactosylceramide and the like is well known, but there is a problem in that the operation is complicated and dangerous, and many by-products are generated. Methods using transferases have also been performed, but these enzymes are unstable and have low specific activity, and require active sugars as substrates. Further, by using a transferase, it is possible to synthesize a glycolipid having a natural sugar chain structure, but there is also a problem that a new glycolipid cannot be synthesized.

On the other hand, enzymes that hydrolyze saccharides, such as β-galactosidase, are known to give sugars by a transglycosylation reaction, and are used for the synthesis of oligosaccharides and the like.
No attempt has been made to use it for the synthesis of glycolipids.

[0004]

SUMMARY OF THE INVENTION The present inventors have conducted intensive studies on a method for synthesizing a glycolipid having a novel sugar chain structure, and as a result, have found that the transglycosylation of β-galactosidase, which has been conventionally known, has been carried out. Utilizing and using glycolipid and lactose as substrates, it was found that a novel glycolipid could be synthesized, and the present invention was completed.

[0005]

Means for Solving the Problems The first aspect of the present invention is a glycolipid represented by the following general formula (I) obtained by a glycosyltransfer reaction of β-galactosidase using glycolipid and lactose as substrates. General formula Galβ1 → 4 Galβ1 → X Glcβ1 → 1 Cer (I) X = 4 or 6 (In the formula, Gal represents a galactose residue, Glc represents a glucose residue, and Cer represents a ceramide residue. It represents a binding site.) Lactosylceramide can be used as a glycolipid, and lactose can be used as a substrate to give a β-galactosidase by transglycosylation.

Further, the second invention is a glycolipid represented by the following general formula (II) obtained by a glycosyltransfer reaction of β-galactosidase using glycolipid and lactose as substrates. General formula Galβ1 → 4 Galβ1 → 6 Galβ1 → 1 Cer (II) (In the formula, Gal represents a galactose residue and Cer represents a ceramide residue.) Galactosylceramide is used as a glycolipid and β is used as a substrate with lactose as a substrate. -It can be obtained by transglycosylation of galactosidase.

[0007] In the method for synthesizing glycolipids of the present invention, β-galactosidase is allowed to act on a substrate composed of glycolipid and lactose, and a glycolipid is provided with a galactose residue by a transglycosylation reaction. Examples of glycolipids that can be used in the present invention include neutral glycolipids such as glucosylceramide, galactosylceramide, and lactosylceramide. The origin of β-galactosidase that can be used in the present invention is not particularly limited, but β-galactosidase derived from Bacillus circulans is preferably used.

Next, the method for synthesizing the glycolipid of the present invention will be described in detail. Lactose 5% or more, preferably 10%
A neutral lipid such as glucosylceramide, galactosylceramide, or lactosylceramide dissolved using a bile acid-based surfactant such as sodium taurocholate or sodium cholate is added to the above-dissolved solution to prepare a substrate solution. Then, by adding β-galactosidase to this substrate solution and reacting for about 5 to 15 hours, a galactose residue can be provided to the glycolipid. At this time, β
-It is necessary to consider the optimal pH and optimal temperature of galactosidase. Therefore, an appropriate buffer is used for dissolving the substrate. By doing so, a galactose residue can be imparted to the glycolipid.

[0009] The glycolipid thus produced was separated by the following method. (1) Silica Gel Chromatography of Glycolipids Purified glycolipids were fractionated by a conventional method using silica gel chromatography with a mixed system of chloroform, methanol and water. (2) Silica gel chromatography of acetylated glycolipids Using pyridine-acetic anhydride, each glycolipid fractionated by silica gel chromatography of glycolipids in (1) was acetylated by heating at 80 ° C. for 30 minutes. Then, the acetylated glycolipid was passed through a column filled with silica gel, and eluted with a solvent system of dichloroethane: acetone = 85: 15 to obtain each fraction. This fraction was deacetylated using a methanol solution containing 0.1 N sodium hydroxide.

[0010] The structure analysis of the formed glycolipid was performed by the following method. (1) Confirmation of sugar chain binding by proton nuclear magnetic resonance spectrum, edited by Tokyo Kagaku Doujin, “Seizoku Chemistry Experiment Course 4 Complex Carbohydrate Research”
II ”, Chapter 8, Nuclear Magnetic Resonance, 8.3 One-Dimensional PM of Glycolipids
According to the method described in R 1, a proton nuclear magnetic resonance spectrum of the formed glycolipid was measured. As a result, it was confirmed that all the sugar chain structures of the formed glycolipid were β-linked. (2) Determination of the sugar chain binding position by methylation analysis, edited by Tokyo Kagaku Doujin, “Seismic Chemistry Experiment Course 4
II ”, Chapter 6, Determination of Sugar Chain Structure, 6.4 Determination of Carbon Position by Methylation Method, and the generated glycolipids were subjected to methylation analysis. (3) Quantification of constituent sugars by hydrolysis with hydrochloric acid According to the method described in Tokyo Kagaku Dojin, “Biochemistry Experiment Course 4 Chemistry of Carbohydrates (below)”, 11 Gas Chromatography, 11.2 Gas Chromatography of Monosaccharides Quantitation was performed.

[0011]

Next, the present invention will be described in more detail with reference to Examples. Example 1 4 mg of galactosylceramide as a glycolipid was dissolved in 0.1 ml of a 20% sodium taurodeoxycholate solution,
M acetic acid-sodium acetate buffer (pH 5.5) 0.1 ml, 40%
After adding 0.5 ml of lactose solution and 5 IU of β-galactosidase derived from Bacillus circulans (manufactured by Daiwa Kasei Co., Ltd.), water was added to make the total volume of the reaction solution 1 ml. Then, the mixture was maintained at 37 ° C. and reacted for 14 hours.

After the completion of each reaction, the reaction solution was separated by Seppak C ₁₈
A cartridge (trade name) was treated to remove sodium taurocholate, enzymes, lactose, and a buffer to obtain a formed glycolipid. The results of thin-layer chromatography of the glycolipid thus obtained are shown in FIG. In the figure, the leftmost lane is the total fraction, and from the top, the starting material galactosylceramide, CD which is a disaccharide transfer reaction product
H, surfactant sodium taurodeoxycholate. A few bands of light coloration were observed in the vicinity of the sodium taurodeoxycholate band, suggesting the formation of a glycolipid having a sugar chain of three or more sugars. 1 to 9
Is obtained by further separating and purifying the total fraction, and is almost a single band. Developing solution: chloroform / methanol / water (65: 25: 4 (volume)), coloring: orcinol-sulfuric acid.

The results of NMR spectrum analysis of the fractions corresponding to Nos. 4, 6, 7, and 8 in FIG. 1 are shown below. Fraction Structure ppm (Hz) 4 Galβ1 → 4 GalCer 4.07 (7.7) 4.27 (7.9) 6 Galβ1 → 6 GalCer 4.18 (7.9) 4.38 (7.9) 7 Galβ1 → 4 Galβ1 → 4 GalCer 4.07 (7.7) 4.29 (7.5) 4.26 (7.7) 8 Galβ1 → 4 Galβ1 → 6 GalCer 4.10 (7.9) 4.29 (7.9) 4.26 (7.8) Regarding the fraction corresponding to No. 8 in Fig. 1, from the results of quantification of constituent sugars by hydrochloric acid hydrolysis It consists of three molecules of galactose. From the result of methylation analysis, galactose is 1 →
It was found that there were 4 bonds and 1 → 6 bonds. Therefore, the structural formula of this glycolipid is Galβ1 → 4 Galβ1 → 6 G
alβ1 → 1 Cer, which proved to be a novel glycolipid.

Example 2 4 mg of lactosylceramide as a glycolipid was dissolved in 0.1 ml of a 20% sodium taurodeoxycholate solution, 0.1 ml of 0.5 M acetic acid-sodium acetate buffer (pH 5.5), 0.5 ml of a 40% lactose solution After adding 5 IU of β-galactosidase derived from Bacillus circulans (manufactured by Daiwa Kasei Co., Ltd.), water was added to adjust the total volume of the reaction solution to 1 ml. Then, the mixture was maintained at 37 ° C. and reacted for 14 hours.

After the completion of each reaction, the reaction solution was added to Seppak C ₁₈
A cartridge (trade name) was treated to remove sodium taurocholate, enzymes, lactose, and a buffer to obtain a formed glycolipid. The result of the thin-layer chromatography of the glycolipid thus obtained is shown in FIG. In the figure, the lane on the left is the total fraction, from the top, galactosylceramide as a degradation product, lactosylceramide as a starting material, and sodium taurodeoxycholate as a surfactant. A few bands of light coloration were observed in the vicinity of the sodium taurodeoxycholate band, suggesting the formation of a glycolipid having a sugar chain of three or more sugars. 1 to 5
Is obtained by further separating and purifying the generated glycolipid having a sugar chain of three or more sugar chains, and is almost a single band.
Developing solution: chloroform / methanol / water (65: 25: 4 (volume)), coloring: orcinol-sulfuric acid.

The results of NMR spectrum analysis of the fractions corresponding to Nos. 1 and 4 in FIG. 2 are shown below. Fraction Structure ppm (Hz) 1 Galβ1 → 4 Galβ1 → 4 GlcCer 4.20 (7.9) 4.35 (7.9) 4.27 (7.7) 4 Galβ1 → 4 Galβ1 → 6 GlcCer 4.11 (7.9) 4.27 (7.9) 4.21 (7.9) Regarding the substance of the fraction corresponding to No. 1, from the result of quantification of constituent sugars by hydrolysis with hydrochloric acid, it was composed of one molecule of glucose and two molecules of galactose, and the result of methylation analysis showed that galactose and galactose had 1 → 4 bonds, It was found that galactose and glucose were linked 1 → 4.
Therefore, the structural formula of this glycolipid is Galβ1 → 4 Galβ
1 → 4 Galβ1 → 1 Cer, which proved to be a novel glycolipid.

The fraction corresponding to No. 4 in FIG. 2 is composed of one molecule of glucose and two molecules of galactose from the results of quantification of constituent sugars by hydrolysis with hydrochloric acid, and galactose and galactose from the results of methylation analysis. Was 1 → 4 bond, and galactose and glucose were 1 → 6 bond. Therefore, the structural formula of this glycolipid is Galβ
1 → 4 Galβ1 → 6 Galβ1 → 1 Cer, which proved to be a novel glycolipid.

Examples 3 and 4 Galactosylceramide 800 mg (Example 3) and lactosylceramide 800 mg (Example 4) were used as glycolipids.
The reaction was carried out in the same manner as in Examples 1 and 2 except that the operation was performed on a scale of 00 times, and fractionation was performed by silica gel chromatography. Table 1 shows the yield of each fraction.

[0019]

[Table 1] Structural analysis was performed on each fraction in the same manner as in Examples 1 and 2, and similar results were obtained.

[0020]

The novel glycolipid of the present invention contributes to medical and biochemical research. Moreover, this glycolipid can be produced in large quantities under mild conditions, and can be supplied at low cost as medicines and reagents.

[Brief description of the drawings]

FIG. 1 shows the results of thin-layer chromatography of the glycolipid obtained in Example 1.

FIG. 2 is a view showing the results of thin-layer chromatography of the glycolipid obtained in Example 2.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C07H 15/10 C12P 19/00-19/64 BIOSIS (DIALOG) CA (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A glycolipid represented by the following general formula (I) obtained by a glycosyltransfer reaction of β-galactosidase using glycolipid and lactose as substrates. General formula Galβ1 → 4 Galβ1 → X Glcβ1 → 1 Cer (I) X = 4 or 6 (In the formula, Gal represents a galactose residue, Glc represents a glucose residue, and Cer represents a ceramide residue. Represents a binding site.) 2. A glycolipid represented by the following general formula (II) obtained by a glycosyltransfer reaction of β-galactosidase using glycolipid and lactose as substrates. General formula Galβ1 → 4 Galβ1 → 6 Galβ1 → 1 Cer (II) (In the formula, Gal represents a galactose residue, and Cer represents a ceramide residue, respectively.)