JPH06298806A - Novel branched cyclodextrin and method for producing the same - Google Patents

Abstract

(57) [Summary] [Structure] A novel galactosyl-cyclodextrin in which a galactosyl group is bonded to the 2- or 3-position hydroxyl group of the glucosyl group of cyclodextrin by an α bond, and a solution containing cyclodextrin and an α-galactosyl sugar compound A method for producing a novel galactosyl-cyclodextrin in which a galactosyl group is bonded to the 2- or 3-position hydroxyl group of the glucosyl group of cyclodextrin by an α bond, characterized by allowing α-galactosyl transferase to act on. According to the present invention, the galactosyl group is bound to the secondary hydroxyl group of the glucosyl group in the CD molecule, that is, the 2- or 3-position hydroxyl group by an α bond by utilizing the glycosyl transfer action of α-galactosyltransferase. It is possible to efficiently obtain the new branched CD. The novel branched CD of the present invention is expected to be widely used in the fields of foods, cosmetics, etc. in addition to the fields of medicines.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel branched cyclodextrin and a method for producing the same. The present invention also relates to a novel galactosyl-cyclodextrin and a method for producing the novel galactosyl-cyclodextrin utilizing the glycosyl transfer activity of an enzyme.

[0002]

BACKGROUND OF THE INVENTION Cyclodextrin (hereinafter abbreviated as CD) is a cyclic dextrin in which glucose is linked by α-1,4 bonds.
The α-, β-, and γ-CDs consisting of 6, 7 and 8 glucoses are well known. Recently, in order to improve the solubility of CD, branched CDs in which a glucosyl group or a maltosyl group is bonded to these CDs by α-1,6 bonds have been synthesized.

These CDs and branched CDs have cavities inside the molecules, and since the insides of the cavities are made hydrophobic, they have an inclusion effect and have the property of incorporating various oily substances. CDs and branched CDs have such properties and are therefore widely used in the fields of food industry, cosmetics industry, pharmaceutical industry, and the like.

Recently, in the field of the pharmaceutical industry, in order to reduce side effects of drugs, attention is paid to the cell recognizability of sugars, and this is used as a sensor for labeled cells of drug carriers of drug delivery systems. Research is actively conducted. In particular, it is well known that galactose has a strong affinity for liver tissue and mannose has a strong affinity for liver parenchymal cells, non-parenchymal cells, and macrophages.

Previously, in view of the above-mentioned situation, we have developed a galactosyl-branched CD or a mannosyl-branched CD in which a galactosyl group or a mannosyl group is bonded to a glucosyl group of a branched cyclodextrin side chain, and a glucosyl group of CD. Have succeeded in developing galactosyl-CD and mannosyl-CD in which a galactosyl group or a mannosyl group is bonded to the 6-position hydroxyl group of the above.

[0006] Therefore, the present inventors utilize the inclusion property of CD and this characteristic of galactose to apply it to a drug delivery system.
Secondary hydroxyl group of glucosyl group of ring, ie 2- or 3-position
An attempt was made to synthesize galactosyl-CD in which a galactosyl group was directly bonded to a hydroxyl group at a position. As a result, a commercially available α-galactosyltransferase synthesizes a galactosyl-CD from an α-galactosyl sugar compound by transferring the galactosyl group to the secondary hydroxyl group of the glucosyl group of α-, β- and γ-CD by α bond. I found that Among these, α-galactosyltransferase derived from unripe coffee beans is
It has been found that galactosyl-CD in which a galactosyl group is transfer-bonded to the secondary hydroxyl groups of glucosyl groups of −, β- and γ-CD by an α bond is efficiently synthesized.

[0007]

That is, the present invention is a CD
Of a novel galactosyl-CD in which a galactosyl group is bound to the 2- or 3-position hydroxyl group of the glucosyl group of α-galactosyl group and a solution containing CD and an α-galactosyl sugar compound with α-galactosyltransferase The present invention provides a method for producing a novel galactosyl-CD in which the galactosyl group is bonded to the 2- or 3-position hydroxyl group of the glucosyl group of CD by α bond.

The novel branched CD according to the present invention can be represented by structural formulas I to III shown in FIG.

The novel branched CD of the present invention can be obtained by allowing an α-galactosyltransferase to act on a solution containing CD and an α-galactosyl sugar compound. In the present invention, CD includes α-CD, β-CD and γ.
-CD and any of these branched CDs, or a mixture thereof.

The α-galactosyl sugar compound (hereinafter referred to as a sugar donor) used in the present invention includes, for example, melibiose, raffinose, phenyl-α-galactoside, paranitrophenyl-α-galactoside, α-galactooligosaccharide and the like. Glycosides containing α-galactosyl groups, oligosaccharides or polysaccharides, partial degradation products thereof, and mixtures thereof can also be used.

The α-galactosyltransferase used in the present invention decomposes a sugar donor when it is allowed to act on a solution containing an α-galactosyl sugar compound and CD, and its α-
The galactosyl group is converted to the secondary hydroxyl group of the glucosyl group of CD α
Any compound can be used as long as it is transferred by binding to synthesize α-galactosyl-CD.

The α-galactosyltransferase used in the present invention is widely distributed in nature. For example,
Enzymes derived from plants such as unripe coffee beans, and enzymes derived from microorganisms such as Aspergillus niger, Escherichia coli, and Mortierella vinase are well known.

In the reaction system of the present invention, the solution (aqueous solution or suspension) containing CD and the sugar donor has a CD concentration of about 1 to 50% (W / W) and a sugar donor concentration of about 1%. ~ 90%
(W / W) is desirable, and the ratio (weight) of sugar donor to CD varies depending on the type of sugar donor used, but is in the range of 0.1 to 50 times, preferably 0.3 to
It is suitable to set the range twice. Also, water and hydrophilic organic solvents such as dimethyl sulfoxide, methanol,
A mixed solution with acetone or the like can also be used.

The pH of the reaction solution is 3 to 10, preferably 4 to
9. The temperature is adjusted to 20 to 70 ° C., preferably 30 to 60 ° C., and the reaction is appropriately performed. Since the amount of enzyme used is closely related to the reaction time, the reaction is usually 5-100.
The amount of enzyme may be such that it is completed in a time, preferably 5 to 20 hours, but the amount is not limited thereto.

The liquid obtained by reacting by the above method is subjected to high performance liquid chromatography to fractionate and collect transfer products to CD, and then to enzymatic decomposition method, FAB-M.
As a result of molecular weight measurement by S and structural analysis by nuclear magnetic resonance (NMR), it was confirmed to be a branched CD represented by Structural Formulas I to III shown in FIG.

[0016]

EXAMPLES Next, the present invention will be specifically described by way of examples, but the present invention is not limited to these. Example 1 (1) Transfer Reaction 1 g of melibiose and 0.5 g of α-CD were added to 0.4 ml of a 50 mM acetate buffer (pH 6.
5) After being dissolved in 4.0 ml, 16 units of unripe coffee bean α-galactosyltransferase (manufactured by Sigma) was added,
The reaction was carried out at 40 ° C for 48 hours. The result of having analyzed a part of reaction liquid by a high performance liquid chromatography is shown in FIG.

After the reaction was completed, the solution in which the enzyme was inactivated by heat was subjected to high performance liquid chromatography using an amino column to separate the transfer product A. Isolated transfer product A
Was analyzed by high performance liquid chromatography using an ODS-based column, and it was separated into two peaks (Fig. 3). As a result, each of the transfer products A1 was 13
7 mg and 8 mg of transfer product A2 were obtained.

(2) Structural analysis The transfer product A1 isolated in the above (1) is FAB-M.
From the S analysis and ¹³ C-NMR analysis, it was found that the present invention has a structure in which one molecule of a galactosyl group is α-bonded to the 6-position hydroxyl group of the glucosyl group in the CD ring of α-CD previously found by the present inventor ( Japanese Patent Application No. 4-114304). Further, the transfer product A2 was found to have a molecular weight of 1135 by FAB-MS analysis (FIG. 4).

Further, as shown in FIG. 5, it was completely decomposed into unequal molar amounts of galactose and α-CD by α-galactosidase of unripe coffee beans. From these, the transfer product A2 has one molecule of galactosyl group in α-CD.
It turned out to be a combination. Furthermore, since the transfer product A2 has a retention time different from that of the transfer product A1 according to the analysis by high performance liquid chromatography using an ODS-based column, the 2-position of the glucosyl group in the CD ring of α-CD or It was found that one molecule of galactosyl group was α-bonded to the 3-position hydroxyl group.

Example 2 (1) Transfer Reaction 1 g of melibiose and 0.4 g of β-CD were added to a 50 mM acetate buffer solution (pH 6.4) containing 0.4 ml of dimethyl sulfoxide.
5) After being dissolved in 4.0 ml, 16 units of unripe coffee bean α-galactosyltransferase (manufactured by Sigma) was added,
The reaction was carried out at 40 ° C for 48 hours. The result of having analyzed a part of reaction liquid by a high performance liquid chromatography is shown in FIG.

After the completion of the reaction, the heat-inactivated solution of the enzyme was subjected to high performance liquid chromatography using an amino column to fractionate the transfer product B. Isolated transfer product B
Was analyzed by high performance liquid chromatography using an ODS-based column, and it was separated into two peaks (Fig. 7). As a result, each of the transfer products B1 was 10
5 mg and 6 mg of the transfer product B2 were obtained.

(2) Structural analysis The transfer product B1 isolated in the above (1) is FAB-M.
From the S analysis and ¹³ C-NMR analysis, it was found that the present invention has a structure in which one molecule of a galactosyl group is α-bonded to the 6-position hydroxyl group of the glucosyl group in the CD ring of β-CD previously found by the present inventor ( Japanese Patent Application No. 4-114304). The transfer product B2 was found to have a molecular weight of 1297 by FAB-MS analysis.

Further, as shown in FIG. 8, it was completely decomposed into unequal molar amounts of galactose and β-CD by α-galactosidase of unripe coffee beans. From these results, the transfer product B2 has one molecule of galactosyl group in α-CD with α
It turned out to be a combination. Furthermore, the transfer product B2 has a retention time different from that of the transfer product B1 according to an analysis by high performance liquid chromatography using an ODS-based column, and therefore, the 2-position of the glucosyl group in the CD ring of β-CD or It was found that one molecule of galactosyl group was α-bonded to the 3-position hydroxyl group.

Example 3 (1) Transfer Reaction 1 g of melibiose and 0.5 g of γ-CD were added to a 50 mM acetate buffer solution (pH 6.4) containing 0.4 ml of dimethyl sulfoxide.
5) After dissolving in 4.0 ml, α derived from unripe coffee beans
-Galactosyl transferase (manufactured by Sigma) was added in 16 units and reacted at 40 ° C for 48 hours. The result of having analyzed a part of reaction liquid by a high performance liquid chromatography is shown in FIG.

After the reaction was completed, the solution in which the enzyme was inactivated by heat was subjected to high performance liquid chromatography using an amino column to fractionate the transfer product C. Isolated transfer product C
Was analyzed by high performance liquid chromatography using an ODS column, and two peaks were separated (Fig. 1
0), the isolation of each resulted in the transfer product C1 being 14
6 mg and 9 mg of the transfer product C2 were obtained.

(2) Structural analysis The transfer product C1 isolated in the above (1) is FAB-M.
From the S analysis and ¹³ C-NMR analysis, it was found that the present invention has a structure in which one molecule of a galactosyl group is α-bonded to the 6-position hydroxyl group of the glucosyl group in the CD ring of γ-CD found by the present inventor ( Japanese Patent Application No. 4-114304). The transfer product C2 was found to have a molecular weight of 1459 by FAB-MS analysis.

Further, as shown in FIG. 11, it was completely decomposed into unequal molar amounts of galactose and γ-CD by α-galactosidase of unripe coffee beans. Than these things,
It was found that the transfer product C2 was one in which one molecule of galactosyl group was α-bonded to γ-CD. Furthermore, the transfer product C2 has a retention time different from that of the transfer product C1 according to an analysis by high performance liquid chromatography using an ODS-based column. It was found that one molecule of galactosyl group was α-bonded to the 3-position hydroxyl group.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, by utilizing the glycosyl transfer activity of α-galactosyltransferase, α is attached to the secondary hydroxyl group of the glucosyl group in the CD molecule, that is, the 2- or 3-position hydroxyl group.
It is possible to efficiently obtain a novel branched CD having a galactosyl group bonded by the bond. Novel branched CD of the present invention
Is expected to be widely used in the fields of food, cosmetics, etc. in addition to the field of medicine.

[Brief description of drawings]

FIG. 1 shows the structure of a branched CD obtained according to the present invention.

2 is a high performance liquid chromatogram of the reaction liquid of Example 1. FIG.

FIG. 3 is a high performance liquid chromatogram of the transfer product A of Example 1.

FIG. 4 is a FAB-MS analysis of the transfer product A2 of Example 1.

5 is a high performance liquid chromatogram of the enzymatic decomposition solution of the transfer product A2 of Example 1. FIG.

FIG. 6 is a high performance liquid chromatogram of the reaction liquid of Example 2.

FIG. 7 is a high performance liquid chromatogram of the transfer product B of Example 2.

FIG. 8 is a high performance liquid chromatogram of the enzymatic decomposition solution of the transfer product B2 of Example 2.

FIG. 9 is a high performance liquid chromatogram of the reaction liquid of Example 3.

10 is a high performance liquid chromatogram of the transfer product C of Example 3. FIG.

FIG. 11 is a high performance liquid chromatogram of the enzymatic decomposition solution of the transfer product C2 of Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kyoko Koizumi 3-14-3 Kasugaoka, Fujiidera City, Osaka Prefecture (72) Nobuhiro Kuwahara 13-46 Daikokucho, Tsurumi-ku, Yokohama-shi, Kanagawa Shimizu Port Sugar Co., Ltd. Within

Claims (2)

[Claims] 1. A glucosyl group of cyclodextrin of 2
A novel galactosyl-cyclodextrin in which a galactosyl group is bonded to the hydroxyl group at the 3-position or the 3-position by an α bond. 2. A galactosyl by an α bond to the 2- or 3-position hydroxyl group of the glucosyl group of cyclodextrin, which is characterized in that an α-galactosyltransferase is allowed to act on a solution containing cyclodextrin and an α-galactosyl sugar compound. A method for producing a novel galactosyl-cyclodextrin having a group bonded thereto.