JP2018030828A - Method of producing galactooligosaccharide, and intermediate thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing a galactooligosaccharide that may cause the onset of allergic reaction, and an intermediate thereof.SOLUTION: A method of producing a galactooligosaccharide represented by the general formula (1) comprises addition of the 1-position of protected galactobiose to the 6-position hydroxy group of protected lactose of formula (5), and subsequent deprotection of a hydroxy group protecting group.SELECTED DRAWING: Figure 5

Description

  The present invention relates to a method for producing a galactooligosaccharide capable of causing an allergic reaction and an intermediate thereof.

  Galacto-oligosaccharide (GOS) is a general term for oligosaccharides in which a plurality of D-galactoses are glycoside bonded to the non-reducing end of lactose. Natural GOS is present in human breast milk and cow colostrum, and its physiological function is to improve the balance of intestinal microflora, promote intestinal function, promote mineral absorption, immunomodulate, and inflammation. It has been reported to prevent and improve sexual bowel disease. In particular, the prebiotic effect that promotes the growth of intestinal bacteria such as Bifidobacterium and Lactobacillus is attracting a great deal of attention and is widely used in infant formula, lactic acid bacteria beverages, and the like. Certified in Japan for FOSHU (Food for Specified Health Uses), GRAS certified by the US Food and Drug Administration (FDA) in the United States (GRAS Notice Nos. GRN 233, 236, 285, 286 , 334, 484, 489, 495, 518, 569). Generally, GOS is produced by transglycosylation of β-galactosidase (EC.3.2.1.23) produced by microorganisms.

  Many microorganisms (Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, Kluyveromyces fragilis, Sporobolomyces singularis, Escherichia coli, Lactobacillus reuter, Bifidobacterium bifidum, etc.) producing β-galactosidase have been reported so far. These β-galactosidases have different GOS binding modes due to differences in the three-dimensional structure of the enzyme, molds such as Aspergillus have β1-6 binding (6'-GOS), bacteria such as Bacillus and S. singularis have It is known to preferentially create β1-4 bonds (4'-GOS). In addition, GOS produced by β-galactosidase produced by B. circulans is sold as Vivinal (registered trademark) GOS by Friesland Campina (Netherlands).

  However, several papers on these allergic symptoms of GOS have been reported. The beginning was reported in Japan in 1996, and several employees working at an oyster farm in Hiroshima Prefecture drank a lactic acid bacteria beverage containing galactooligosaccharide (6'-GOS) produced by mold-derived lactase Later he developed allergic symptoms. The relationship with ascidian asthma has been pointed out, and it is speculated that there may be a common part between 6'-GOS and ascidian antigen. According to Non-Patent Document 1 reported by Kaneko et al. In 2014, a sugar that induces a particularly strong allergy among 4'-GOS derived from Bacillus was clarified. In particular, from the results of the histamine release test, it was a neutral branched tetrasaccharide called “4P-X” that was most regarded as a problem.

K. Kaneko, Y. Watanabe, K. Kimura, K. Matsumoto, T. Mizobuchi, M. Onoue.Development of hypoallergenic galacto-oligosaccharides on the basis of allergen analysis.Bioscience, Biotechnology, and Biochemistry, 78, 100-108 ( 2014) Sander S. van Leeuwen, Bas JH Kuipers, Lubbert Dijkhuizen, Johannis P. Kamerling.1H NMR analysis of the lactose / β-galactosidase-derived galacto-oligosaccharide components of Vivinal GOS up to DP5. Carbohydrate Research, 400, 59-73 ( 2014)

  In Non-Patent Document 1, after sugar transfer reaction with β-galactosidase, derivatization of oligosaccharide with pyridylamine (PA) and three kinds of HPLC columns (Shodex KS-802, Shimadzu STR-ODS-II, Shimadzu ODS) are combined. Although 4P-X is isolated, the analytical system is very complicated, and the obtained 4P-X is derivatized with pyridylamine (Non-patent Document 1).

  On the other hand, in Non-Patent Document 2 reported by Leeuwen et al., By using ion chromatography (Dionex ICS-3000), separation between structural isomers is generally possible without derivatization, and NMR data has been published. . However, regarding the target 4P-X, data of a single product (high-purity product) that is a mixture with other isomers and has no isomers has not yet been acquired. 4P-X is a highly dangerous and important oligosaccharide, and it is considered highly important to acquire a high-purity target for further study in the future.

  That is, the method for producing the target galactooligosaccharide (4P-X) according to the present invention as a highly pure product has not been established in the prior art. Therefore, the conventional production method requires a complicated isolation and purification step, which is not easy and has a problem of purification cost. On the other hand, when various synthesis methods are combined, there is a problem in that the yield and the number of steps increase. Therefore, this invention makes it a subject to provide the novel manufacturing method with few steps, and high purity of the target galactooligosaccharide obtained, and its intermediate body.

で示されるガラクトオリゴ糖の製造方法であって、
一般式（２）：

（式中、Ｒ、Ｒ^１及びＲ^２は、それぞれ独立して水酸基の保護基から選択され、Ｒ、Ｒ^１及びＲ^２は２種以上が同一であってもよい）で示される化合物の水酸基の保護基を脱保護する工程を含むことを特徴とするガラクトオリゴ糖の製造方法である。
本発明［２］は、
本発明［１］に記載のガラクトオリゴ糖の製造方法であって、
一般式（３）：

［式中のＲ^２は、水酸基の保護基から選択され、Ｙは、‐Ｆ、‐Ｃｌ、‐Ｂｒ、‐Ｉ、‐ＳＣＨ_３、‐ＳＣＨ_２ＣＨ_３、‐ＳＣＨ（ＣＨ_３）_２、‐ＳＣ_６Ｈ_５及び一般式（４）：

（Ｘが、‐Ｆ、‐Ｃｌ、‐Ｂｒ、又は、‐Ｉである場合、Ｘ^１は‐Ｈであり、Ｘが‐Ｈである場合、Ｘ^１は‐ＣＨ_３であり、Ｘが‐Ｓ‐ＣＨ_２Ｃ_６Ｈ₄ＣＦ_３である場合、Ｘ^１は‐Ｃ_６Ｈ_５ＣＦ_３である）から選択される］で示される化合物と、一般式（５）：

（式中のＲ、及びＲ^１は、それぞれ独立して水酸基の保護基から選択され、ＲとＲ^１は同一であってもよい）で示される化合物と、を反応させ、糖鎖付加物として、一般式（２）：

（式中Ｒ、Ｒ^１及びＲ^２は、それぞれ独立して、水酸基の保護基から選択され、Ｒ、Ｒ^１及びＲ^２は２種以上が同一であってもよい）で示される化合物を得る工程を含むことを特徴とするガラクトオリゴ糖の製造方法である。
本発明［３］は、
本発明［１］又は［２］に記載のガラクトオリゴ糖の製造方法であって、
ガラクトースとラクトースとから、糖転移反応によってガラクトビオースを得る工程を含むことを特徴とする、ガラクトオリゴ糖の製造方法である。
本発明［４］は、
前記糖転移反応によって生成された、前記ガラクトビオースを含む混合物を精製し精製物を得る第１精製工程と、
第１精製工程によって得られた、前記精製物に含まれる水酸基を保護し保護体を得る保護工程と、
前記保護工程によって得られた、前記保護体を精製する第２精製工程と、
を含む、本発明［３］に記載のガラクトオリゴ糖の製造方法である。
本発明［５］は、
一般式（２）：

（式中Ｒ、Ｒ^１及びＲ^２は、それぞれ独立して、水酸基の保護基から選択され、Ｒ、Ｒ^１及びＲ^２は２種以上が同一であってもよい）で示される化合物である。 The inventor has found a novel production method excellent in the yield and the number of steps and the purity of the galactooligosaccharide of the present invention obtained through an intermediate which is a novel compound, and has completed the present invention. It is.
That is,
The present invention [1]
General formula (1):

A method for producing a galactooligosaccharide represented by
General formula (2):

(Wherein R, R ¹ and R ² are each independently selected from hydroxyl-protecting groups, and two or more of R, R ¹ and R ² may be the same). A method for producing a galactooligosaccharide, comprising a step of deprotecting the protecting group.
The present invention [2]
The method for producing a galactooligosaccharide according to the present invention [1],
General formula (3):

[Wherein R ² is selected from a protecting group for a hydroxyl group, and Y is -F, -Cl, -Br, -I, -SCH ₃ , -SCH ₂ CH ₃ , -SCH (CH ₃ ) ₂ ,- SC ₆ H ₅ and general formula (4):

(When X is -F, -Cl, -Br, or -I, X ¹ is -H, and when X is -H, X ¹ is -CH ₃ and X is -S. -CH ₂ C ₆ H ₄ CF ₃ , X ¹ is selected from -C ₆ H ₅ CF ₃ ) and a compound represented by the general formula (5):

(Wherein R and R ¹ are each independently selected from a hydroxyl-protecting group, and R and R ¹ may be the same), and reacted with a compound as a sugar chain adduct General formula (2):

(Wherein R, R ¹ and R ² are each independently selected from hydroxyl protecting groups, and two or more of R, R ¹ and R ² may be the same). It is a manufacturing method of the galactooligosaccharide characterized by including a process.
The present invention [3]
The method for producing a galactooligosaccharide according to the present invention [1] or [2],
A method for producing a galactooligosaccharide, comprising a step of obtaining galactobiose from galactose and lactose by a sugar transfer reaction.
The present invention [4]
A first purification step of purifying a mixture containing the galactobiose produced by the transglycosylation reaction to obtain a purified product;
A protective step for protecting the hydroxyl group contained in the purified product obtained by the first purification step to obtain a protector;
A second purification step for purifying the protector obtained by the protection step;
Is a method for producing a galactooligosaccharide according to the present invention [3].
The present invention [5]
General formula (2):

(Wherein R, R ¹ and R ² are each independently selected from hydroxyl-protecting groups, and two or more of R, R ¹ and R ² may be the same). .

  According to the present invention, it is possible to provide a novel production method and an intermediate thereof excellent in the yield and the number of steps and the purity of the resulting galactooligosaccharide of the present invention through an intermediate which is a novel compound. It becomes possible.

In the description of this specification or the claims, the “hydroxyl group” means “—OH”.
In the description of the present specification or the claims, the “hydroxyl-protecting group” (in the description of the present specification or the claims, R, R ¹ , R ² ), unless otherwise specified. And a known protecting group known as a protecting group for a hydroxyl group. For example, acyl protecting groups (acetyl group, benzoyl group, pivaloyl group, chloroacetyl group, lebuinoyl group, etc.), ether protecting groups (benzyl group, paramethoxybenzyl group, allyl group, silyl ether group, trityl group, etc.), Methyl group substituted with 1 to 3 aryl groups, alkyl, alkoxy, halogen atom, or 1 to 3 aryls optionally substituted with 1 to 3 substituents selected from cyano and nitro A methyl group substituted with a group or a silyl group, and more preferably an acyl protecting group or an ether protecting group. Most preferred is an acyl protecting group, and among them, an acetyl group, a pivaloyl group and a benzoyl group are preferred.

FIG. 1 shows the 1H NMR spectrum of Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-D-glucopyranoside . Figure 2 shows 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-α-D-glactopyranosyl 2,2 1 shows the 1H NMR spectrum of 2-trichloroacetimidate. Fig. 3 shows Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-β-D-galactopyranosyl- ( 1 → 6)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4)]-2,3-di-O-acetyl-β-D-glucopyranoside 1H NMR spectrum is shown. FIG. 4 shows β-D-galactopyranosyl- (1 → 4) -β-D-galactopyranosyl- (1 → 6)-[β-D-galactopyranosyl- (1 → 4)]-β-D-glucopyranose (4P- 2 shows the 1H NMR spectrum of X). FIG. 5 shows a 1H NMR spectrum in which the main peak portion of the 1H NMR spectrum of FIG. 4 is enlarged.

で示される、中性分岐四糖のオリゴ糖であり、ラクトースに対して、ガラクトースがβ1,6-結合し、そのガラクトースに対して、更にガラクトースがβ1,4-結合した構造を有する（或いは、グルコースに対して、ガラクトースがβ1,4-及びβ1,6-結合し、β1,6-結合したガラクトースに対して、更にガラクトースがβ1,4-結合した構造を有する）。前述のとおり、本化合物4P-XはB. circulans由来の酵素を用いて乳糖からGOSを製造した際に生成することが分かっている。従って、4P-Xを含んだGOSを乳飲料、ヨーグルト、育児用粉乳など各種乳製品に添加することにより、食品中へ混入する危険性が考えられる。尚、4P-Xが高純度であるかについては、NMRスペクトルやマススペクトル等によって確認が可能である。 <<< Galacto-oligosaccharide according to the present invention >>>
The final product of the production method of the present invention, galactooligosaccharide (4P-X), has the following general formula (1):

The oligosaccharide of a neutral branched tetrasaccharide represented by the formula: and having a structure in which galactose is β1,6-linked to lactose and further galactose is β1,4-linked to the galactose (or It has a structure in which galactose is β1,4- and β1,6-linked to glucose, and galactose is further β1,4-linked to β1,6-linked galactose). As described above, this compound 4P-X is known to be produced when GOS is produced from lactose using an enzyme derived from B. circulans. Therefore, adding GOS containing 4P-X to various dairy products such as dairy drinks, yogurt, and infant formula can be considered to be mixed into food. Whether 4P-X is highly pure can be confirmed by NMR spectrum, mass spectrum or the like.

（式中Ｒ、Ｒ^１及びＲ^２は、それぞれ独立して、水酸基の保護基から選択され、Ｒ、Ｒ^１及びＲ^２は２種以上が同一であってもよい）で示される、ガラクトオリゴ糖（4P-X；ラクトースに対して、ガラクトースがβ1,6-結合し、そのガラクトースに対して、更にガラクトースがβ1,4-結合した構造を有する）の水酸基が保護された化合物である。その原料、及び合成経路によって、Ｒ、Ｒ^１及びＲ^２の水酸基の保護基は相違し得る。また、当該中間体におけるＲ、Ｒ^１及びＲ^２が全て同一の場合であっても、２以上の工程により合成されることが好適である。１の工程としては、例えば、4P-Xを出発物質として、水酸基の保護基を導入する場合が挙げられる。 <<< Intermediate of galactooligosaccharide according to the present invention >>>
The galactooligosaccharide that is an intermediate of the production method of the present invention has the following general formula (2):

(Wherein R, R ¹ and R ² are each independently selected from hydroxyl-protecting groups, and two or more of R, R ¹ and R ² may be the same), It is a compound in which the hydroxyl group of 4P-X; galactose is β1,6-linked to lactose and galactose is further β1,4-linked to the galactose is protected. The protecting groups for the hydroxyl groups of R, R ¹ and R ² can be different depending on the raw material and the synthesis route. Moreover, even if R, R ¹ and R ² in the intermediate are all the same, it is preferable that they are synthesized by two or more steps. Examples of the process 1 include a case where a hydroxyl protecting group is introduced using 4P-X as a starting material.

<<< Method for Producing Galactooligosaccharide According to the Present Invention >>>
A method for producing a galactooligosaccharide according to the present invention will be described. The galactooligosaccharide can be synthesized from a commercially available raw material by the following production example or a method analogous thereto. For example, it can be produced by the following method.

  The production method according to the present invention is a step of obtaining a galactooligosaccharide (4P-X). In order to efficiently synthesize 4P-X, the inventors synthesized a lactose moiety and a galactobiose moiety, and studied a route for synthesizing a tetrasaccharide by a coupling reaction between disaccharides. That is, it was found that 4P-X can be synthesized by a coupling reaction between a lactose protector and a galactobiose protector. It was found that the lactose protector used could be synthesized by glycosylation of a glucose protector synthesized from glucose and a commercially available galactose protector. It was found that a protected galactobiose can be synthesized from galactobiose prepared by transglycosylation with β-galactosidase using lactose and galactose as raw materials. As described above, an efficient synthesis of a protected galactobiose can be achieved by combining an enzymatic synthesis method and a chemical synthesis method with the synthesis of a protected galactobiose.

  A preferred example of the method for producing the galactooligosaccharide (4P-X) of the present invention is shown below. The galacto-oligosaccharide of the present invention can be roughly classified into the synthesis of a lactose-protected body (Scheme 1), the synthesis of a galactobiose-protected body (Scheme 2), and the coupling between the lactose-protected body and the galactobiose-protected body. This is a process (Scheme 3) for synthesizing sugar (4P-X). Details will be described below.

<< Synthesis of lactose protector >>
[Scheme 1]

<Step 1-1>
This step is a step for obtaining a glucose protector II in which glucose I is a starting material and the hydroxyl groups at positions 4 and 6 are selectively protected. General methods such as isopropylidene, benzylidene, paramethoxybenzylidene and the like can be used as a method for selectively protecting the hydroxyl groups at positions 4 and 6. However, from the viewpoint of the yield of subsequent glycosylation, benzylidene protection is possible. Is preferably performed. As the solvent, DMF, acetonitrile, chloroform or the like can be used. The reaction can be carried out at 20-80 ° C. for 1-24 hours.

<Step 1-2>
Next, as a method for protecting the hydroxyl groups at the 1, 2 and 3 positions of glucose protector II, acyl protecting groups (acetyl group, benzoyl group, pivaloyl group, chloroacetyl group, levinoyl group, etc.) and ether protecting groups ( General methods such as benzyl group, paramethoxybenzyl group, allyl group, silyl ether group, and trityl group) can be used. From the viewpoint of the number of steps, reaction rate, and yield, protection with an acyl-based protecting group can be used. It is preferable to carry out (specifically, in the step 3-2, since all protecting groups can be deprotected using a base in one step), pyridine can be used as the solvent. The reaction can be carried out at 0 to 30 ° C. for 0.5 to 10 hours to obtain glucose protector III.

<1-3 process>
By applying a reducing agent (such as triethylsilane or sodium cyanoborohydride) to glucose protector III under acidic conditions, benzylidene acetal can be deprotected selectively to synthesize sugar receptor IV. From the viewpoint of yield and reaction rate, triethylsilane is preferred as the reducing agent. As the solvent, dichloromethane or THF can be used. The reaction can be carried out at -5 to 30 ° C for 1 to 10 hours.

<Step 1-4>
Glycosylation of synthesized sugar acceptor IV and commercially available sugar donor V can yield lactose protector VI. As the activator to be used, Lewis acids such as BF3 · OEt ₂ (boron trifluoride-ether complex) and TMSOTf (trimethylsilyl trifluoromethanesulfonate) can be used, but in the glucosylation reaction using imidate sugar From the viewpoints of reaction rate, yield, etc., TMSOTf is preferred. As the solvent, dichloromethane, chloroform and the like can be used. The reaction can be carried out at -50 to 0 ° C for 0.5 to 10 hours.

<Step 1-5>
By deprotecting the benzyl group of the obtained lactose protector VI, lactose protector VII can be synthesized. As the solvent, acetonitrile or alcohols such as methanol, ethanol, and 2-propanol can be used. The reaction can be carried out at 0 to 60 ° C. for 1 to 30 hours.

<< Synthesis of protected galactobiose >>
[Scheme 2]

<Step 2-1>
(Glycosyltransferase reaction by enzyme)
The production method of the present invention can include a step of transglycosylation using an enzyme derived from a microorganism. As a preferred example for obtaining galactobiose protector XI, a method of synthesizing GOS containing galactobiose X by transglycosylation of β-galactosidase derived from B. circulans using galactose VIII as an acceptor and lactose IX as a donor Is mentioned. As another method, it can also be prepared by a glucosylation reaction by an enzymatic reaction between galactoses.

(Enzyme type)
The enzyme to be used is not limited as long as it has an ability to produce galactobiose, but an enzyme that produces galactobiose from galactose and lactose is preferable.

  Examples of such enzymes include phosphorylase and other glycosyltransferases in addition to the aforementioned β-galactosidase. In particular, an enzyme that preferentially creates a β1,4 glycosidic bond of galactobiose X can be suitably used. However, β-galactosidase is preferred from the viewpoint of easy availability of the enzyme and the ease of the reaction system. Moreover, as long as the said enzyme can be collect | recovered, it may be derived from an animal, a plant, and microorganisms, and is not specifically limited. Examples of such microorganisms include Bacillus circulans, Kluyveromyces lactis, Aspergillus oryzae, Kluyveromyces fragilis, Sporobolomyces singularis, Escherichia coli, Lactobacillus reuter, and Bifidobacterium bifidum. From the viewpoint of reaction efficiency and availability, Bacillus circulans is preferred. Further, it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like. The form of glycosyltransferase is not particularly limited, and a dried product of enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.

(Enzyme consumption)
The amount of glycosyltransferase used varies depending on the conditions of the glycosyltransferase reaction, the type of sugar, etc. For example, in the case of β-galactosidase, 0.1 to 10 LU / mL is preferable. Here, the activity is measured by the following method. Enzyme is added to lactose (final concentration 10%), and the amount of glucose after reaction is measured at pH 6.0 and 40 ° C to quantify. The amount of enzyme required to produce 1 μmol of glucose per minute is defined as 1 unit (LU).

(Reaction conditions)
As the conditions for the transglycosylation reaction, the reaction temperature and the pH of the reaction solution can be selected in accordance with the characteristics of the enzyme used. For example, the pH is preferably 5 to 8, more preferably 6 to 7. Is preferred.

  The reaction temperature is preferably 30 to 70 ° C, more preferably 40 to 60 ° C, from the viewpoint of the stability of glycosyltransferase and the improvement of the reaction rate of the glycosyltransferase reaction. Furthermore, it is most preferable to set it as 45-55 degreeC.

  The reaction time varies depending on the type of glycosyltransferase and the like, but from the viewpoint of improving reaction efficiency, for example, 1 to 100 hours is preferable, 12 to 60 hours is more preferable, and 24 to 48 hours. Most preferably.

<Step 2-2>
A mixed fraction of galactobiose X and lactose (galactobiose: lactose = 3.3: 1) can be recovered with an activated carbon column (sometimes referred to as a first purification step), and the hydroxyl group of the mixture can be acetylated. As a method for protecting a hydroxyl group, an acyl protecting group (acetyl group, benzoyl group, pivaloyl group, chloroacetyl group, lebuinoyl group, etc.) or an ether protecting group (allyl group, silyl ether group, trityl group), etc. The method can be used. In step 2-3, deprotection of the anomeric position can be selectively performed, and therefore, from the viewpoint of the number of steps, reaction rate, and yield, it is preferable to perform protection with an acyl protecting group, and the solvent is pyridine. Can be used. The reaction can be carried out at 0 to 40 ° C. for 0.5 to 24 hours.
The acetylated form XI of galactobiose can be separated from the acetylated form of lactose by a purification operation using the silica gel column after this reaction (sometimes referred to as a second purification step).

  In particular, at the end of the enzymatic reaction by the glycosyltransferase in <2-1 step>, a mixture of 6 or more components of monosaccharide (glucose, galactose), disaccharide (galactobiose, lactose), trisaccharide, tetrasaccharide is there. This mixture is used to obtain a disaccharide fraction using an activated carbon column (first purification step). In <2-2 step>, the mixture of this disaccharide fraction is acetylated, and then the acetylated galacto is purified using a silica gel column. It is preferable to separate into biose and acetylated lactose (second purification step). This is because separation between monosaccharides, disaccharides, trisaccharides and tetrasaccharides is easier with an activated carbon column, while disaccharides are more easily separated with a silica gel column after acetylation. Therefore, by combining two stepwise purification methods in the two steps, it becomes possible to efficiently obtain a target product with high purity. Moreover, in <2-1 process>, the usage-amount of reagents, a silica gel, and an organic solvent, such as an acetylating agent, can also be reduced by including the process refine | purified with an activated carbon column. The purification method used in the first purification step is not limited to the activated carbon column, and may be any purification method that can separate the disaccharide fraction from a mixture of monosaccharide, disaccharide, trisaccharide, tetrasaccharide and the like. Furthermore, the purification method used in the second purification step is not limited to silica gel chromatography as long as it is a purification method capable of efficiently separating acetylated disaccharides.

  In addition, the process by chemical synthesis can be taken as a process of obtaining the galactobiose protector XI mentioned above. However, when synthesizing disaccharide XI, according to a general method by chemical synthesis, for example, six steps are required. An example is shown below. Specifically, a galactose protected body i in which the hydroxyl groups at positions 4 and 6 are selectively protected with benzylidene can be obtained using galactose as a starting material (step A-1). Furthermore, the galactose protector ii can be obtained by protecting the 1, 2, and 3 position hydroxyl groups of the galactose protector i with an acetyl group (step A-2). By reacting the galactose protector ii with triethylsilane under acidic conditions, the benzylidene acetal can be regioselectively deprotected and the sugar acceptor iii can be synthesized (step A-3). A glycosylated reaction of the synthesized sugar acceptor iii and a commercially available sugar donor V can be carried out to obtain a protected galactobiose iv (step A-4). The galactobiose protector v can be obtained by deprotecting the benzyl group of the obtained galactobiose protector iv (step A-5). Furthermore, by protecting the hydroxyl group with an acetyl group, an acetylated form XI of galactobiose can be obtained (step A-6).

[Scheme A]

  However, in addition to the number of steps of six steps, the step includes a step requiring a particularly strict condition setting (step A-3), which is not preferable in terms of yield and number of steps. On the other hand, a process using the glycosyltransferase of the present invention is relatively easy to isolate and is advantageous from the viewpoint of yield. Therefore, the number of steps can be reduced, and it is preferable to employ a step including a transglycosylation reaction from the viewpoint of speeding up all steps.

<Step 2-3>
The acetylated form of galactobiose XI is allowed to react with a secondary amine such as piperidine or N-methylpiperazine to selectively deprotect the anomeric acetyl group, thereby protecting the galactobiose protected form. XII can be synthesized. As the solvent, THF can be used. The reaction can be carried out at 0 to 30 ° C. for 1 to 48 hours.

<Step 2-4>
By substituting the hydroxyl group at the anomeric position of the protected galactobiose XII, a protected galactobiose XIII represented by the following general formula (3) as a sugar donor can be synthesized. The sugar donor is not particularly limited as long as it can be applied to a coupling reaction (glycosylation reaction) in Scheme 3 described below. For example, as an imidate sugar, an imidate sugar, a thioformimidate sugar, a glycosyl halide, or a thiol Examples include glycosides. It is not limited to the substituents listed below as long as the effect of the present invention also Y, X, and X ¹ and the like shown below.

［式中のＲ^２は、水酸基の保護基から選択され、Ｙは、‐Ｆ、‐Ｃｌ、‐Ｂｒ、‐Ｉ、‐ＳＣＨ_３、‐ＳＣＨ_２ＣＨ_３、‐ＳＣＨ（ＣＨ_３）_２、‐ＳＣ_６Ｈ_５及び一般式（４）：

（Ｘが、‐Ｆ、‐Ｃｌ、‐Ｂｒ、又は、‐Ｉである場合、Ｘ^１は‐Ｈであり、Ｘが‐Ｈである場合、Ｘ^１は‐ＣＨ_３であり、Ｘが‐Ｓ‐ＣＨ_２Ｃ_６Ｈ_４ＣＦ_３である場合、Ｘ^１は‐Ｃ_６Ｈ_５ＣＦ_３である）から選択される］ General formula (3):

[Wherein R ² is selected from a protecting group for a hydroxyl group, and Y is -F, -Cl, -Br, -I, -SCH ₃ , -SCH ₂ CH ₃ , -SCH (CH ₃ ) ₂ ,- SC ₆ H ₅ and general formula (4):

(When X is -F, -Cl, -Br, or -I, X ¹ is -H, and when X is -H, X ¹ is -CH ₃ and X is -S. If a _{-CH 2 C 6 H 4 CF 3} , X 1 is selected from a a) _-C 6 _H 5 CF _3]

(Imidate sugar)
As a more preferred example of the protected galactobiose XIII, an imidate sugar represented by the following general formula (6) can be synthesized by a general production method. Dichloromethane can be used as the solvent. The reaction can be carried out at 0 to 30 ° C. for 1 to 10 hours.

（式中のＲ^２は、水酸基の保護基から選択され、Ｘが、‐Ｆ、‐Ｃｌ、‐Ｂｒ、及び‐Ｉから選択される場合、Ｘ^１は‐Ｈであり、Ｘが‐Ｈである場合、Ｘ^１は‐ＣＨ_３であり、Ｘが‐Ｓ‐ＣＨ_２Ｃ_６Ｈ_４ＣＦ_３である場合、Ｘ^１は‐Ｃ_６Ｈ_４ＣＦ_３である） General formula (6):

(Wherein R ² is selected from hydroxyl protecting groups, and when X is selected from —F, —Cl, —Br, and —I, X ¹ is —H, X is —H; In some cases, X ¹ is —CH ₃ and when X is —S—CH ₂ C ₆ H ₄ CF ₃ , X ¹ is —C ₆ H ₄ CF ₃ )

(Glycosyl halide or thioglycoside)
Further, the sugar donor such as the galactobiose protector XIII is not limited to imidate sugar, and glucosyl halide and thioglycoside can be synthesized by a general production method, and can also be used as a sugar donor. Specifically, in the case of a glycosyl halide, a method of reacting a halogen with an unprotected sugar, a method of reacting a hydrogen halide with an acylated sugar, trimethylsilyl bromide, titanium tetrachloride, titanium tetrabromide, chloride It can be synthesized by a method of reacting aluminum. In the case of thioglycosides, thiols and Lewis acids can be reacted with acylated sugars, and thiols (PhSH, MeSH, EtSH, iPrSH, etc.) and Lewis acids (BF ₃ OEt ₂ , TMSOTf, MeOTf etc.) can be used with acylated sugars. Method of reacting, method of reacting thioalkylating agent (Bu ₃ SnSMe, Me ₃ SiSMe, etc.) and sulfurous heavy metal salt (SnCl ₄ ), thioalkylating agent (Bu ₃ SnSMe, Me ₃ SiSMe, etc.) and Lewis acid (BF ₃ · OEt ₂ , TMSOTf, MeOTf, etc.).

<< Coupling of lactose protector and galactobiose protector >>
[Scheme 3]

(Step 3-1)
A tetrasaccharide XIV can be synthesized by performing a coupling reaction between the synthesized lactose protector VII and galactobiose protector XIII. As in the step 1-3, the activating agent used may be a Lewis acid such as BF ₃ · OEt ₂ (boron trifluoride-ether complex) or TMSOTf (trimethylsilyl trifluoromethanesulfonate). In the case of a glucosylation reaction using an imidate sugar, TMSOTf is preferable from the viewpoint of reaction rate, yield, and the like. As the solvent, dichloromethane or chloroform can be used. The reaction can be carried out at -50 to 0 ° C for 0.5 to 10 hours.

As described above, a glucosylation reaction using a sugar donor other than the imidate sugar is also possible, and in that case, an activator different from the imidate sugar can be used. For example, when thioformimidate sugar is used as a sugar donor, a Bronsted acid such as TfOH can be used as an activator. When glycosyl halides are used as sugar donors, quaternary ammonium salts (Bu ₄ NCl, etc.), mercury salts (HgBr ₂ , Hg (CN) ₂ etc.), silver salts (AgOSO) are used as activators. ₂ CF ₃ , AgClO ₄ etc. ”In particular, in the above general formula (3), when Y = F, LiClO ₄ or strong Bronsted acid (TfOH etc.) may be used. When thioglycosides are used as donors, as activators, mercury salts (HgBr ₂ , Hg (CN) ₂ etc.), silver salts (AgOSO ₂ CF ₃ , AgClO ₄ etc.), Lewis acids (BF _3. OEt ₂ , TMSOTf, MeOTf, etc.), N-iodosuccinimide / trifluoromethanesulfonic acid can be used.

(Step 3-2)
Galactooligosaccharide XV (4P-X) can be synthesized by deprotecting the acetyl group of the synthesized tetrasaccharide XIV. A base such as sodium methoxide, potassium methoxide, sodium hydroxide, potassium hydroxide or the like can be used as a reagent for deprotection. Water or methanol can be used as the solvent. The reaction can be carried out at 0 to 30 ° C. for 1 to 24 hours.
The total yield from glucose I to galactooligosaccharide XV (4P-X) was 5.4%.

<< Uses of Galactooligosaccharides and Intermediates According to the Present Invention >>
The galactooligosaccharide according to the present invention is useful, for example, as a research material for elucidating a preparation for evaluating the allergenicity of food or the onset mechanism of the allergic reaction. In addition, the intermediate of the galacto-oligosaccharide of the present invention can be used to quickly and comprehensively create various oligosaccharides and polysaccharides containing 4P-X analogs and 4P-X structures by using this compound as a starting material. Become. The knowledge gained from the structure-activity relationship studies of compounds containing these 4P-X analogs includes detailed elucidation of the mechanism of GOS-derived allergy, research on the development of highly safe glycosyltransferases and GOS, and sugar chain-related pharmaceuticals It is considered useful for development and can be expected to be used in the academic field, food-related industry and pharmaceutical industry.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to the aspect described in these Examples at all.

  Reagents were used without purification unless otherwise specified. Biolacta FN5 (Amano Enzyme) was used as β-galactosidase. The progress of the reaction was followed by analytical thin layer chromatography (Merck TLC Silica gel 60 F254 Glass plate 20 × 20 cm). Anisaldehyde was used for coloring. Silica gel chromatography used Silica Gel 60N (Spherical, neutral, 63-210 μm) manufactured by Kanto Chemical Co., or Silica Gel 60N (Spherical, 40-100 μm) manufactured by Kanto Chemical Co., Ltd. was used. For activated carbon chromatography, activated carbon (acid-treated) manufactured by Wako Pure Chemical Industries, Ltd. was used. 1H nuclear magnetic resonance spectrum was measured using JEOL JNM-ECX-400P (400 MHz) or Varian NMR System 600 (600 MHz). Chemical shift values in 1H nuclear magnetic resonance spectrum were trimethylsilane (0.00 ppm) as an internal standard in deuterated chloroform solvent and acetone (2.225 ppm) as an internal standard in deuterated solvent.

  In the following description, the production process of 4P-X, the process of producing a lactose protector (Example 1), the process of producing a galactobiose protector (Example 2), and producing a galactooligosaccharide (4P-X) The process will be described separately (Example 3).

[Example 1]
Synthesis of Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-D-glucopyranoside (1-1) 4,6 Synthesis of -O-Benzyliden-D-glucopyranose Glucose (1.00 g, 5.55 mmol) was added to an N, N-dimethylformamide solution (11.0 mL) and heated to 60 ° C to dissolve. Benzaldehyde dimethyl acetal (1.24 mL, 8.33 mmol) and p-toluenesulfonic acid monohydrate (10 mg) were added thereto, and the mixture was stirred at 60 ° C for 6 hours. Depressurization was performed every hour for 10 minutes. After 6 hours, the solvent of the reaction solution was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and 4,6-O-Benzyliden-D-glucopyranose (799.5 mg, 2.98 mmol, yield) was obtained as white crystals from the eluate of methanol / ethyl acetate (1/10). 53.7%). The glucose used was D-(+)-glucose (manufactured by Kanto Chemical Co., Inc.).

  The structure of the target product was confirmed by 1H NMR.

(1-2) Synthesis of Acetyl 2,3-di-O-acetyl-4,6-O-benzyliden-D-glucopyranoside
Acetic anhydride (7.5 mL, 79.2 mmol) and 4-dimethylaminopyridine (193.0 mg, 1.58 mmol) were added to a pyridine solution (26 mL) of 4,6-O-Benzyliden-D-glucopyranose (1.416 g, 5.28 mmol). And stirred at room temperature for 1 hour. After 1 hour, the solvent of the reaction solution was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography. From the eluate of ethyl acetate / hexane (1/1), Acetyl 2,3-di-O-acetyl-4,6-O-benzyliden-D-glucopyranoside ( 1.954 g, 4.955 mmol, 93.8% yield).

  The structure of the target product was confirmed by 1H NMR.

(1-3) Synthesis of Acetyl 2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside
Molecular Sieves 4A was added to a dichloromethane solution (26 mL) of Acetyl 2,3-di-O-acetyl-4,6-O-benzyliden-D-glucopyranoside (1.012 g, 2.57 mmol) and stirred at room temperature for 10 minutes. Then, triethylsilane (4.09 mL, 25.7 mmol) was added. The reaction solution was cooled to 0 ° C., and trifluoroacetic acid (1.97 mL, 25.7 mmol) was slowly added over 5 minutes. The reaction solution was stirred at room temperature for 4.5 hours. After 4.5 hours, triethylamine (4.0 mL) and water (40 mL) were added at 0 ° C. The aqueous layer and dichloromethane layer of the solution were separated, the aqueous layer was extracted with ethyl acetate (50 mL × 2), and the organic layer was dried over sodium sulfate. After filtering the organic layer, the solvent was distilled off under reduced pressure. The residue was subjected to flash silica gel column chromatography.From the eluate of ethyl acetate / hexane (1/1), as a colorless oily substance, Acetyl 2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside ( 621.9 mg, 1.569 mmol, yield 61.0%).

  The structure of the target product was confirmed by 1H NMR.

(1-4) Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-6-O-benzyl-D- Synthesis of glucopyranoside
Acetyl 2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside (200.7 mg, 0.506 mmol) and 2,3,4,6-Tetra-O-acetyl-α-D-galactopyranosyl 2,2 , 2-Trichloroacetimidate (373.9 mg, 0.759 mmol) (Tokyo Chemical Industry Co., Ltd.) was dissolved in a toluene solution (2 mL), and the solvent was distilled off under reduced pressure. Acetyl 2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside and 2,3,4,6-Tetra-O-acetyl-α-D-galactopyranosyl 2,2,2-Trichloroacetimidate Removed. A dichloromethane solution (2 mL) and molecular sieves 4A were added to the crystals after the solvent was distilled off, and the mixture was stirred at room temperature for 30 minutes. Then, TMSOTf in dichloromethane (50.6 mM, 1 mL, 50.6 μmol) was added dropwise over 2 minutes at −40 ° C. (prepared by cooling the acetone solution with dry ice), and stirring was continued. Thirty minutes after the start of the reaction, a solution of TMSOTf in dichloromethane (506 mM, 0.2 mL, 101 μmol) was added dropwise and stirring was continued. One hour after the start of the reaction, the reaction temperature was increased from -40 ° C to -20 ° C. Stirring was continued at -20 ° C for 2 hours. Three hours after the start of the reaction, a dichloromethane solution (10 mL) was added at −20 ° C. to dilute the reaction solution, and stirring was continued for 30 minutes. This solution was added to a separately prepared saturated aqueous sodium hydrogen carbonate solution (20 mL) at 0 ° C. and stirred. The aqueous layer and dichloromethane layer of the solution were separated, the aqueous layer was extracted with ethyl acetate (50 mL × 2), and the organic layer was dried over sodium sulfate. After filtering the organic layer, the solvent was distilled off under reduced pressure. The residue was subjected to flash silica gel column chromatography. From the eluate of ethyl acetate / hexane (1/1 → 2/1), Acetyl 2,3,4,6-tetra-O-acetyl-β-D -galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside (195.4 mg, 0.269 mmol, yield 53.1%) was obtained. As a result of NMR analysis, the glycosylation reaction proceeds, and Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3- Di-O-acetyl-6-O-benzyl-D-glucopyranoside was obtained, and the yield was 53.1%.

  The structure of the target product was confirmed by 1H NMR.

(1-5) Synthesis of Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-D-glucopyranoside
Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside (150.6 mg, To a methanol solution (4.1 mL) of 75.4 μmol) was added palladium carbon (palladium 5% supported, 55% water wet product, 150.6 mg) under a nitrogen atmosphere. The nitrogen gas in the flask was replaced with hydrogen gas, and the mixture was stirred at room temperature for 22 hours. After 22 hours, palladium carbon in the reaction solution was filtered through celite. At this time, celite was washed with ethyl acetate (10 mL × 4) and methanol (10 mL × 4). The filtrate was collected and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography. From the eluate of ethyl acetate / hexane (2/1 → 4/1 → 1/0), Acetyl 2,3,4,6-tetra-O-acetyl was obtained as a colorless oil. -β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-D-glucopyranoside (122.9 mg, 0.1932 mmol, yield 93.3%) was obtained.

The structure of the target product was confirmed by 1H NMR. The 1H NMR spectrum is shown in FIG.
(α: β = 3: 1): 1H-NMR (400 MHz, CDCl ₃ ): σ6.27 (d, J = 3.7 Hz, 0.75H), 5.69 (d, J = 8.2 Hz, 0.25H), 5.47 (t, J = 9.9 Hz, 0.75H), 5.35 (dd, J = 3.24, 3.20Hz, 1H), 5.24 (t, J = 9.4 Hz, 0.25H), 5.15-5.08 (m, 1H), 5.05- 4.95 (m, 2H), 4.64 (d, J = 8.2 Hz, 0.75H), 4.63 (d, J = 8.2 Hz, 0.25H), 4.17-4.05 (m, 2.25H), 4.03-3.89 (m, 2.25 H), 3.87-3.74 (m, 2.5H), 3.54 (d, J = 9.6 Hz, 0.25H), 2.18 (s, 2.25H), 2.16 (s, 2.25H), 2.15 (s, 0.75H), 2.10 (s, 0.75H), 2.06 (s, 7.5H), 2.052 (s, 0.75H), 2.048 (s, 0.75H), 2.03 (s, 0.75H), 2.02 (s, 2.25H), 1.971 ( s, 2.25H), 1.968 (s, 0.75H);

[Example 2]
2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-α-D-glactopyranosyl 2,2,2-trichloroacetimidate (2-1) Synthesis of β-D-galactopyranosyl- (1 → 4) -D-galactopyranose Galactobiose was synthesized by a transglycosylation reaction of β-galactosidase. The acceptor was galactose, the donor was lactose, and β-galactosidase was biolacta FN5 derived from B. circulans. Add enzyme to 0.01 ml to 10 ml of substrate solution [galactose 25% (2.5 g, 13.9 mmol), lactose 25% (2.50 g, 7.30 mmol), 100 mM sodium phosphate buffer (pH 6.5)] Shake gently for 24 hours at 50 ° C. Thereafter, the enzyme was inactivated by heating at 100 ° C. for 5 minutes, and the supernatant was collected by centrifugation (15,000 rpm, 5 min, 4 ° C.). Apply the supernatant to an activated carbon column (φ4.6 cm × 20 cm), and gradually increase the ethanol concentration to obtain galactobiose (β-D-galactopyranosyl- (1 → 4) -D-galactopyranose) and lactose. The contained ethanol fraction was concentrated to recover a mixture of galactobiose and lactose (1.13 g, yield 45% as a mixture, galactobiose: lactose = 3.3: 1) as white crystals. The mixing ratio of galactobiose and lactose was confirmed by HPLC. As the HPLC column, CARBOSep CHO-620CA manufactured by Toransgenomic was used. In addition, galactose used special grade galactose (made by Wako Pure Chemical Industries), and lactose used lactose monohydrate (made by Kanto Chemical Co., Inc.).

(2-2) Synthesis of Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-D-glactopyranoside Galacto Acetic anhydride (2.3 mL, 24.3 mmol) and 4-dimethylaminopyridine (19.5 mg, 0.16 mmol) are added to a pyridine solution (16 mL) of a mixture of biose and lactose (554.7 mg, 1.62 mmol), and the mixture is stirred at room temperature for 3 hours. Stir. Three hours later, acetic anhydride (2.3 mL, 24.3 mmol) and 4-dimethylaminopyridine (19.5 mg, 0.16 mmol) were added, and the mixture was stirred at room temperature for 18.5 hours. After completion of the reaction, the solvent was distilled off from the reaction solution under reduced pressure. The residue was subjected to silica gel column chromatography, ethyl acetate / hexane (1 / 1.5 → 1/1 → 1/0) → methanol / ethyl acetate (1/10) from the eluate, as white crystals, Acetyl 2,3, 4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-D-glactopyranoside (748.4 mg, 1.10 mmol, yield 67.9%) Obtained.

  The structure of the target product was confirmed by 1H NMR.

(2-3) Synthesis of 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-D-glactopyranose
Of Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-D-glactopyranoside (86.7 mg, 0.128 mmol) To the tetrahydrofuran solution (2.4 mL) was added piperidine in tetrahydrofuran (2 M, 0.25 mL, 0.500 mmol), and the mixture was stirred at room temperature for 30 hours. Thereafter, 1 N hydrochloric acid (1 mL) and water (10 mL) were added in order at 0 ° C., and the mixture was stirred. The reaction solution was extracted with ethyl acetate (30 mL × 2), and the organic layer was dried over sodium sulfate. After filtering the organic layer, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography, and 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl as white crystals from the eluate of ethyl acetate / hexane (2/1 → 4/1) -(1 → 4) -2,3,6-tri-O-acetyl-D-glactopyranose (61.9 mg, 0.0973 mmol, yield 76.0%) was obtained.

  The structure of the target product was confirmed by 1H NMR.

(2-4) 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-α-D-glactopyranosyl 2, Synthesis of 2,2-trichloroacetimidate
2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-D-glactopyranose (270.2 mg, 0.425 mmol) in dichloromethane To the solution (8.4 mL) was added trichloroacetonitrile (17.2 mL, 17.0 mmol) and 1,8-diazabicyclo [5.4.0] -7-undecene in dichloromethane (0.85 mM, 0.3 mL, 0.255 mmol), and then at room temperature for 4 hours. Stir. Thereafter, the reaction solution was concentrated under reduced pressure, and the solvent was distilled off. The residue was subjected to silica gel column chromatography.From the eluate of ethyl acetate / hexane (2/1) containing 0.5% triethylamine, 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl was obtained as white crystals. -(1 → 4) -2,3,6-tri-O-acetyl-α-D-glactopyranosyl 2,2,2-trichloroacetimidate (285.7 mg, 0.366 mmol, yield 86.1%) was obtained.

The structure of the target product was confirmed by 1H NMR. The 1H NMR spectrum of the target product is shown in FIG.
(α: β = 1: 0): 1H-NMR (400 MHz, CDCl3): σ8.64 (s, 1H), 6.51 (d, J = 3.7 Hz, 1H), 5.40-5.35 (m, 2H), 5.28 (dd, J = 2.8, 11.0 Hz, 1H), 5.20 (dd, J = 7.8, 10.5 Hz, 1H), 5.00 (dd, J = 3.2, 10.5 Hz, 1H), 4.43 (d, J = 8.2 Hz , 1H), 4.38 (dd, J = 4.1, 11.9 Hz, 1H), 4.31 (d, J = 3.2 Hz, 1H), 4.29 (d, J = 4.6 Hz, 1H), 4.15 (dd, J = 7.3, 11.5 Hz, 1 H), 4.10 (d, J = 6.4 Hz, 2H), 3.86 (t, J = 6.6 Hz, 1H), 2.17 (s, 3H), 2.14 (s, 3H), 2.12 (s, 3H ), 2.05 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H);

[Example 3]
Synthesis of β-D-galactopyranosyl- (1 → 4) -β-D-galactopyranosyl- (1 → 6)-[β-D-galactopyranosyl- (1 → 4)]-β-D-glucopyranose (4P-X) (3-1) Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-β-D-galactopyranosyl- (1 → 6)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4)]-2,3-di-O-acetyl-β-D-glucopyranoside Composition
Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3-di-O-acetyl-D-glucopyranoside (26.0 mg, 40.9 μmol) and 2, 3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-α-D-glactopyranosyl 2,2,2-trichloroacetimidate (47.9 mg, 61.4 μmol) was added toluene solution (2 mL), and the solvent was distilled off under reduced pressure. A dichloromethane solution (1 mL) and molecular sieves 4A (0.5 g) were added to the crystals after the solvent was distilled off, and the mixture was stirred at −40 ° C. for 30 minutes. Then, TMSOTf in dichloromethane (4.09 mM, 1 mL, 4.09 μmol) was added dropwise at -40 ° C (prepared by cooling the acetone solution with dry ice) over 3 minutes, and stirring was continued. After 2 hours, a dichloromethane solution (10 mL) was added at room temperature to dilute the reaction solution. The diluted reaction solution was added to a 100 mL eggplant-shaped flask containing a separately prepared saturated sodium hydrogen carbonate aqueous solution (30 mL) at 0 ° C. and stirred for 10 minutes. The aqueous layer and dichloromethane layer of the solution were separated, the aqueous layer was extracted with ethyl acetate (50 mL × 2), and the organic layer was dried over sodium sulfate. After filtering the organic layer, the solvent was distilled off under reduced pressure. The residue was subjected to flash silica gel column chromatography. From the eluate of ethyl acetate / hexane (4/1 → 1/0), Acetyl 2,3,4,6-tetra-O-acetyl-β- D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-β-D-galactopyranosyl- (1 → 6)-[2,3,4,6-tetra-O-acetyl-β -D-galactopyranosyl- (1 → 4)]-2,3-di-O-acetyl-β-D-glucopyranoside (22.3 mg, 17.8 μmol, yield 43.4%) was obtained.

The structure of the target product was confirmed by 1H NMR. The 1H NMR spectrum of the target product is shown in FIG.
(α: β = 4: 1): 1H-NMR (400 MHz, CDCl3): σ6.25 (d, J = 3.7 Hz, 0.8H), 5.62 (d, J = 8.2 Hz, 0.2H), 5.42 ( dd, J = 7.8, 8.3 Hz, 1H), 5.37 (t, J = 3.6 Hz, 2H), 5.22-5.05 (m, 4.8H), 5.03-4.97 (m, 2.2H), 4.89 (dd, J = 3.2, 10.3 Hz, 1H), 4.66 (d, J = 7.3 Hz, 0.8H), 4.60-4.56 (m, 1H), 4.46-4.37 (m, 1H), 4.23-4.05 (m, 6.4H), 4.00 (t, J = 7.4 Hz, 1H), 3.94-3.85 (m, 4.8 H), 3.72 (dd, J = 5.0, 6.0 Hz, 1H), 2.19 (s, 3H), 2.16 (s, 3H), 2.150 (s, 3H), 2.146 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.054 (s, 3H), 2.047 ( s, 3H), 2.002 (s, 3H), 2.000 (s, 3H), 1.96 (s, 3H);

(3-2) Synthesis of β-D-galactopyranosyl- (1 → 4) -β-D-galactopyranosyl- (1 → 6)-[β-D-galactopyranosyl- (1 → 4)]-β-D-glucopyranose
Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-acetyl-β-D-galactopyranosyl- (1 → 6) -[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl- (1 → 4)]-2,3-di-O-acetyl-β-D-glucopyranoside (21.9 mg, 17.4 μmol ) Was added to a 45% aqueous methanol solution (1 mL) of sodium methoxide (34.8 mM, 1 mL, 34.8 μmol), and the mixture was stirred at room temperature for 9 hours. After 9 hours, the solvent was distilled off under reduced pressure. Dissolve the residue in MQ (2 mL) and use a dialysis membrane (Spectra, Biotech dialysis membrane, MWCO: 0.1-0.5 kDa, FW: 31 mm, Dia: 20 mm, vol / L: 3.1 mL / cm) Then, dialysis was performed for 6 hours against MQ water (1 L). The purified solution was lyophilized to obtain 4P-X (9.5 mg, 14.25 μmol, 81.9%) as white crystals.

The structure of the target product was confirmed by 1H NMR. The 1H NMR spectrum of the target product is shown in FIG. 4 and its enlarged view is shown in FIG.
(α: β = 1: 1.5); 1H-NMR (600 MHz, D2O): σ5.23 (d, J = 4.2 Hz, 0.4H), 4.68 (d, J = 8.4 Hz, 0.6H), 4.59 ( d, J = 7.8 Hz, 1H), 4.50 (d, J = 7.8 Hz, 1H), 4.49 (d, J = 7.8 Hz, 0.6H), 4.48 (d, J = 7.8 Hz, 0.4H), 4.28 ( dd, J = 11.1, 1.8 Hz, 0.6H), 4.20 (dd, J = 11.1, 2.4 Hz, 0.4H), 4.18 (d, J = 3.6 Hz, 1H), 4.10-4.07 (m, 0.4H), 3.96 (dd, J = 11.4, 3.6 Hz, 0.4H), 3.93-3.52 (m, 20.6H), 3.30 (dd, J = 6.2, 5.2 Hz, 0.6H);

  The present inventors have succeeded in synthesizing allergenic tetrasaccharide 4P-X for the first time. The total yield from glucose was 5.4%. Until now, it has been difficult to purify the galactooligosaccharide (4P-X) of the present invention from tetrasaccharides having other structures by preparation using only enzymatic reaction. There is no precedent for a production method that can be efficiently obtained without derivatizing 4P-X by pyridylamination or the like. In the future, it is highly expected to establish a rapid and accurate quantitative analysis system for various GOS-containing foods on the market by using 4P-X of the high purity product of the present invention.

  It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and thus are within the scope of the claims appended hereto.

Claims (5)

General formula (1):

A method for producing a galactooligosaccharide represented by
General formula (2):

(Wherein R, R ¹ and R ² are each independently selected from hydroxyl-protecting groups, and two or more of R, R ¹ and R ² may be the same). The manufacturing method of the galactooligosaccharide characterized by including the process of deprotecting said protecting group. A method for producing a galactooligosaccharide according to claim 1,
General formula (3):

[Wherein R ² is selected from a protecting group for a hydroxyl group, and Y is -F, -Cl, -Br, -I, -SCH ₃ , -SCH ₂ CH ₃ , -SCH (CH ₃ ) ₂ ,- SC ₆ H ₅ and general formula (4):

(When X is -F, -Cl, -Br, or -I, X ¹ is -H, and when X is -H, X ¹ is -CH ₃ and X is -S. -CH ₂ C ₆ H ₄ CF ₃ , X ¹ is selected from -C ₆ H ₅ CF ₃ ) and a compound of the general formula (5):

(Wherein R and R ¹ are each independently selected from a hydroxyl-protecting group, and R and R ¹ may be the same), and reacted with a compound as a sugar chain adduct General formula (2):

(Wherein R, R ¹ and R ² are each independently selected from hydroxyl protecting groups, and two or more of R, R ¹ and R ² may be the same). The manufacturing method of the galactooligosaccharide characterized by including a process. A method for producing a galactooligosaccharide according to claim 1 or 2,
A method for producing a galactooligosaccharide, comprising a step of producing galactobiose from galactose and lactose by a sugar transfer reaction. A first purification step of purifying a mixture containing the galactobiose produced by the transglycosylation reaction to obtain a purified product;
A protective step for protecting the hydroxyl group contained in the purified product obtained by the first purification step to obtain a protector;
A second purification step for purifying the protector obtained by the protection step;
The manufacturing method of the galactooligosaccharide of Claim 3 containing this. General formula (2):

Wherein R, R ¹ and R ² are each independently selected from hydroxyl protecting groups, and two or more of R, R ¹ and R ² may be the same.

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