JP2007314585A - Method for producing oligosaccharide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an oligosaccharide having a more complex structure efficiently under mild conditions. <P>SOLUTION: Provided is a method for producing an oligosaccharide comprising subjecting a saccharide donor represented by formula 1 (wherein ring A denotes a monosaccharide or a derivative thereof; S is bonded to the carbon at the anomeric position of ring A; n is 1 or 2; and R denotes a monovalent alkyl group or a monovalent aryl group) and a saccharide acceptor comprising xylooligosaccharide of an average degree of polymerization of 2-20 to glycosylation. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing oligosaccharides efficiently and industrially at low cost. In particular, the present invention relates to a method for producing an oligosaccharide having a more complicated structure efficiently under mild conditions.

  With the recent shift to a rapidly aging society and an increase in health consciousness, foods are required to have bioregulatory functions related to health maintenance / promotion and disease prevention and recovery. Under such circumstances, various functional foods are developed, and oligosaccharides having functions such as low calorie, intestinal regulation, and dental caries prevention are also attracting attention in carbohydrates.

  Most saccharides are homochiral (optically active), and in order to utilize this property, saccharides are used as a raw material for synthesizing functional substances or used as homochiral optical elements. For example, it is known that a cholesteric liquid crystal of a hydroxypropyl derivative of cellulose may be applied to the display field by changing its pitch by electrical stimulation (see Non-Patent Document 1). Furthermore, it is known that a circularly polarized light scattering film can be obtained by fixing the structure of such a cholesteric liquid crystal. Therefore, in recent years, development of a simple and versatile method for producing oligosaccharides has been progressing in order to obtain oligosaccharides having various functions.

  As means for obtaining oligosaccharides, extraction from main constituent components of plants such as wheat bran and corn cob is widely performed. For example, xylooligosaccharides can be recovered and purified from a hemicellulase treatment solution of paper pulp (see Patent Document 1). However, 60% or more of xylo-oligosaccharides obtained by this method are neutral saccharides, and acidic xylo-oligosaccharides that are expected to have a physiological effect cannot be efficiently obtained in less than 40% of the total. If glucuronic acid can be chemically bound to neutral xylo-oligosaccharides, the xylose component obtained from wood can be used more effectively, but no efficient method for advancing this chemical reaction has been established so far.

  A conventional oligosaccharide production method includes a glycosylation reaction, which is a condensation reaction of a sugar having a leaving group at the anomeric position (sugar donor) and a sugar having a free hydroxyl group (sugar acceptor). In normal oligosaccharide chemical synthesis, a protecting group at the anomeric position of a new sugar obtained by glycosylation is removed, a leaving group is introduced to form a sugar donor, and then condensed with the next sugar acceptor. Or removing the hydroxyl protecting group of a new sugar to give a sugar acceptor and condensing with the next sugar donor. By repeating these steps, the sugar chain is elongated.

  Conventionally, sugar derivatives in which the anomeric carbon of a sugar is chlorinated or brominated have been used for this reaction as a sugar donor (see Non-Patent Document 2). These are the corresponding 1-O-acylated sugars reacted with hydrogen halide, and this sugar donor can be used to generate a glycosidic bond. However, the method using hydrogen halide cannot be applied to a compound having a protecting group sensitive to an acid, and requires highly dehydrated reaction conditions. In addition, when a sugar derivative in which the carbon at the anomeric position of the sugar is chlorinated or brominated is used when applied to an oligosaccharide, the glycosidic bond may be cleaved, making industrialization difficult.

  As a method for solving these problems, recently, reactions using glycosylimidates, thioglycosides, and glycosyl fluorides as sugar donors have been developed (see Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). . Common in these methods is that they can be prepared in much more stable and relatively mild conditions compared to chloro or brominated glycosyl halides, and the reagents required for activation are also compared to glycosyl halides. It is diverse, and may be applicable to substrates that cannot be glycosylated by conventional methods.

  Among these methods, the method using thioglycoside as a sugar donor is not affected by the introduction or deprotection of protecting groups under acid and basic conditions, and the conditions of glycosylation reactions that are usually used. When combined with other glycosylation reactions, complex oligosaccharides can be synthesized efficiently. It has also been reported that the yield is significantly increased by using dithioglycoside (see Non-Patent Document 6). However, when synthesizing oligosaccharides having a high degree of polymerization, it is necessary to carry out the reaction in multiple stages, which is costly. There was a problem that it was bulky.

As a method for efficiently synthesizing oligosaccharides, there is a One-Pot glycosylation reaction. The One-Pot glycosylation reaction utilizes the difference in reactivity of sugar donors or sugar acceptors, and by using a plurality of combinations of leaving groups of sugar donors and activators, it can be continuously produced in one flask. Although it is a reaction for forming a glycosidic bond, there is a problem that a variety of drugs are used and the process is complicated and requires precise control (see Patent Document 2, Non-Patent Document 7, and Non-Patent Document 8).
JP 2000-333692 A JP 2002-522598 A R. Chiba et al., Macromolecules., Vol. 36, pp. 1706-1712 (2003) HGFletcher et al., Methods Carbohydr. Chem., Vol. 2, pp. 226 (1963) AHHaines., Adv. Carbohydr. Chem. Biochem., Vol. 33, pp. 11 (1976) D. Horton., Adv. Carbohydr. Chem., Vol. 18, pp. 123, (1963) KCNicolaou et al., Journal of American Chemica Society., Vol.106, pp.4189-4192 (1984) l EJ Grayson et al., Journal of Organic Chemistry, vol. 70, pp9740-9754, (2005) Z, Zhang et al., Journal of American Chemical Society., Vol. 121, pp. 734-753 (1999) H, Yamada et al., Tetrarhedron Letter., Vol 40, 4581 (1999)

  An object of the present invention is to provide a method for efficiently producing an oligosaccharide having a more complicated structure under mild conditions.

  As a result of diligent research to solve the above problems, the inventors of the present invention have used a monosaccharide or monosaccharide derivative provided with a sulfide bond or disulfide bond as a sugar donor, and an oligosaccharide having a polymerization degree of 2 to 20 as a sugar. The inventors have found that the glycosylation reaction proceeds particularly efficiently and simply when reacted as an acceptor, and have completed the present invention. The present invention includes the following inventions.

（式中、環Ａは単糖類又はその誘導体を表わし、Ｓは環Ａのアノマー位の炭素に結合しており、ｎは１もしくは２であり、Ｒは１価のアルキル基もしくは１価のアリール基を表わす）で表される糖供与体と平均重合度が２〜２０のキシロオリゴ糖からなる糖受容体とをグリコシル化反応させることを特徴とするオリゴ糖の製造方法。 (1) General formula 1

(In the formula, ring A represents a monosaccharide or a derivative thereof, S is bonded to the anomeric carbon of ring A, n is 1 or 2, and R is a monovalent alkyl group or monovalent aryl. And a sugar acceptor comprising a xylo-oligosaccharide having an average polymerization degree of 2 to 20 is subjected to a glycosylation reaction.

(2) The method for producing an oligosaccharide according to (1), wherein the ring A in the general formula 1 is at least one selected from glucose, xylose, and glucuronic acid.

(3) The xylo-oligosaccharide having an average degree of polymerization of 2 to 20 as the sugar acceptor is a xylo-oligosaccharide in which all hydroxyl groups not involved in the reaction are protected (1) or (2) A method for producing the oligosaccharide according to 1.

(4) The method for producing an oligosaccharide according to any one of items (1) to (3), wherein the xylooligosaccharide having an average degree of polymerization of 2 to 20 is an acetalized xylo-oligosaccharide .

(5) The sugar donor comprises a monosaccharide or a derivative thereof in which ring A in the general formula 1 represents a neutral monosaccharide or a neutral derivative thereof, and converts an alcoholic hydroxyl group to a carboxyl group after the glycosylation reaction. The method for producing an oligosaccharide according to any one of items (1) to (4), wherein an oxidation reaction is performed.

(6) A xylo-oligosaccharide in which the sugar donor in the glycosylation reaction is glucopyranosylmethane sulfite or glucopyranosylmethane disulfite, and the sugar acceptor is protected at a hydroxyl group not involved in the glycosylation reaction The method for producing an oligosaccharide according to any one of items (1) to (5), wherein

  The method of the present invention is stable because the sugar donor used in the reaction is stable and easy to prepare, and the yield is high due to the high reactivity, and the oligosaccharide is used as a raw material, and thus the process is multistage. An industrially advantageous method for producing an oligosaccharide that can omit synthesis is provided.

The present invention will be specifically described below.
A monosaccharide and a sugar derivative which are sugar donors in the glycosylation reaction of the present invention are represented by the following general formula 1.

  In the above general formula 1, A is a sugar or a sugar derivative, and the cyclic structure of the sugar may be a 5-membered ring or a 6-membered ring. Usually aldose is used, but ketose can also be used. Specifically, as these sugars, tetraoses such as erythrose and throse, pentoses such as ribose, arabinose and xylose, hexoses such as glucose, mannose, galactose, allose and talose, or 2-deoxy Examples include sugars in which a part of these sugars are deoxylated such as glucose and 2-deoxyribose, amino sugars such as 2-acetamido-2-deoxyglucose, sialic acid, and glucuronic acid. Examples of the derivatized sugar include glucuronic acid derivatives such as 4-0-methylglucuronic acid in which the hydroxyl group at the 4-position of glucose is methyl etherified. These sugars exist in D-form and L-form, either of which can be used as a mixture. n may be either 1 or 2, and R may be an alkyl group such as a methyl group or an ethyl group or an aromatic group such as a phenyl group, but is not limited by a substituent.

  Needless to say, these sugars can be used with all hydroxyl groups other than the anomeric positions protected, but may be derivatives having a free hydroxyl group that is protected to the minimum necessary for solubility or the like. A conventionally well-known thing can be used as a protective group of a hydroxyl group. Specifically, a method of protecting with an acyl group such as acetyl, trifluoroacetyl, trichloroacetyl, benzoyl, p-nitrobenzoyl group, a method of acetalizing with acetaldehyde, acetone, etc., methyl, benzyl, triphenylmethyl group, etc. Examples thereof include a method of etherification with an alkyl group, a method of silylation with a t-butyldimethylsilyl, t-butyldiphenylsilyl group and the like.

  As an oligosaccharide serving as a sugar acceptor, there is no limitation on the type of sugar to be constructed as long as the average degree of polymerization is 2 to 20, but xylose and glucose are particularly abundant as wood resources and are suitable. . When the degree of polymerization of the oligosaccharide exceeds 20, the steric structure constituting the oligosaccharide becomes complicated and the glycosylation reaction does not proceed easily. As a means for obtaining xylooligosaccharide, it can be efficiently obtained from wood pulp by the method described in Patent Document 1. When producing xylo-oligosaccharides by this production method, the degree of oligosaccharide polymerization can be easily adjusted by changing the type of xylanase used, the amount of enzyme added, the acid treatment conditions, and the like.

  Oligosaccharides that serve as sugar receptors have many hydroxyl groups, but all hydroxyl groups that are not involved in the reaction must be protected. A conventionally well-known thing can be used as a protective group of a hydroxyl group. Specifically, a method of protecting with an acyl group such as acetyl, trifluoroacetyl, trichloroacetyl, benzoyl, p-nitrobenzoyl group, a method of acetalizing with acetaldehyde, acetone, etc., methyl, benzyl, triphenylmethyl group, etc. Examples thereof include a method of etherification with an alkyl group, a method of silylation with a t-butyldimethylsilyl, t-butyldiphenylsilyl group and the like. In particular, when protecting the hydroxyl group of the xylooligosaccharide receptor, acetalization with acetaldehyde, acetone, etc. will leave one hydroxyl group that is not acetalized at the terminal xylose unit constituting the oligosaccharide. And can be used as a reactive group.

Next, the glycosylation reaction will be described. In the present invention, thioglycosides and dithioglycosides are used as sugar donors, but they can be synthesized by the following known methods.
First, the synthesis method of thioglycoside will be described. First, a Vilsmeier reagent is reacted with a derivative in which the sugar hydroxyl group is protected to convert it to a glycosyl halide. The reaction temperature is preferably 5 to 10 ° C. A thioglycoside is obtained by reacting the resulting glycosyl halide with sodium methanethiosulfonate in a solvent such as dioxane. The solvent is preferably a polar solvent, particularly an aprotic polar solvent, but is not particularly limited. The reaction temperature is preferably 50 ° C to 100 ° C. Sodium methanethiosulfonate is obtained by reacting methanesulfonyl chloride with sodium sulfide nonahydrate in water.

  Known methods can also be applied to the synthesis of dithioglycosides. Dithioglycoside is obtained by reacting the thioglycoside obtained by the above reaction with ethanethiol in the presence of triethylamine.

  Glycosylation is carried out by reacting the above thioglycoside or dithioglycoside with an acetalized oligosaccharide.

  Examples of the activator include those generally used for glycosylation of thioglycosides. That is, mercury salts such as mercury acetate and mercury nitrate, silver salts such as silver trifluoromethanesulfonate, NBS, N-iodosuccinimide (NIS) -trifluoromethanesulfonate, bromine, iodine, trimethylsilyl triflate, triethylsilyl triflate , Boron trifluoride ether complex, tin (IV) chloride and the like.

  The solvent used for the reaction may be a well-known aprotic organic solvent such as ether, benzene, toluene, dichloromethane, chloroform, acetonitrile, dimethyl sulfoxide, and is not particularly limited. The reaction temperature and reaction time vary depending on the catalyst and solvent used, and are not particularly limited. However, 0 to 25 ° C. and 10 minutes to 1 hour are suitable, respectively. Although there is no restriction | limiting in particular in the usage-amount of an activator, Usually, it adds in the range of 1.0-2.0 equivalent with respect to 1-thioglycoside derivative. It is preferable to use an oligosaccharide in excess relative to the 1-thioglycoside derivative, and usually 2.0 to 5.0 equivalents are more preferable.

  Although there is no restriction | limiting in particular also as reaction temperature, Usually, it is the range of -80 degreeC to the boiling point of a solvent, and the range of -10 degreeC-room temperature is preferable when operativity etc. are considered.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. Unless otherwise indicated,% shown below means mass%, and the addition ratio of pulp is the ratio of mass to the absolute dry mass of pulp. In addition, each measuring method is as follows.

(1) Quantification of total sugar amount The total sugar amount is determined by preparing a calibration curve using D-xylose (Wako Pure Chemical Industries) and using the phenol-sulfuric acid method (published by the "Reducing Sugar Quantification Method" Society Publishing Center). did.

(2) Quantification of the amount of reducing sugar The amount of reducing sugar was prepared by using a calibration curve using D-xylose (Wako Pure Chemical Industries) and the Sommoji-Nelson method (published by the "Reducing Sugar Determination Method" published by the Japan Society for Publications). Quantified.

(3) Quantitative determination of uronic acid The uronic acid was quantified using a carbazole sulfate method (published by the "Reducing Sugar Quantification Method" published by the Japan Society for the Determination of Reducing Sugars) using a calibration curve of D-glucuronic acid (Wako Pure Chemical Industries). did.

(4) Determination of average degree of polymerization Keep the sample sugar solution at 50 ° C., centrifuge at 15000 rpm for 15 minutes to remove insoluble matter, and use the total sugar amount in the supernatant as the reducing sugar amount (both converted to xylose). The average degree of polymerization was determined by dividing.

(5) Quantitative method of acidic xylooligosaccharide The quantitative ratio of uronic acid residue and xylose residue in one molecule of acidic oligosaccharide was determined using a nuclear magnetic resonance apparatus (hereinafter referred to as NMR, manufactured by JEOL Ltd.). The sample was measured after freeze-drying and deuterium substitution. 1H NMR, 13C NMR, and HMQC measurements determined the proton chemical shift between the xylose 1-position (4.409 ppm) and uronic acid 1-position (5.226 ppm), which are glycosidic bonds. The area of each signal corresponding to the amount ratio of hydrogen was calculated to determine the ratio of glucuronic acid and xylose. In addition, high-resolution molecular weight measurement was performed using a high performance liquid chromatography mass spectrometer (Applied Biosystems) to determine the number of uronic acid residues per oligosaccharide molecule.

(6) Definition of enzyme titer Kaxylan (manufactured by Sigma) was used for measuring the activity of xylanase used as an enzyme. Enzyme titer is defined by measuring the reducing power of reducing sugars obtained by xylanase degrading xylan using the DNS method (published by the “Reducing Sugar Quantitative Method” Society Publishing Center), 1 micromole per minute. The amount of enzyme that produces a reducing power corresponding to xylose was 1 unit.

(7) Analysis by ion chromatograph An ion chromatograph (Dionex) was used for analysis of xylooligosaccharides. For the analysis, Carbo Pac PA-10 (Dionex) was used as a column suitable for the analysis of saccharides.

Example 1
(Xylooligosaccharide production process)
(Enzyme treatment process)
Using unmixed hardwood chips consisting of 70% domestic hardwood chips and 30% eucalyptus wood as raw materials, unbleached pulp made in a factory with a copper number of 20.1 and a pulp viscosity of 41 cps was obtained by kraft cooking. Subsequently, oxygen delignification was performed to obtain an oxygen delignification pulp having a copper number of 9.6 and a pulp viscosity of 25.1 cps. This pulp is filtered with a 100 mesh filter cloth, washed, the pulp concentration is adjusted to 10%, diluted sulfuric acid is added to adjust to pH 8, and then Bacillus sp. Research Institute Patent Biological Deposit Center Deposited xylanase produced by the deposited strain FERM BP-5264) was added to 1 unit / g of pulp and treated at 60 ° C. for 120 minutes. After the treatment, it was filtered through a 100 mesh filter cloth to separate pulp residues and the like, and a 1050 liter treatment liquid (3900 g as the total sugar amount) containing a total sugar concentration of 3700 mg / l was obtained. Subsequently, the mixture was concentrated 40 times by volume using an NF membrane (Nitto Denko: NTR-7450, membrane quality: sulfonated polyethersulfone, salt rejection 50%). This concentrated solution had a total sugar amount of 2700 g, and the total sugar recovery rate was 70%.

(Acid hydrolysis treatment process)
Sulfuric acid was added to 1,000 ml of the concentrated sugar solution obtained in the enzyme treatment step to adjust the pH to 3.5, and then this concentrated sugar solution was reacted at 121 ° C. for 1 hour. As a result of analyzing the reaction product by ion chromatography using an ion chromatography column (Dionex: PA-10), it was found to contain a high concentration of xylo-oligosaccharides (dimer to 10-mer).

(Xylooligosaccharide purification / separation process)
Column filled with 10 ml of xylo-oligosaccharide sugar solution (117 mg / ml) prepared in the acid hydrolysis treatment step and 1.2 g as the total sugar amount with a strongly acidic ion exchange resin (Rohm and Haas: Amberlite 200C) (Inner diameter 36 mm, length 150 mm). After collecting the xylo-oligosaccharide that passed through the column, it was loaded onto a similar column packed with a weakly basic ion exchange resin (Rohm and Haas: IRA67). The xylo-oligosaccharide obtained through the column was concentrated, and then 80 mg of activated carbon (manufactured by Wako Pure Chemicals: product number 037-02115) was added to the xylo-oligosaccharide solution and stirred at 60 ° C. for 1 hour for decolorization. . After stirring, the activated carbon was filtered through a 0.22 μm membrane filter to obtain a purified xylooligosaccharide solution. Absorption at wavelengths of 280 nm and 250 nm was not observed in the purified xylo-oligosaccharide solution, and the ultraviolet absorbing substance contained in the xylo-oligosaccharide solution after acid treatment was removed. The residual rate of ash was 0.1% or less with respect to the acid-treated xylo-oligosaccharide solution as a starting material. The recovery rate of xylo-oligosaccharide was 70.2%. The average degree of polymerization of xylo-oligo and the like was 5.0.

(Preparation of acetalized products such as xylo-oligo)
4 g of the xylo-oligosaccharide obtained as described above was dissolved in 50 ml of tetrahydrofuran, 500 ml of dry acetone and 660 mg of (±) camphorsulfonic acid were added to the solution, and one day at room temperature in a 40 ° C. hot water bath for 19 hours. Reacted. 4 ml of triethylamine was added and the solution was stirred for a while to stop the reaction. Next, the mixture was concentrated and added to a silica gel column (developing solvent: toluene: ethyl acetate = 1: 1) for purification, and then the solvent was distilled off to obtain an acetalized product of xylooligosaccharide.

(Preparation of monosaccharide derivatives)
(Preparation of 2,3,4,6-tetrabenzylglucopyranosyl bromide)
2 g of 2,3,4,6-tetrabenzylglucose was dissolved in a mixed solution of 12 ml of dichloromethane and 0.8 ml of dimethylformamide in the presence of argon gas. Next, oxalyl bromide (6 ml, dissolved in 12 ml DMF at 2 M) was added to the benzyl glucose solution and stirred for 1 hour.

After the solvent was distilled off, the residue was dissolved in 50 ml of dichloromethane. The solution was washed twice with 20 ml of saturated sodium sulfate solution. After adding a magnesium sulfate solution to the dichloromethane solution and drying, the magnesium sulfate was removed by a filter, and the dichloromethane solution was removed by an evaporator to obtain a solid content.
NMR results (numbers are in ppm)
δ: 3.57 (dd, 1H), 3.68 (dd, 1H), 3.79-3.84 (m, 2H, H-4, H-6 '), 4.07 (t, 1H), 4.07-4.11 (m, 1H)
4.47-4.62 (m, 3H), 4.74 (s, 2H), 4.84-4.89 (m, 2H), 5.10 (d, 1H), 6.46 (d, 1H), 7.15-7.40 (m, 20H)
As a result of 1 H-NMR, it was confirmed that 2,3,4,6-tetrabenzylglucose was substituted with 2,3,4,6-tetrabenzylglucopyranosyl bromide.

(Preparation of sodium methanethiosulfonate)
Sodium sulfide 9 hydrate [Kanto Chemical Co., Ltd., special grade, 72.1 g, 0.3 mol] was dissolved in 80 ml of water and heated to 60 ° C. The solution was cooled to 0 to 5 ° C. and distilled methanesulfonyl chloride [Kanto Chemical Co., Ltd., special grade, 23.3 ml, 34.5 g, 0.3 mol] was added dropwise to the sodium sulfide nonahydrate solution over 1 hour. did. The resulting mixed solution was heated under reflux for 18 hours and cooled. The solution was dried with an evaporator, and the solid content was dried in calcium chloride with a vacuum dryer for 24 hours, and then dried with a vacuum dryer for 24 hours. The obtained powder was washed 15 times with 100 ml of ethanol and filtered for each washing. As a result, 32.5 g of sodium thiosulfonate was obtained.

(Preparation of 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl methanethiosulfonate)
Sodium methanethiosulfonate (1.48 g, 11.04 mmol) was added to tetrabenzylglucopyranosyl bromide (2.28 g, 3.7 mmol), and 15 ml of dioxane solution was added. The solution was heated at 70 ° C. for 15 hours and dried with an evaporator to obtain an oily substance. The obtained oil was purified by silica gel column chromatography (hexane / acetic acid ether = 8: 2). The anomeric ratio of purified sugar (76% yield) was β: α = 7.5: 1. The obtained sugar isomers were further separated on a silica gel column (hexane / ethyl acetate = 9: 1), and 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosylmethanethio was obtained. Obtained sulfonate

NMR results (numbers are in ppm)
δ: 3.36 (s, 3H), 3.60 (dd, 1H), 3.45 (m, 4H), 3.78 (pt, 1H), 4.49 (d, 1H), 4.51 (d, 1H)
4.58 (d, 1H), 4.74 (d, 1H), 4.78 (d, 1H), 4.85 (d, 1H), 4.88 (d, 1H), 4.93 (d, 1H), 5.12 (d, 1H)
7.19-7.22,7.31-7.37 (m, 20H)

(Preparation of 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosylmethane disulfide)
2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosylmethanethiosulfonate obtained by the above production method was dissolved in 15 ml of dichloromethane, and triethylamine (221 μL, 1.59 mmol) was dissolved. Was added and dissolved to 0 ° C. A diluted ethanethiol solution [Kanto Chemical Co., Ltd., special grade, 39.7 ml, 0.04 M, 1.59 mmol] was added dropwise to the above solution with stirring over 40 minutes. After dropping, the temperature was returned to room temperature and further stirred for 2 hours. The solution was dried by an evaporator, and the residue was purified by silica gel chromatography (hexane / acetic acid ether = 9: 1) to obtain 2,3,4,6-tetra-O-benzyl-β-D-glucopyrano. Silmethane disulfide was obtained.

NMR results (numbers are in ppm)
δ: 3.30 (s, 3H), 3.51 (t, 1H), 3.59 (t, 1H), 3.63 (dd, 1H), 3.70 (dd, 1H), 3.95 (dd, 1H)
4.10 (ddd, 1H), 4.46 (d, 1H), 4.49 (d, 1H), 4.52 (d, 1H), 4.61 (d, 1H), 4.75 (d, 1H)
4.78 (d, 1H), 4.84 (d, 1H), 4.93 (d, 1H), 6.25 (d, 1H), 7.15-7.19,7.26-7.39 (m, 20H)

(Glycosylation reaction)
Acetalized product of 0.081 mmol of 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosylmethane disulfide, 0.81 mmol of N-succinimide and xylooligosaccharide (average polymerization degree) 5.0, molecular weight 0.081 mmol) was dissolved in 5 ml of dichloromethane in the presence of molecular sieve 4A (Kanto Chemical), stirred at room temperature for 1 hour in an argon atmosphere, and then cooled to 0 ° C.

  0.041 mmol of triethylsilyl methanesulfonate [Kanto Chemical Co., Ltd.] solution was stirred at 0 ° C. for 1 hour, 15 ml of dichloromethane was added, and the washing operation was repeated with a saturated sodium sulfate solution. The mixture was concentrated by an evaporator, and the obtained product was separated by a silica gel column. The resulting solution was concentrated with an evaporator. 1 g of the obtained powder was dissolved in 50 ml of THF, Pd / C [Kanto Chemical Co., Ltd., 50 mg] was added, and the benzyl group was deprotected under a hydrogen atmosphere for 12 hours. The solvent was distilled off from the obtained solution with an evaporator. Further, 4 g of oligosaccharide was taken, and 200 ml of methanol and 40 ml of dichloromethane were added and dissolved. 700 mg (3 mmol) of (±) -10-camphorsulfonic acid was added thereto, and the reaction system was adjusted to pH = 1. A calcium chloride tube was attached to the reaction system and reacted at room temperature for 24 hours. Thereafter, 0.4 ml of triethylamine was added to stop the reaction. It was then concentrated and diluted with 400 ml of ethyl acetate. The extract was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated sodium chloride, and dehydrated with magnesium sulfate. This was filtered and concentrated, purified by adding to a silica gel column (developing solvent: toluene: ethyl acetate = 2: 1), and the solvent was distilled off to obtain 2,3,4,6-tetra-O-benzyl. An oligosaccharide composed of a condensate of -β-D-glucopyranosylmethane disulfide and an xylooligosaccharide acetalized product having an average polymerization degree of 5.0 was obtained.

(Oxidation reaction)
Obtained by 100 g of an aqueous solution containing 0.02 g of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 0.24 g of sodium bromide by the above glycosylation reaction of 0.5 g in terms of absolute dryness. An oligosaccharide consisting of the resulting condensate was added. Thereafter, 4.6 g of sodium hypochlorite [trade name Antiformin, manufactured by Wako Pure Chemical Industries, Ltd.] was added and reacted at room temperature for 50 minutes. After completion of the reaction, ethanol was added to the reaction solution to precipitate the reaction product, and the product was recovered by centrifugation. The collected product was subjected to washing and collecting operations three times using a 70% aqueous ethanol solution, washed and collected with 99% ethanol, and then dried in vacuum to obtain acidic oligosaccharide powder as an oxidation reaction product.

(Polymerization degree of acidic oligosaccharide)
The acidic oligosaccharide powder was dissolved in ultrapure water to prepare a 1% aqueous solution. The average degree of polymerization was determined by measuring the total sugar amount by the phenol-sulfuric acid method, and then measuring the reducing sugar amount of the 1% aqueous solution by the Somogene Nelson method. In any measurement, a calibration curve was prepared using D-xylose. The average degree of polymerization was determined by dividing the total amount of sugar per ml by the amount of reducing sugar per ml. As a result, the average degree of polymerization of the oligosaccharide produced by the above method was 5.2.

(Analysis with nuclear magnetic resonance apparatus and mass spectrometer)
Using the acidic oligosaccharide powder as a pure water solution, the polymerization degree and the quantitative ratio of uronic acid residues and xylose residues in one oligosaccharide molecule were analyzed by NMR method. The proton chemical shift between xylose 1-position (4.409 ppm) and uronic acid 1-position (5.226 ppm) was determined, and the area of each signal corresponding to the amount ratio of hydrogen was calculated. As a result, the area ratio was 5: 1. there were. Therefore, it was found that most of them were acidic xylooligosaccharides in which one glucuronic acid was bonded to a xylose pentamer.
When this aqueous solution was subjected to mass spectrometry and the molar ratio of xylose and glucuronic acid was determined, it was 5: 1, which was consistent with the NMR results. The yield was 85%.

Example 2
In Example 1, xylobiose [manufactured by Wako Pure Chemical Industries, Ltd., M.I. W. 282.25] was used, and oligosaccharides were synthesized in the same manner as in Example 1. The yield was 93%. When analyzed by NMR in the same manner as in Example 1, the area ratio of proton chemical shift between xylose 1-position (4.409 ppm) and uronic acid 1-position (5.226 ppm) was measured, and the area ratio was 2: 1. It was. Moreover, when the molar ratio of xylose and glucuronic acid was calculated | required with the mass spectrometer about the synthetic | combination by mass spectrometry, it was 2: 1 and corresponded with the measurement result of NMR.

Example 3
In Example 1, except that the glycosylation reaction was carried out using 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosylmethanethiosulfonate, the same as Example 1 was carried out. Oligosaccharides were synthesized. The yield was 65%. When analyzed by NMR in the same manner as in Example 1, the area ratio of proton chemical shift between the 1st position of xylose (4.409 ppm) and the 1st position of uronic acid (5.226 ppm) was measured. As a result, the area ratio was 5: 1. It was. Moreover, when the molar ratio of xylose and glucuronic acid was calculated | required with the mass spectrometer about the compound by mass spectrometry, it was 5: 1 and corresponded with the measurement result of NMR.

  According to the present invention, there is provided a method capable of stably producing an acidic oligosaccharide having a low calorie and having functions such as an intestinal regulating action and a caries-preventing action under relatively mild conditions. Acidic xylooligosaccharides, which are expected to have physiological effects from sugar, can be produced in high yield, and the use of xylooligosaccharides is dramatically expanded.

Claims (6)

General formula 1

(In the formula, ring A represents a monosaccharide or a derivative thereof, S is bonded to the anomeric carbon of ring A, n is 1 or 2, and R is a monovalent alkyl group or monovalent aryl. And a sugar acceptor comprising a xylo-oligosaccharide having an average polymerization degree of 2 to 20 is subjected to a glycosylation reaction. The method for producing an oligosaccharide according to claim 1, wherein the ring A in the general formula 1 is at least one selected from glucose, xylose, and glucuronic acid. The oligosaccharide according to claim 1 or 2, wherein the xylo-oligosaccharide having an average degree of polymerization of 2 to 20 as the sugar acceptor is a xylo-oligosaccharide in which all hydroxyl groups not involved in the reaction are protected. Method. The method for producing an oligosaccharide according to any one of claims 1 to 3, wherein the xylo-oligosaccharide having an average degree of polymerization of 2 to 20 is an acetalized xylo-oligosaccharide. The sugar donor is composed of a monosaccharide or a derivative thereof in which the ring A in the general formula represents a neutral monosaccharide or a neutral derivative thereof, and an oxidation reaction for converting an alcoholic hydroxyl group to a carboxyl group is performed after the glycosylation reaction. The method for producing an oligosaccharide according to any one of claims 1 to 4, wherein: The sugar donor in the glycosylation reaction is glucopyranosyl methane sulfite or glucopyranosyl methane disulfite, and the sugar acceptor is a xylooligosaccharide with a protected hydroxyl group that does not participate in the glycosylation reaction The method for producing an oligosaccharide according to any one of claims 1 to 5, wherein: