JP7598148B2 - Method for producing repeating disaccharides and their oligomers in oligosaccharide synthesis - Google Patents

Description

The present invention relates to a synthetic intermediate for glycans and a method for producing the same.

Glycosylation-related muscular dystrophy, a type of muscular dystrophy, is caused by abnormalities in the synthesis of the glycoprotein dystroglycan, and is associated with central nervous system disorders such as brain malformations and mental retardation, as well as damage to muscle cells. Recent research has revealed that abnormalities in the sugar chains of dystroglycan present in muscle cells are the cause of glycosylation-related muscular dystrophy. The sugar chain portion of dystroglycan that has affinity for laminin is called matriglycan. Matriglycan connects muscle cells to the basement membrane via laminin, and abnormalities in the synthesis of this sugar chain cause glycosylation-related muscular dystrophy.

Research has been conducted on the overall structure of the glycan from Ser/Thr onwards, including matriglycan, and it has been found to be an O-mannosylglycan in which matriglycan extends via ribitol phosphate. Matriglycan contains a glycan with repeating disaccharides consisting of α-xylose and β-glucuronic acid at its non-reducing end (Non-Patent Document 1), and it has been found that this glycan with repeating disaccharides is necessary for binding to laminin (Non-Patent Document 2).

Improving the genes that regulate the glycan biosynthetic enzymes involved in the biosynthesis of O-mannosylglycan is seen as a promising treatment for glycosylation-related muscular dystrophy. However, there are many types of genes involved, and it has been reported that overexpression of these genes can worsen the condition, making gene therapy for glycosylation-related muscular dystrophy not easy.

Kanagawa, M. et al., Cell Rep., 14, 2209-2223 (2016) Goddeeris, M. et al., Nature 503, 136-140 (2013)

If we could reconstruct matriglycan on dystroglycan, which connects muscle cells to the basement membrane via laminin, we could suppress the onset of glycosylation-related muscular dystrophy. Therefore, the problem to be solved by the present invention is to reconstruct matriglycan.

The present inventors have conducted extensive research to solve the above problems, and have succeeded in synthesizing a disaccharide unit (Xylα1-3GlcAβ) for synthesizing a glycan having repeating disaccharides composed of α-xylose and β-glucuronic acid, synthesizing a tetrasaccharide in which the disaccharide units are linked together, and synthesizing a tetrasaccharide portion that connects the glycan having the repeating disaccharides and the glycan extending from α-dystroglycan, demonstrating the possibility of reconstructing matriglycan. These findings led the present inventors to complete the present invention.

Thus, the present invention provides the following:
[1] Formula (1):

[In the formula, X ¹ is a leaving group or a protected hydroxyl group, Y ¹ is a carboxyl group or a hydroxymethyl group or a group which is a precursor thereof, and R ¹ to R ⁵ are each independently hydrogen or a protecting group for a hydroxyl group]
A disaccharide derivative represented by the formula:

[2] The disaccharide derivative according to [1], wherein ^X1 is a group selected from the group consisting of trichloroacetimidoyloxy, alkylthio, ^arylthio , halogen, pentenyloxy, alkoxy, and phenoxy, ^Y1 is ^COOZ1 or _CH2OZ2 , ^Z1 is an alkyl group, ^Z2 is a protecting group for a hydroxyl group, and ^R1 to ^R5 are each independently a group selected from the group consisting of benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, isopropylidene, benzylidene, and trialkylsilyl.

[3] Formula (2):

[wherein ^X2 is a leaving group, Y is a group that fixes the conformation of the xylose residue, and ^R6 is a group selected from the group consisting of p-alkyloxybenzyl, 3,4-dialkyloxybenzyl, immobilized p-alkyloxybenzyl, immobilized 3,4-dialkyloxybenzyl, naphthyl, naphthylmethyl, -CH= _CH2 , -C( _CH3 )= _CH2 , _-CH2 -CH= _CH2 , -CH= _CH - _CH3 , -CH=C=CH2, and hydrogen]
and a xylose derivative represented by the formula:
Formula (3):

[wherein ^X3 is a leaving group or a protected hydroxyl group, ^R7 and ^R8 are protecting groups for the hydroxyl group, and ^Y4 is a group which is a precursor of a carboxyl group or a hydroxymethyl group]
From a glucose derivative or a glucuronic acid derivative represented by
Formula (4):

[In the formula, ^X3 , ^R7 , ^R8 and ^Y4 are as defined above.]
The method for producing an α-glycoside represented by the following steps 1) to 3):
Step 1) reacting a xylose derivative represented by formula (2) with a glucose derivative or a glucuronic acid derivative represented by formula (3) to produce a mixed acetal derivative via the ^OR6 group of the xylose derivative and the unprotected hydroxyl group of the glucose derivative or the glucuronic acid derivative;
Step 2) The mixed acetal derivative obtained in step 1) is reacted by activating the leaving group ^X2 to produce an α1→3 disaccharide derivative, and then
Step 3) Deprotecting the disaccharide derivative obtained in step 2) to obtain the compound represented by formula (4).

[4] The method according to [3], wherein ^X2 is a group selected from the group consisting of trichloroacetimidoyloxy, alkylthio, halogen, arylthio, and pentenyloxy, Y is a group selected from the group consisting of acetal, carbonate, silylene acetal, and stannylene acetal, ^R6 is a group selected from the group consisting of p-methoxybenzyl and naphthylmethyl, ^X3 is a group selected from the group consisting of trichloroacetimidoyloxy, alkylthio, arylthio, halogen, pentenyloxy, alkoxy, and phenoxy, ^R7 and ^R8 are each independently a group selected from the group consisting of benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, and ^{trialkylsilyl} , ^Y4 is ^COOZ2 or _CH2OZ3 , ^Z2 is an alkyl group, and ^Z3 is a protecting group for a hydroxyl group.

[5] The method according to [4], wherein R ⁷ and R ⁸ are groups selected from the group consisting of benzoyl including substituted benzoyl, acetyl including substituted acetyl, and levulinoyl.

[6] Formula (2'):

[In the formula, X ² ' is a leaving group, Y' is a group that fixes the conformation of the xylose residue, and R ⁶ ' is a protecting group for a hydroxyl group that does not have neighboring group participation ability]
and a xylose derivative represented by the formula:
Formula (3'):

[In the formula, X ³ ' is a leaving group or a protected hydroxyl group, R ⁷ ' is a protecting group for a hydroxyl group having an adjacent group participation ability, R ⁸ ' is a protecting group for a hydroxyl group, and Y ⁴ ' is a group which is a precursor of a carboxyl group or a hydroxymethyl group]
From a glucose derivative or a glucuronic acid derivative represented by
Formula (4'):

[In the formula, X ³ ', R ⁷ ', R ⁸ ' and Y ⁴ ' are as defined above.]
The method for producing an α-glycoside represented by the following steps 1) to 3):
Step 1) directly condensing a xylose derivative represented by formula (2') with a glucose derivative or a glucuronic acid derivative represented by formula (3') to obtain a mixture of α1→3 glycosides and β1→3 glycosides;
Step 2) The mixture obtained in step 1) is deprotected, and then
Step 3) isolating the α-glycoside from the mixture deprotected in step 2).

[7] The method according to [6], wherein the condensation in step 1) is carried out using NIS-AgOTf, NIS-TfOH, MeOTf, CuBr ₂ -AgOTf-nBu ₄ NI, or NBS-AgOTf as a condensation agent.

[8] The method according to [6] or [7], wherein the deprotection in step 2) is carried out by hydrolysis with trifluoroacetic acid (TFA) to form a triol having hydroxyl groups at the 2-, 3-, and 4-positions of the xylose residue.

[9] The method according to any one of [6] to [8], wherein the separation in step 3) is carried out by dissolving the β-glycoside in chloroform to remove the β-glycoside.

[10] A method for producing a compound represented by the formula (5):

[In the formula, ^X4 is a leaving group or a protected hydroxyl group, and n is an integer of 1 or more]
A method for producing an XG unit oligomer represented by the formula:

[11] Using a disaccharide derivative represented by formula (1) as a sugar donor,

[In the formula, ^Y5 is a group which is a precursor of a carboxyl group or a hydroxymethyl group, ^X5 is a leaving group or a protected hydroxyl group, and ^R9 to ^R12 are protecting groups for the hydroxyl groups.]
The method according to claim 10, comprising reacting a disaccharide derivative represented by the following formula (1):

[12] The method according to [11], ^{wherein Y5} is ^COOZ3 or _CH2OZ4 , ^Z3 is an alkyl group, ^Z4 is a protecting group for a hydroxyl group, ^X5 is a group selected from the group consisting of trichloroacetimidoyloxy, alkylthio, arylthio, halogen, pentenyloxy, alkoxy and phenoxy, and ^R9 to ^R12 are each independently a group selected from the group consisting of benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl and trialkylsilyl.

[13] The method according to [12], wherein R ⁹ is a group selected from the group consisting of benzoyl including substituted benzoyl, acetyl including substituted acetyl, and levulinoyl.

[14] Formula (9):

or a derivative thereof.

[15] Formula (7):

[In the formula, X ¹ is a leaving group or a protected hydroxyl group, X ² to X ⁶ are protecting groups for the hydroxyl groups, and X ⁷ is an alkyl group.]
With a disaccharide donor represented by formula (8):

[In the formula, X ⁸ to X ¹³ are protecting groups for hydroxyl groups, and X ¹² and X ¹³ may combine together to form an acetal]
and then the tetrasaccharide derivative is deprotected to give the disaccharide acceptor of formula (9):

A method for producing a tetrasaccharide represented by formula (9), comprising obtaining a tetrasaccharide represented by formula (9):

[16] The method according to [15], ^{wherein X1} is trichloroacetimidoyloxy, ^X2 to ^X6 are acetyl, ^X7 is methyl, X8 is allyl, ^X9 and ^X10 are benzyl, ^X11 is TBDPS, and ^X12 and ^X13 together form isopropylidene acetal.

According to the present invention, a disaccharide unit consisting of α-xylose, which may be derivatized, and β-glucuronic acid, which may be derivatized, is provided. The post-phosphate glycan can be reconstructed using the disaccharide unit. This makes it possible to suppress the onset of glycosylation-induced muscular dystrophy.

1. Disaccharide Derivative In one aspect, the present invention relates to a disaccharide derivative represented by formula (1):

[In the formula, X ¹ is a leaving group or a protected hydroxyl group, Y ¹ is a carboxyl group or a hydroxymethyl group or a group which is a precursor thereof, and R ¹ to R ⁵ are each independently hydrogen or a protecting group for a hydroxyl group]
The present invention provides a disaccharide derivative represented by the formula:

As X ¹ , a known leaving group or a protected hydroxyl group can be used, and examples thereof include leaving groups commonly used in sugar chain synthesis, such as trichloroacetimidoyloxy, alkylthio, arylthio, halogen, pentenyloxy (4-pentenyloxy), etc., but are not limited thereto. The same applies to leaving groups in other places in this disclosure. Preferred leaving groups X ¹ include, but are not limited to, those that form thioglycosides, such as trichloroacetimidoyloxy, alkylthio, and phenylthio. Protecting groups for hydroxyl groups are well known to those skilled in the art. That is, protected hydroxyl groups are well known to those skilled in the art. X ¹ may be alkoxy or phenoxy.

Y ¹ is a carboxyl or hydroxymethyl group or a group that is a precursor thereof. Groups that are precursors of carboxyl or hydroxymethyl groups are well known to those skilled in the art and represent groups that can be converted to carboxyl or hydroxymethyl groups, typically in one step, for example by oxidation/reduction reactions, reactions such as hydrolysis, heating, etc. Precursors of carboxyl or hydroxymethyl groups are usually also derivatives of carboxyl or hydroxymethyl groups. Examples of Y ¹ groups include, but are not limited to, COOZ ¹ or CH ₂ OZ ^2. Here, Z ¹ is an alkyl group, preferably an alkyl group having 1 to 3 carbon atoms. A more preferred Z ¹ group is methyl. Z ² is a commonly used hydroxyl protecting group. Hydroxyl protecting groups are known, and specific examples are described herein.

R ¹ to R ⁵ are each independently hydrogen or a protecting group for a hydroxyl group. The protecting group in the present disclosure is preferably a group that can be removed after glycosylation. In the present disclosure, when there are multiple protecting groups for a hydroxyl group in a compound, the multiple protecting groups may be different from each other or may be the same type. By using protecting groups of different types, selective deprotection can be promoted. Such protecting groups for a hydroxyl group are known, and examples thereof include protecting groups described in Chapter 2 of Greene's Protective Groups in Organic Synthesis, 5th Edition (Wiley). Specific examples thereof include, but are not limited to, benzyl, p-methoxybenzyl, p-methoxyphenyl, p-nitrobenzyl, benzoyl, p-methylbenzoyl, benzylidene, acetyl, pivaloyl, levulinoyl, allyl, methoxymethyl, tert-butyldimethylsilyl, triisopropylsilyl, isopropylidene, benzylidene, allyloxycarbonyl, and trialkylsilyl. Preferred R ¹ groups include, but are not limited to, benzoyl including substituted benzoyl with neighboring group participation ability, acetyl including substituted acetyl, and levulinoyl. Further examples of protecting groups with neighboring group participation ability are described for the R ⁷ ' group below, and they can be used for the R ¹ group. Preferred R ² to R ⁵ groups include, but are not limited to, protecting groups commonly used in sugar chain synthesis, such as benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, and trialkylsilyl. It is preferred that at least one of R ¹ to R ⁵ (e.g., R 1 or any of R ² to R ⁵ ) is a protecting group, and in that case ^{, the remaining group is hydrogen. All of R 1 to} R ⁵ may be protecting groups.

In a further aspect, the present invention relates to a compound of formula (2):

[wherein ^X2 is a leaving group, Y is a group that fixes the conformation of the xylose residue, and ^R6 is a group selected from the group consisting of p-alkyloxybenzyl, 3,4-dialkyloxybenzyl, immobilized p-alkyloxybenzyl, immobilized 3,4-dialkyloxybenzyl, naphthyl, naphthylmethyl, -CH= _CH2 , -C( _CH3 )= _CH2 , _-CH2 -CH= _CH2 , -CH=CH- _CH3 , -CH=C= _CH2 , and hydrogen]
and a xylose derivative represented by the formula:
Formula (3):

[wherein ^X3 is a leaving group or a protected hydroxyl group, ^R7 and ^R8 are protecting groups for the hydroxyl group, and ^Y4 is a group which is a precursor of a carboxyl group or a hydroxymethyl group]
From a glucose derivative or a glucuronic acid derivative represented by
Formula (4):

[In the formula, ^X3 , ^R7 , ^R8 and ^Y4 are as defined above.]
The method for producing an α-glycoside represented by the following steps 1) to 3):
Step 1) reacting a xylose derivative represented by formula (2) with a glucose derivative or a glucuronic acid derivative represented by formula (3) to produce a mixed acetal derivative via the ^OR6 group of the xylose derivative and the unprotected hydroxyl group of the glucose derivative or the glucuronic acid derivative;
Step 2) The mixed acetal derivative obtained in step 1) is reacted by activating the leaving group ^X2 to produce an α1→3 disaccharide derivative, and then
Step 3) Deprotecting the disaccharide derivative obtained in step 2) to obtain a compound represented by formula (4).

In the above-mentioned method for producing α-glycosides, the compound of formula (2) serves as a sugar donor and the compound of formula (3) serves as a sugar acceptor.

In the compound of formula (2), ^X2 is a leaving group. ^X2 is the same as the leaving group ^X1 described above, and examples thereof include leaving groups commonly used in sugar chain synthesis, such as trichloroacetimidoyloxy, alkylthio, arylthio, phenylsulfinyl, halogen, and pentenyloxy, but are not limited thereto. Examples of ^X2 preferable as a leaving group include those that form thioglycosides, such as trichloroacetimidoyloxy, alkylthio, and arylthio, but are not limited thereto. The alkylthio group described as a leaving group in this disclosure may be, for example, an alkylthio group having 1 to 20 carbon atoms, but is not limited thereto. The arylthio group described as a leaving group in this disclosure may be, for example, a phenylthio or tolylthio, but is not limited thereto.

In the sugar donor of formula (2), Y is a group that can fix the xylose residue in the indicated conformation and prevent ring inversion. In formula (2), the two oxygen atoms shown on the left side are included in the Y group. Specifically, Y can be an acetal, carbonate, silylene acetal, or stannylene acetal. Acetals include diacetals. It should be understood that the oxygen atoms that define these structures correspond to the two oxygen atoms shown on the left side of formula (2) above. More specifically, Y can be a structure represented by formula (2a) below.

In formula (2a), Z is C, Si, or Sn, and when Z=C and Y is an acetal, n is an integer of 0 to 2 (preferably 0 or 1), except for the above, n is 0. The two Ls bonded to each Z are each independently alkyl, phenyl, or hydrogen. It is preferable that either one or both of the two Ls are alkyl or phenyl. When n is 1 or more, one of the two Ls bonded to each Z may further be alkyloxy (e.g., methoxy) or phenyloxy. Alternatively, when Z=C and Y is a carbonate, the two Ls bonded to Z together represent "O=". The alkyl group in alkyl and alkyloxy may be, for example, an alkyl group having 1 to 6 carbon atoms, but is not limited thereto.
Preferred specific examples of the sugar donor of formula (2) are shown below. In the following examples, (2-1) and (2-2) represent embodiments in which Y is an acetal with n=0, (2-3) represents an embodiment in which Y is an acetal with n=1, and (2-4), (2-5), and (2-6) represent embodiments in which Y is a carbonate, a silylene acetal, and a stannylene acetal, respectively. The L groups shown here are merely examples, and the L group is not limited thereto.

R ⁶ is selected from the group consisting of p-alkyloxybenzyl, 3,4-dialkyloxybenzyl, immobilized p-alkyloxybenzyl, immobilized 3,4-dialkyloxybenzyl, naphthyl, naphthylmethyl, -CH=CH ₂ , -C(CH ₃ )=CH ₂ , -CH ₂ -CH=CH ₂ , -CH=CH-CH ₃ , -CH=C=CH ₂ , and hydrogen. These R ⁶ are groups that generate a mixed acetal linking the 2-position of the glycosyl donor and the 3-position (the only unprotected hydroxyl group) of the glycosyl acceptor by the IAD (intramolecular aglycon delivery) method. The IAD method itself is known to those skilled in the art and has been reviewed by Cumpstey in Carbohydrate Research, 343, 1553-1573 (2008). For example, when R ⁶ is p-alkyloxybenzyl, 3,4-dialkyloxybenzyl, immobilized p-alkyloxybenzyl, immobilized 3,4-dialkyloxybenzyl, naphthyl, or naphthylmethyl, a mixed acetal is generated between the sugar acceptor shown in formula (3) (via the unprotected hydroxyl group at the 3-position of the sugar acceptor) under oxidation conditions using 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). As will be understood by those skilled in the art, in addition to DDQ, different reagents can be used to generate mixed acetals depending on the type of R ⁶ (for example, but not limited to, acid catalysts such as tosylic acid, N-iodosuccinimide (NIS), and base catalysts such as imidazole and 4-dimethylaminopyridine (DMAP)). In particular, when R ⁶ is hydrogen, Me ₂ SiCl ₂ can be added to the reaction with the sugar acceptor to participate in the formation of a silylene mixed acetal. As described in the above review by Cumpstey, specific reagents for producing mixed acetals by the IAD method are known to those skilled in the art and can be appropriately selected by those skilled in the art. The alkyloxy group in the above R ⁶ group may be, for example, one having 1 to 6 carbon atoms, but is not limited thereto. Preferred R ⁶ groups include, but are not limited to, p-methoxybenzyl and naphthylmethyl.

In the sugar acceptor of formula (3), X ³ is a leaving group or a protected hydroxyl group, and R ⁷ and R ⁸ are protecting groups for the hydroxyl group. X ³ is the same as X ¹ described above, and R ⁷ and R ⁸ are the same as R ¹ to R ⁵ , particularly R ¹ and R ^2, described above. X ³ is preferably a leaving group that is commonly used in sugar chain synthesis, such as trichloroacetimidoyloxy, alkylthio, arylthio, halogen, and pentenyloxy. Preferred R ⁷ and R ⁸ include protecting groups that are commonly used in sugar chain synthesis, such as benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, and trialkylsilyl. Particularly preferred R ⁷ includes, but is not limited to, benzoyl including substituted benzoyl with neighboring group participation ability, acetyl including substituted acetyl, and levulinoyl. Further examples of protecting groups capable of anchimeric participation are described below for the R ⁷ ' group and can be used for the R ⁷ group.

^Y4 is the same as ^Y1 described above, and examples thereof include, but are not limited to, ^COOZ2 or _CH2OZ3 . Here, ^Z2 is an alkyl group, preferably an alkyl group having 1 to ³ carbon atoms. A more preferred ^Z2 group is methyl. ^Z3 is a commonly used hydroxyl-protecting group. Hydroxyl-protecting groups are known.

The xylose derivative (sugar donor) represented by formula (2) is condensed with the glucose derivative or glucuronic acid derivative (sugar acceptor) represented by formula (3) to obtain the α-glycoside represented by formula (4). The steps for obtaining the α-glycoside include the following steps 1) to 3). Steps 1) to 3), especially steps 1) and 2), are based on the IAD method.
Step 1) reacting a xylose derivative represented by formula (2) with a glucose derivative or a glucuronic acid derivative represented by formula (3) to produce a mixed acetal derivative in which the ^OR6 group at the 2-position of the xylose derivative is linked to the unprotected hydroxyl group at the 3-position of the glucose derivative or the glucuronic acid derivative;
Step 2) The mixed acetal derivative obtained in step 1) is reacted by activating the leaving group ^X2 to produce an α1→3 disaccharide derivative, and then
Step 3) The disaccharide derivative obtained in step 2) is deprotected to obtain the compound of formula (4).

The reaction in step 1) can be carried out, for example, in the presence of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). The mixed acetal derivative produced in step 1) is a mixed acetal in which the 2-position carbon of the glycosyl donor and the 3-position carbon of the glycosyl acceptor are bonded to two oxygen atoms of the acetal, respectively. The disaccharide derivative obtained in step 2) is a disaccharide in which the 1-position carbon of the glycosyl donor and the 3-position carbon of the glycosyl acceptor are bonded to the same oxygen atom, as in formula (4). A variety of condensing agents (glycosylation promoters) capable of activating different types of leaving groups ^X2 are known to those skilled in the art, and are described in detail in Cumpstey's review as part of the IAD method. The reaction in step 2) can be carried out in the presence of a condensing agent (glycosylation promoter) such as NIS-AgOTf, NIS-TfOH, MeOTf, CuBr ₂ -AgOTf-nBu ₄ NI, or NBS-AgOTf. Step 3) is a deprotection step. Although the deprotection method varies depending on the type of protecting group, various deprotection methods for protecting groups are known. For example, in the case of a benzyl group, deprotection may be carried out by hydrolysis with an acid or alkali. Trifluoroacetic acid is particularly preferably used for deprotection. The Y group is also removed by deprotection, leaving hydroxyl groups at the 3- and 4-positions of xylose.

A specific example of the method for producing the above-mentioned α-glycoside is shown in Scheme 1 below.

[In the above scheme, Lev is -C(=O) _C2H4C (=O) _CH3 , Bz is -C(= _O ) _C6H5 , MBn is _-CH2C6H4OCH3 ( _{p )} , MP is _-C6H4OCH3 (p ₎ , Me is _-CH3 , Ac is _{-C (} =O) _CH3 and MBz is -C(= _{O ) C6H4CH3} ( _p )]

In a further aspect, the present invention provides a method for producing a disaccharide (xylose α1-3 glucuronic acid, in which the xylose residues and glucuronic acid residues may be derivatized), which comprises reacting a xylose derivative represented by the above formula (2) with a glucose derivative or a glucuronic acid derivative represented by the above formula (3) in the presence of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) or the like to obtain a mixed acetal derivative, condensing the mixed acetal derivative in the presence of a condensing agent, and then, if desired, deprotecting the condensate.
Optionally, deprotection refers to removing some (at least one) or all of the protecting groups. Derivatization of sugars is known to those skilled in the art. For example, the disaccharide may contain a known protecting group and may contain a known leaving group. The protecting group and leaving group of the disaccharide include, but are not limited to, those described above.

The reaction between a xylose derivative (sugar donor) corresponding to formula (2) and a glucose derivative or glucuronic acid derivative (sugar acceptor) corresponding to formula (3) has the following problems:
- Since neighboring group participation is not available for α-xylose, stereocontrol at the anomeric position is difficult.
-Xylose is prone to ring inversion.
These problems may result in the α-glycosidic bond not being formed.
Therefore, the present inventors have made it possible to perform α-selective glycosylation by protecting the 2-hydroxyl group of xylose with a protecting group that induces intramolecular aglycon transfer and condensing it with a sugar acceptor (glucuronic acid derivative), thereby causing an intramolecular aglycon transfer reaction. As described above, examples of protecting groups that induce intramolecular aglycon transfer include p-methoxybenzyl and naphthyl groups.
Furthermore, the present inventors have decided to prevent ring inversion of xylose by protecting the hydroxyl groups at the 3- and 4-positions of xylose as a diacetal using 2,3-butanedione or the like and fixing the ring.
By utilizing these ingenuity, the present inventors have solved the above problems and succeeded in stereoselectively synthesizing the α-glycoside represented by formula (4).

The hydroxyl groups of the disaccharide of formula (4) obtained by the above method can be appropriately protected to obtain the disaccharide derivative of the present invention represented by formula (1). The selection and attachment/detachment of the protecting groups are within the skill of a person skilled in the art.

In the method described in paragraphs 0014 to 0025 above, although a disaccharide product can be obtained stereoselectively, the yield is low. Therefore, the present inventors directly condensed a xylose derivative (sugar donor) represented by formula (2') with a glucose derivative or glucuronic acid derivative (sugar acceptor) represented by formula (3') without using a method involving mixed acetal (intramolecular aglycon transfer) to obtain a mixture containing α-glycosides and β-glycosides represented by formula (4'). Isolation of the desired α-glycosides was difficult. However, as a result of intensively studying the solubility of a diastereomeric mixture containing the desired α-glycoside obtained by removing the protecting group by hydrolysis with trifluoroacetic acid (TFA) shown in Scheme 2 below, the inventors discovered that the desired α-glycoside is soluble in methanol but insoluble in chloroform, while the by-product β-glycoside is soluble in chloroform. By utilizing this characteristic, the present invention has succeeded in obtaining only the desired α-glycoside in a higher yield than the methods described in paragraphs 0014 to 0025 above.

Thus, in a further aspect, the present invention provides a method for producing a method for treating a pulmonary arthritis, comprising the steps of:
Formula (2'):


[In the formula, X ² ' is a leaving group, Y' is a group that fixes the conformation of the xylose residue, and R ⁶ ' is a protecting group for the hydroxyl group]
and a xylose derivative represented by the formula:
Formula (3'):

[wherein X ³ ' is a leaving group or a protected hydroxyl group, R ⁷ ' is a protecting group for a hydroxyl group, R ⁸ ' is a protecting group for a hydroxyl group, and Y ⁴ ' is a group which is a precursor of a carboxyl group or a hydroxymethyl group]
From a glucose derivative or a glucuronic acid derivative represented by
Formula (4'):

[In the formula, X ³ ', R ⁷ ', R ⁸ ' and Y ⁴ ' are as defined above.]
The method for producing an α-glycoside represented by the following steps 1) to 3):
Step 1) directly condensing a xylose derivative represented by formula (2') with a glucose derivative or a glucuronic acid derivative represented by formula (3') to obtain a mixture of α1→3 glycosides and β1→3 glycosides;
Step 2) The mixture obtained in step 1) is deprotected, and then
Step 3) isolating the α-glycoside from the mixture deprotected in step 2).

The glycosyl donor 103 used in the condensation gives the products 208a and 208b in the same yield regardless of whether it is α or β (Method 1 and Method 2 in Scheme 2 below). Therefore, the stereochemistry of the glycosyl donor 103 used is not limited, and a mixture thereof may also be used (Method 3 in Scheme 2 below).

In the sugar donor represented by formula (2'), X ² ' and Y' are the same as X ² and Y described in relation to formula (2), respectively. In formula (2'), the two oxygen atoms shown on the left side are included in the Y' group. R ⁶ ' is a hydroxyl-protecting group. It is more preferable that R ⁶ ' is a hydroxyl-protecting group that does not have neighboring group participation ability, and an acyl group that has neighboring group participation ability can exclusively produce β-glycosides. Particularly suitable specific examples of R ⁶ ' include, but are not limited to, a benzyl group, a p-methoxybenzyl group, a p-methoxyphenyl group, a p-nitrobenzyl group, an allyl group, a methoxymethyl group, a tert-butyldimethylsilyl group, and a triisopropylsilyl group. A preferred example of R ⁶ ' is, but is not limited to, a p-methoxybenzyl group.
In the present disclosure, a hydroxyl-protecting group capable of neighboring group participation (Chemical Encyclopedia, Kyoritsu Shuppan) refers to a hydroxyl-protecting group generalized as -C(=O)R, as will be understood by those skilled in the art, and referring to the example of formula ( ² '), it is a protecting group that has the function of exclusively allowing attack of an acceptor from the opposite side (trans side) by forming a ring structure formed by the oxygen atom attacking a carbocation generated on the 1-position carbon atom after the 1-position leaving group (X 2 ') is eliminated, thereby covering the same face (cis face) as the 2-position hydroxyl group. Protecting groups that do not fall under this description are understood to be protecting groups without neighboring group participation ability.

In the sugar acceptor represented by formula (3'), R ⁷ ' is a hydroxyl-protecting group, but in order to form a β-glycoside in the condensation between sugars, R ⁷ ' is preferably a hydroxyl-protecting group capable of neighboring group participation. Examples of hydroxyl-protecting groups capable of neighboring group participation include, but are not limited to, those that form an acyl group as -C(=O)R, such as an acetyl group where R is an alkyl or aryl, an acetyl group substituted with 1 to 3 halogen atoms, a benzoyl group, a benzoyl group substituted with 1 to 5 halogens, a nitro group, and/or an alkyl group having 1 to 3 carbon atoms, and a levulinoyl group, as well as a benzyloxycarbonyl group, an allyloxycarbonyl group, and a 2,2,2-trichloroethoxycarbonyl group. Preferred R ⁷ ' include, but are not limited to, an acetyl group, an acetyl group substituted with 1 to 3 halogen atoms, a benzoyl group, a benzoyl group substituted with 1 to 5 halogens, a nitro group, and/or an alkyl group having 1 to 3 carbon atoms. X ³ ', R ⁸ ' and Y ⁴ ' are the same as X ³ , R ⁸ and Y ⁴ described in relation to formula (3), respectively. For example, X ³ ' may be a hydroxyl group protected by p-methoxyphenyl, R ⁷ ' and R ⁸ ' may be acetyl groups, and Y ⁴ ' may be a methyl carboxylate group.

The condensation reaction in step 1) can be carried out using a known condensation agent (glycosylation promoter). Known condensation agents include NIS-AgOTf, NIS-TfOH, MeOTf, CuBr ₂ -AgOTf-nBu ₄ NI, and NBS-AgOTf, with NIS-AgOTf being preferred. The leaving group X ² ' is activated in the presence of these condensation agents. Direct condensation refers to a bond formed between the substituent of the sugar donor and the substituent of the sugar acceptor without the intervention of another compound or group. In particular, in the present disclosure, direct condensation refers to a condensation that forms a 1→3 glycosidic bond from the beginning, in contrast to the mode via intramolecular aglycon transfer.

The deprotection in step 2) can be carried out by a known method. Preferably, the 2-, 3-, and 4-positions of the xylose residue are deprotected to give a triol. Among such triols, α-glycosides are disaccharide derivatives of formula (4'). Moreover, the disaccharide derivatives of formula (4') can be easily separated using a solvent to be described later. An acid is used as the agent for such deprotection. As such acids, trifluoroacetic acid (TFA), trichloroacetic acid, etc. are preferred, with TFA being particularly preferred.

In step 3), α-glycosides are separated from the deprotected glycoside mixture (diastereomeric mixture of deprotected triol) obtained in step 2). As a method for separating diastereomers (α-glycosides and β-glycosides), a method using chromatography, a solvent extraction method, etc. can be used. When the separated α-glycoside is different from the disaccharide derivative of formula (4'), the disaccharide derivative of formula (4') may be obtained by protecting and/or deprotecting it by a known method. In particular, the α-glycoside (disaccharide derivative of formula (4')) obtained as a triol by deprotecting the 2-, 3-, and 4-positions of the xylose residue is insoluble in chloroform but soluble in methanol, and the β-glycoside is soluble in chloroform (its solubility in methanol is relatively low). By utilizing this property, the desired α-glycoside can be separated by treating the diastereomeric mixture with chloroform and removing the dissolved β-glycosides. Alternatively, the desired α-glycoside may be isolated via treatment of the diastereomeric mixture with methanol and recovery of the dissolved α-glycoside.

A specific example of the above-mentioned new method for producing α-glycosides is shown in Scheme 2 below.

[In the above scheme, MBn is _-CH2C6H4OCH3 ( _p ), MP is _-C6H4OCH3 ( _{p )} , Me is _-CH3 , _Ac is -C(=O) _CH3 , NIS is N _- iodosuccinimide, AgOTf is silver trifluoromethanesulfonate, and TFA is trifluoroacetic acid]

In a further aspect, the present invention provides a method for producing a disaccharide (xylose α1-3 glucuronic acid, in which the xylose residues and glucuronic acid residues may be derivatized), which comprises directly condensing a xylose derivative represented by the above formula (2') with a glucose derivative or a glucuronic acid derivative represented by the above formula (3') to obtain a mixture of α-glycosides and β-glycosides, optionally deprotecting the mixture, and then separating the α-glycosides from the mixture.
Optionally, deprotection refers to removing some (at least one) or all of the protecting groups. Derivatization of sugars is known to those skilled in the art. For example, the disaccharide may contain a known protecting group and may contain a known leaving group. The protecting group and leaving group of the disaccharide include, but are not limited to, those described above.

2. Oligomers of disaccharide derivatives The disaccharide derivatives of the present invention can be used as building blocks (sometimes referred to as XG units in this specification) for the synthesis of post-phosphate glycans on dystroglycan, which connects muscle cells to the basement membrane via laminin. Post-phosphate glycans can be reconstructed by oligomerizing the disaccharide derivatives. The disaccharide derivatives of the present invention are essential synthetic units for reconstructing post-phosphate glycans.

A tetrasaccharide derivative can be obtained by condensing disaccharide derivatives together. A hexasaccharide derivative can be obtained by condensing a tetrasaccharide derivative with a disaccharide derivative and optionally deprotecting the derivative. An octasaccharide derivative can be obtained by condensing the hexasaccharide derivative with a disaccharide derivative and optionally deprotecting the derivative. Alternatively, a tetrasaccharide derivative can be condensed with another tetrasaccharide derivative and optionally deprotected to obtain an octasaccharide derivative. In this manner, an oligomer of a disaccharide derivative can be synthesized.

Thus, in a further aspect, the present invention relates to a method for the preparation of a disaccharide derivative of formula (1) as an intermediate (XG unit), comprising the steps of:

[In the formula, ^X4 is a leaving group or a protected hydroxyl group, and n is an integer of 1 or more]
The present invention provides a method for producing an oligomer of XG units represented by the formula: Since the ligation reaction of the oligomer can be simply repeated, it will be understood by those skilled in the art that there is no particular upper limit to n. n can be, for example, 300 or less, 200 or less, 100 or less, 50 or less, 20 or less, etc.

When ^X4 in the compound represented by formula (5) is a leaving group, ^X4 is a leaving group similar to X1 described above, etc. When ^X4 is a protected hydroxyl group, ^X4 may contain a protecting group similar to ^R1 described above ^, etc.

Specifically, a disaccharide derivative represented by the formula (1) is used as a sugar donor, and a compound represented by the formula (1'):

[In the formula, ^Y5 is a group which is a precursor of a carboxyl group or a hydroxymethyl group, ^X5 is a leaving group or a protected hydroxyl group, and ^R9 to ^R12 are protecting groups for the hydroxyl groups.]
A tetrasaccharide derivative can be obtained by condensing the disaccharide derivative represented by formula (1) with a tetrasaccharide derivative, or by condensing a tetrasaccharide derivative with a disaccharide derivative represented by formula (1'), or by repeating the condensation of these oligomers with oligomers and/or disaccharide derivatives. These oligomer derivatives can be deprotected by a known method to obtain an oligomer represented by formula (5) with a longer chain length.

In formula (1'), ^Y5 is a group similar to ^Y1 described above. Examples of ^Y5 include, but are not limited to, ^COOZ3 or _CH2OZ4 . Here, ^Z3 is an alkyl group, preferably an alkyl group having 1 to ³ carbon atoms. A more preferred ^Z3 group is methyl. ^Z4 is a normal hydroxyl protecting group. ^X5 is a leaving group or a protected hydroxyl group. ^R9 to ^R12 are hydroxyl protecting groups, which are the same groups as R1 described above. Preferred ^X5 includes, but is not limited to, leaving groups commonly used in sugar chain synthesis, such as trichloroacetimidoyloxy, alkylthio, arylthio, halogen, and pentenyloxy. Preferred ^R9 to ^R12 include, but are not limited to, protecting groups commonly used in sugar chain synthesis, such as benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including ^substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, and trialkylsilyl. Preferred ^{R9 groups} include, but are not limited to, benzoyl, including substituted benzoyl, acetyl, including substituted acetyl, levulinoyl, etc. Further examples of protecting groups capable of neighboring group participation are described above for the ^R7 ' group and can be used for the ^R9 group.

A specific example of a procedure for synthesizing a tetrasaccharide derivative by condensing a disaccharide derivative represented by formula (1) as a sugar donor and a disaccharide derivative represented by formula (1') as a sugar acceptor is shown in the following scheme 3. The oligomer 206b obtained in the specific example below can be deprotected by a known method to obtain the oligomer represented by formula (5) (n=1).

[In the above scheme, Lev is -C(=O) _C2H4C (=O) _CH3 , Bz is -C(= _O ) _C6H5 , _MP is _{-C6H4OCH3 (} p), Me is _-CH3 and MBz is -C(= _O ) _C6H4CH3 ( _p ) _]

The oligomer 206b obtained in the above specific example can be deprotected by a known method, for example by treating it with a base such as an aqueous sodium hydroxide solution, to obtain the oligomer (n=1) shown in formula (5).

In a further aspect, the present invention provides a method for producing an optionally derivatized tetrasaccharide represented by formula (5), which comprises reacting a disaccharide derivative represented by formula (1) as a sugar donor with a disaccharide derivative represented by formula (1') as a sugar acceptor, and then, if desired, deprotecting the obtained tetrasaccharide derivative.
Optionally, deprotection refers to removing some (at least one) or all of the protecting groups. Derivatization of sugars is known to those skilled in the art. For example, the tetrasaccharide may contain known protecting groups and may contain known leaving groups. The protecting groups and leaving groups of the tetrasaccharide include, but are not limited to, those described above.

3. Synthesis of a tetrasaccharide linking a xylose-glucuronic acid repeat structure of a post-phosphate glycan and a glycan extending from α-dystroglycan In a further aspect, the present invention relates to a tetrasaccharide represented by the formula (9):

The present invention provides a tetrasaccharide represented by the formula (9) or a derivative thereof. The tetrasaccharide of formula (9) is a portion that connects the xylose-glucuronic acid repeating structure of the post-phosphate glycan to the glycan extending from α-dystroglycan. The post-phosphate glycan can be reconstructed using the disaccharide derivative, its oligomer, and the tetrasaccharide of formula (9). The reconstructed glycan can be used as a medicine for treating and preventing glycosylation-related muscular dystrophy. Examples of the derivative of formula (9) include, but are not limited to, compounds in which at least a part or all of the hydroxyl groups in formula (9) are protected with protecting groups, and compounds in formula (9) in which the carboxyl groups are alkyl esterified. Specific examples of the derivative of the tetrasaccharide of formula (9) include, but are not limited to, compounds 404 and 405 in the scheme below.

In yet another aspect, the present invention relates to a compound represented by formula (7):

[In the formula, X ¹ is a leaving group or a protected hydroxyl group, X ² to X ⁶ are protecting groups for the hydroxyl groups, and X ⁷ is an alkyl group.]
With a disaccharide donor represented by formula (8):

[In the formula, X ⁸ to X ¹³ are protecting groups for hydroxyl groups, and X ¹² and X ¹³ may combine together to form an acetal]
and then the tetrasaccharide derivative is deprotected to give the disaccharide acceptor of formula (9):

The present invention provides a method for producing a tetrasaccharide represented by the formula:

As the leaving group X ¹ in compound (7), a known one can be used, and examples thereof include leaving groups commonly used in sugar chain synthesis, such as trichloroacetimidoyloxy, alkylthio, arylthio, halogen, and pentenyloxy, but are not limited thereto. Preferred leaving groups X ¹ include those which form thioglycosides, such as trichloroacetimidoyloxy, alkylthio, and phenylthio, but are not limited thereto. Examples of the protecting groups X ² to X ⁶ for the hydroxyl group in compound (7) include protecting groups commonly used in sugar chain synthesis, such as benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, and trialkylsilyl, but are not limited thereto. The alkyl group in compound (7) is preferably an alkyl group having 1 to 3 carbon atoms, and is typically a methyl group. In a specific example of 1, X ¹ in compound (7) is trichloroacetimidoyloxy, X ² to X ⁶ are acetyl, and X ⁷ is methyl.

As the protecting groups X ⁸ to X ¹³ of the hydroxyl groups in compound (8), protecting groups generally used in sugar chain synthesis can be used as long as the 2- and 3-position hydroxyl groups of xylose are protected and the protecting group on the ribitol side is not removed or changed. Examples of such protecting groups include, but are not limited to, benzyl including substituted benzyl, allyl, levulinoyl, benzoyl including substituted benzoyl, acetyl including substituted acetyl, allyloxycarbonyl, trialkylsilyl, and the like. The 2- and 3-position hydroxyl groups of compound (8) may be protected by acetal. In one specific example, X ⁸ of compound (8) is allyl, X ⁹ and X ¹⁰ are benzyl, X ¹¹ is TBDPS, and the 2- and 3-position hydroxyl groups are protected by isopropylidene acetal (i.e., X ¹² and X ¹³ together form an isopropylidene acetal).

The compound of formula (7) may be derived from a disaccharide derivative of formula (1) using known techniques.

The compound of formula (8) can be produced, for example, by a reaction of a compound of formula (10):

[wherein Lev is -C(= _O ) _C2H4C (=O) _{CH3 ,} Bn is _-CH2C6H5 , All is _{-CH2CH = CH2} , and TBDPS is -Si( _{C6H5 ) 2tert - C4H9} ]
It may also be derived from a disaccharide derivative represented by the formula:

A specific example of the above method is shown in Scheme 4 below.

[In the above scheme, Lev is -C(=O) _C2H4C (=O _{) CH3} , Et is _-C2H5 , Ac is -C(=O) _CH3 , _MBz is -C(= _O ) _C6H4CH3 (p), All is _-CH2CH = _CH2 , CSA is camphorsulfonic acid, DMF is _{dimethylformamide, TBDPS is -Si(C6H5 ) 2tert} - _C4H9 , THF is tetrahydrofuran, TMSOTf is trimethylsilyl trifluoromethanesulfonate, All is _{-CH2CH = CH2} , and _TBAF is tetrabutylammonium fluoride]

A compound represented by formula (7) may be obtained by introducing a leaving group such as -OC(NH) _CCl3 into the 1-position of glucuronic acid of a compound in which the protecting group of the hydroxyl group of the disaccharide derivative represented by formula (1) is replaced by an acetyl group. A compound represented by formula (10) may be derived from xylose. The levulinoyl protecting groups at the 2-, 3-, and 4-positions of the compound represented by formula (10) are deprotected, and then a ring is formed between the hydroxyl groups at the 2- and 3-positions and 2-methoxypropene to obtain a compound represented by formula (8). A compound represented by formula (7) (sugar donor) is reacted with a compound represented by formula (8) (sugar acceptor), and then deprotected by a known method to obtain a tetrasaccharide represented by formula (9).

In a further aspect, the present invention provides a process for preparing an optionally derivatized tetrasaccharide of formula (9), which comprises reacting a disaccharide donor of formula (7) above with a disaccharide acceptor of formula (8) above, followed by, optionally, deprotecting the resulting tetrasaccharide derivative.
Optionally, deprotection refers to removing some (at least one) or all of the protecting groups. Derivatization of sugars is known to those skilled in the art. For example, the tetrasaccharide may contain known protecting groups and may contain known leaving groups. The protecting groups and leaving groups of the tetrasaccharide include, but are not limited to, those described above.

The reaction conditions shown in the above description and scheme are illustrative only and a person skilled in the art can modify these reaction conditions as appropriate.

Unless otherwise specified, the meanings of terms used in this specification are the same as those commonly understood in the fields of biochemistry, biology, chemistry, pharmacology, medicine, etc.

The present invention will be explained in more detail and specifically below with reference to the following examples. However, the examples are merely illustrative and do not limit the scope of the present invention.

Example 1 Synthesis of disaccharide derivatives (1) Synthesis of dodecyl 3,4-O-(2,3-dimethoxybutan-2,3-diyl)-1-thio-α-D-xylopyranoside (102a) A known compound (101a, 6.19 g, 18.5 mmol) dissolved in MeOH (94 mL) was added with trimethyl orthoformate (18 mL), 2,3-butanedione (6.5 mL) and a catalytic amount of camphorsulfonic acid, and the mixture was stirred overnight at 50° C. Triethylamine was added and the mixture was concentrated. The concentrated residue was purified by silica gel column chromatography (toluene:ethyl acetate=20:1 to 5:1) to obtain 102a (2.97 g, 27%).

(x) Synthesis of Dodecyl 3,4-O-(2,3-dimethoxybutan-2,3-diyl)-1-thio-β-D-xylopyranoside (102b) The known compound (101b, 2.10 g, 6.28 mmol) dissolved in MeOH (30 mL) was added with trimethyl orthoformate (6 mL), 2,3-butanedione (2.4 mL) and a catalytic amount of camphorsulfonic acid, and stirred overnight at 40° C. Triethylamine was added and the mixture was concentrated. The concentrated residue was purified by silica gel column chromatography (toluene:ethyl acetate=20:1 to 3:1) to obtain 102b (2.52 g, 89%).

(2) Synthesis of 3,4-O-(2,3-dimethoxybutan-2,3-diyl)-2-O-(4-methoxy)benzyl-1-thio-α-D-xylopyranoside (103a) Compound (102a, 941.9 mg, 2.099 mmol) was dissolved in dimethylformamide (11 mL), sodium hydride (55%, 106.3 mg, 2.44 mmol) was added, and the mixture was stirred for 2 hours. 4-Methoxybenzyl chloride (0.57 mL, 5.6 mmol) was added thereto, and the mixture was stirred for 4 hours. Ice and 1M aqueous ammonium chloride solution were added to the reaction solution, and the mixture was extracted with ethyl acetate. The concentrated residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 50:1 to 10:1) to obtain 103a (743.0 mg, 62%).

(2) Synthesis of 3,4-O-(2,3-dimethoxybutan-2,3-diyl)-2-O-(4-methoxy)benzyl-1-thio-β-D-xylopyranoside (103b) Compound (102b, 2.52 g, 5.62 mmol) was dissolved in dimethylformamide (28 mL), sodium hydride (55%, 0.50 g, 11.5 mmol) was added, and the mixture was stirred for 2 hours. 4-Methoxybenzyl chloride (1.6 mL, 8.9 mmol) was added thereto, and the mixture was stirred for 3 hours. Ice and 1M aqueous ammonium chloride solution were added to the reaction solution, and the mixture was extracted with ethyl acetate. The concentrated residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 8:1 to 6:1) to obtain 103b (2.43 g, 76%).

(3) Synthesis of Methyl (4-methoxyphenyl 2,4-di-O-acetyl-β-D-glucopyranoside) uronate (113a) The known compound (111, 22.75 g, 51.66 mmol) dissolved in MeOH (120 mL) was added with 1.0 M NaOH (120 mL) and stirred at room temperature overnight. It was then neutralized with 1.0 M HCl, concentrated, and dried. Ac ₂ O (500 mL) and I ₂ (0.24 g) were added to the obtained dried product and stirred at room temperature for 8 hours. An appropriate amount of MeOH, ice, and 1 M hypo (sodium thiosulfate) were added, extracted with ethyl acetate, and dried over anhydrous MgSO _4. The insoluble matter was filtered, concentrated, and purified by silica gel column chromatography (toluene:ethyl acetate = 6:1) to obtain the product. The obtained product was dissolved in 400 mL of MeOH and heated under reflux for one week. The reaction solution was concentrated and purified by silica gel column chromatography (toluene:ethyl acetate=4:1) to obtain 3.56 g of 113a (3-step yield: 17%).

(4) Synthesis of Methyl (4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranoside) uronate (113b) A known compound (111, 23.92 g, 54.31 mmol) dissolved in a mixed solvent of THF (212 mL) and water (31 mL) was added with 1.25 M LiOH (296 mL) at 0 °C and stirred for 6 hours. Then, it was neutralized with 1.0 M HCl, concentrated, and dried. The obtained dried product was dissolved in DMF (550 mL), and benzoic anhydride (172.24 g, 761.34 mmol) was added and stirred at 79 °C for 3 hours. Then, pyridine (226 mL) and DMAP (3.40 g, 27.8 mmol) were added and stirred at room temperature overnight. An appropriate amount of ice was added, extracted with ethyl acetate, washed with ice-cold 1 M HCl and saturated saline, and the organic layer was dried over anhydrous MgSO ₄ . Insoluble matter was filtered and concentrated, the product was dissolved in MeOH (563 mL), sodium acetate (7.86 g, 95.8 mmol) was added, and the mixture was heated and stirred for 5 hours. The reaction solution was concentrated, extracted with chloroform, and subjected to post-treatment by a conventional method. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=8:1 to 0:1) to obtain 10.9 g of 113b (3-step yield 38%).

(5) Methyl (2,3,4-tri-O-acetyl-α-D-xylopyranosyl)-(1→3)-β-(4-methoxyphenyl2,4-di-O-acetyl-β-D-glucopyranosid )uronate (204a) synthesis
Molecular sieves 4A (1.5 g) was added to a solution of DDQ (477.2 mg, 2.102 mmol) in CH ₂ Cl ₂ (5 mL), and a solution of 113a (535.7 mg, 1.354 mmol) in CH ₂ Cl ₂ (10 mL) was A solution of 103b (967.4 mg, 1.700 mmol) in CH ₂ Cl ₂ (7 mL) was added thereto with stirring at 0° C., and the reaction was continued at room temperature for 4 hours. The reaction mixture was mixed with 0.1M ascorbic acid and filtered through diatomaceous earth, and the filtrate was extracted with chloroform. The organic layer was washed with 0.1M ascorbic acid, saturated sodium bicarbonate solution and saturated saline, and dried over anhydrous _MgSO4 . Insoluble matter was filtered off, and the concentrated residue of the filtrate was purified by silica gel column chromatography (n-hexane:ethyl acetate=6:1 to 1:10) to obtain 1.074 g (83%) of mixed acetal (201a). NIS (525.5 mg, 2.34 mmol) and AgOTf (195.3 mg, 760 μmol) were suspended in CH ₂ Cl ₂ (22 mL) and incubated in the presence of molecular sieves 4A (2.3 g) for 1 hour in the dark. A solution of the mixed acetal (201a, 1.074 g) in CH ₂ Cl ₂ (220 mL) was added dropwise at −20° C. and the mixture was stirred for 2 hours. Water was added, and the mixture was filtered through diatomaceous earth, and the filtrate was extracted with chloroform. The organic layer was washed with 1M hypo, saturated sodium bicarbonate water and saturated saline, and dried over anhydrous MgSO _4. Insoluble matter was filtered off, and the concentrated residue of the filtrate was purified by silica gel column chromatography (n-hexane:ethyl acetate=30: The disaccharide fraction (753.9 mg) was purified with 1:10 to ethyl acetate:methanol = 30:1. 299.7 mg of the disaccharide fraction was added to 90% trifluoroacetic acid (15 mL) at 0°C. The reaction mixture was allowed to warm to room temperature over 2 hours, and the reagents were removed by distillation under reduced pressure. Pyridine (3 mL) and acetic anhydride (3 mL) were added to the concentrated residue, and the mixture was stirred for 3 hours. The reagents were removed by distillation under reduced pressure, and the concentrated residue The residue was purified by silica gel column chromatography (toluene:ethyl acetate=10:1 to 2:3) to obtain 85.6 mg (24%) of 204a.

(6) Synthesis of Methyl α-D-xylopyranosyl-(1→3)-β-(4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranosid)uronate (202ba) A CH 2 Cl 2 (7 mL) solution of 113b (706.3 mg, 1.352 mmol) was added to a CH ₂ Cl ₂ (3 mL) suspension of molecular sieves 4A (1.74 g) and DDQ (474.2 mg, 2.087 mmol) _, and the mixture was stirred at room temperature for 1 hour. _{A CH 2 Cl 2} (8 mL) solution of 103b (962.9 mg, 1.693 mmol) was added thereto at 0° C. with stirring, and the reaction was continued overnight at room temperature. The reaction solution was cooled again to 0° C., DDQ (160.4 mg, 706 μmol) was added, and the mixture was stirred for 5 hours. The mixture was returned to room temperature and treated in the same manner as in the synthesis of 204a. The concentrated residue was purified by gel filtration column chromatography (chloroform:methanol=1:1) and silica gel column chromatography (toluene:ethyl acetate=1:0-2:1) to obtain 435.5 mg of mixed acetal (201b) (yield 30%). NIS (124.6 mg, 553.8 μmol) and AgOTf (61.6 mg, 0.24 mmol) were suspended in CH ₂ Cl ₂ (4 mL) in the dark, and a solution of mixed acetal (201b, 232.9 mg) in CH ₂ Cl ₂ (40 mL) was added dropwise at −20° C. in the presence of molecular sieves 4A (0.43 g) and the mixture was stirred for 3 hours. The mixture was treated in the same manner as in the synthesis of 204a and purified by gel filtration column chromatography (chloroform:methanol=1:1) to obtain the product (156.7 mg). To this was added 90% trifluoroacetic acid (8 mL) at 0° C. and stirred for 2.5 hours. The mixture was returned to room temperature, the reagents were distilled off under reduced pressure, and the mixture was purified by gel filtration column chromatography (chloroform:methanol=1:1) to obtain 202ba (49.4 mg, 75.5 μmol, two-step yield: 35%).

(7) Synthesis of Methyl [2,4-di-O-(4-methyl)benzoyl-α-D-xylopyranosyl]-(1-3)-β-(4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranosid)uronate (203b1) and Methyl [3,4-di-O-(4-methyl)benzoyl-α-D-xylopyranosyl]-(1-3)-β-(4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranosid)uronate (203b2) Compound 202ba (239.2 mg, 365.4 μmol) was dissolved in toluene (13 mL), and dibutyltin(IV) oxide (327.6 mg, 1.32 mmol) was added and reacted for 2.5 hours using a Dean-Stark apparatus. The reaction solution was cooled on ice, 4-methylbenzoyl chloride (122 μL, 923 μmol) was added, and the mixture was stirred overnight at room temperature. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate and post-treated in the usual manner. The concentrated residue was purified by gel filtration column chromatography (chloroform:methanol=1:1), the chloroform solution of the product was washed with 1M HCl and saturated saline, and the concentrated residue of the organic layer (381.9 mg) was dissolved in toluene (12.5 mL) and methanol (2.5 mL). This solution was cooled on ice, 2M TMS diazomethane (660 μL, 11.6 μmol) was added, and the mixture was concentrated under reduced pressure after 1 hour. The residue was purified by gel filtration column chromatography (chloroform:methanol=1:1) and silica gel column chromatography (toluene:ethyl acetate=10:0 to 0:1), and 203b1 (202.9 mg) and 203b2 (42.6 mg) were obtained in yields of 62% and 25%, respectively.
203b1: ESI-HRMS m/z [(M+Na) ⁺ ]: calcd. for C ₄₉ H ₄₆ NaO ₁₆ : 913.2678; found, 913.2678.
203b2: ESI-HRMS m/z [(M+Na) ⁺ ]: calcd. for C ₄₉ H ₄₆ NaO ₁₆ : 913.2678; found, 913.2658.

(8) Synthesis of Methyl [3-O-levulynoyl-2,4-di-O-(4-methyl)benzoyl-α-D-xylopyranosyl]-(1→3)-β-(4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranosid)uronate (204b) Compound (203b1, 111.4 mg, 125 μmol) was dissolved in pyridine (640 μL), and a 1M solution of levulinic acid (325 μl, 0.313 mmol) in 1,2-dichloroethane and a small amount of DMAP were added and stirred at room temperature for 2 hours. The reaction solution was neutralized with saturated saline, and CHCl ₃ extraction and post-treatment were performed by the usual method. The obtained residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 50:1 to 0:1) to quantitatively obtain 204b (124.8 mg).

(9) Synthesis of Methyl (2,3,4-tri-O-acetyl-α-D-xylopyranosyl)-(1→3)-β-(4-methoxyphenyl 2,4-di-O-acetyl-β-D-glucopyranosylimidate)uronate (205a) Compound (204a, 83.4 mg, 127 μmol) was dissolved in a mixture of acetonitrile (4 mL) and H ₂ O (1 mL), and CAN (203.6 mg, 371.4 μmol) was added thereto and stirred at room temperature for 1.5 hours. 0.1 M ascorbic acid was added to the reaction solution, which was extracted with CHCl ₃ and washed with saturated saline, and post-treatment was performed by the usual method. The obtained residue was purified by silica gel column chromatography (toluene:ethyl acetate = 5:1 to 2:3) to obtain the product (73.6 mg). This was dissolved in dichloromethane (2 mL), CCl ₃ CN (127 μL, 1.25 mmol) was added, and the mixture was cooled to 0° C. Then, 2 drops of DBU were added, and the mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction solution was purified by silica gel column chromatography (toluene:ethyl acetate=10:1 to 1:1) to give 205a (72.8 mg) in a yield of 82% (2 steps).
¹ H-NMR δ _H (CDCl ₃ ): 8.72 (s, 1H, NH), 6.67 (d, 1H, J _1,2 =3.54 Hz, GlcA-1), 5.35 (brt, 1H,J=10.08 Hz, Xyl-3), 5.33 (d, 1H, J _1,2 =3.72 _{Hz , ​ ​ 4,5a} =6.42 Hz, _J4,5e =9.60 Hz, Xyl-4), 4.72 (dd, 1H, J _2,3 =10.26 Hz, Xyl-2), 4.35 (brt, 1H,J=9.63 Hz, GlcA-3), 4.35 (d, 1H, GlcA-5), 3.76 (m, 2H, Xyl-5a,e), 3.73 (s, 3H, COOMe), 2.09, 2.04, 2.04, 2.02, 2.01 (each s, 3Hx5, 5Ac)

(10) Synthesis of Methyl [3-O-levulynoyl-2,4-di-O-(4-methyl)benzoyl-α-D-xylopyranosyl]-(1→3)-β-(4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranosylimidate)uronate (205b) Compound (204b, 124.8 mg, 126.2 μmol) was dissolved in a mixture of acetonitrile (5.8 mL) and H ₂ O (1.4 mL), and CAN (362.2 mg, 660.7 μmol) was added thereto and stirred at room temperature for 4.5 hours. 0.1 M ascorbic acid was added to the reaction solution, which was extracted with CHCl ₃ and washed with saturated saline, and post-treatment was performed by the usual method. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=50:1 to 0:1) to obtain the product (110.8 mg). This was dissolved in dichloromethane (1 mL), CCl ₃ CN (125 μL, 1.25 mmol) was added, and the mixture was cooled to 0° C. Then, one drop of DBU was added, and the mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction solution was purified by silica gel column chromatography (toluene:ethyl acetate=50:1 to 0:1) to obtain 205b (109.7 mg) in a yield of 80% (2 steps).
¹ H-NMR δ _H (CDCl ₃ ): 8.70 (s, 1H, NH), 7.97-7.95 (m, 2H, Ar), 7.64-7.58 (m, 4H, Ar), 7.54-7.51 (m, 3H, Ar), 7.33-7.29 (m, 2H, Ar), 7.22 (brt, 1H, J=7.80 Hz, Ar), 7.16 (d, 1H, J=7.98 Hz, Ar), 7.11 (d, 1H, J=7.98 Hz, Ar), 6.84 (d, 1H, J _1,2 =3.78 Hz, GlcA-1), 5.60 (brt, 1H,J=9.90 Hz, Xyl-3), 5.59-5.56 (m, 2H, GlcA-2, 4), 5.50 (d, 1H, J _1,2 =3.90 Hz, Xyl-1), 5.00 (dd, 1H, J _2,3 =10.44 Hz, Xyl-2), 4.97 (m, 1H,Xyl-4), 4.72 (t, 1H, J _2,3 = J _3,4 =9.46 Hz, GlcA-3), 4.52 (d, 1H, J _4,5 =10.20 Hz, GlcA-5), 3.70 (brt, 1H, J=10.92 Hz, Xyl-5a), 3,63 (dd, 1H, J _4,5e =6.18 Hz, J _5a,5e =11.22 Hz, Xyl-5e), 3.52 (s, 3H, COOMe), 2.40 (s, 6H, 2Ph Me ), 2.39-2.16 (m, 4H, 2CH ₂ ), 1.86 (s, 3H, Lev).

(x) Methyl 3,4-O-(2,3-dimethoxybutan-2,3-diyl)-2-O-(4-methoxy)benzyl-α-D-xylopyranosyl-(1→3)-β-( Synthesis of 4-methoxyphenyl 2,4-di-O-acetyl-α and β-D-glucopyranosid)uronate (208a, 208b)

(Condensation using α-thioglycoside: Method 1)
103a (397.8 mg, 0.699 mmol) and 113a (183.5 mg, 0.461 mmol) were dissolved in a mixture of toluene (3.0 mL) and dioxane (3.0 mL) and then cooled to 100° C. for 2 hours at 30° C. for 1 hour. The mixture was stirred at room temperature for 1 hour in the presence of 1M hypo. NIS (238.2 mg, 1.059 mmol) and AgOTf (71.4 mg, 0.278 mmol) were added thereto at -20 ^° C. After 1.5 hours, 1M hypo was added. The reaction was stopped by adding saturated NaHCO ₃ and saturated NaCl, and the filtrate was filtered through diatomaceous earth and extracted with CHCl _3. The organic layer was washed with saturated NaHCO ₃ and saturated NaCl and dried over anhydrous MgSO ₄ . The insoluble matter was filtered off, and the concentrated residue of the filtrate was purified by gel filtration column chromatography (chloroform:methanol=1:1) to obtain a mixture of 208a and 208b (245.9 mg, α 58%, β 12%). Ta.

(Condensation using β-thioglycosides: Method 2)
103b (184.7 mg, 0.325 mmol) and 113a (86.9 mg, 0.218 mmol) were added to a mixture of toluene (3.0 mL) and dioxane (3.0 mL) using molecular sieves 4A (0.33 g). The mixture was stirred at room temperature for 2 hours in the presence of 1M hypo and saturated NaHCO. The reaction was stopped by adding ₃ and saturated NaCl, and the filtrate was filtered through diatomaceous earth and extracted with CHCl _3. The organic layer was washed with saturated NaHCO ₃ and saturated NaCl and dried over anhydrous MgSO ₄ . The insoluble matter was filtered off, and the concentrated residue of the filtrate was purified by gel filtration column chromatography (chloroform:methanol=1:1) to obtain a mixture of 208a and 208b (120.7 mg, α 60%, β 12%). .

(Condensation using α and β thioglycosides: Method 3)
A mixture of 103a and 103b (1.05 g, 1.85 mmol) and 113a (491.4 mg, 1.234 mmol) in a mixture of toluene (10.0 mL) and dioxane (10.0 mL) was subjected to molecular spectrometry. The mixture was stirred at room temperature for 1 hour in the presence of sieves 4A (1.81 g). NIS (588.3 mg, 2.615 mmol) and AgOTf (191.6 mg, 0.7458 mmol) were added thereto at -20°C. After 2 hours, the reaction was stopped by adding 1M hypo, saturated NaHCO ₃ and saturated NaCl, and the filtrate was filtered through diatomaceous earth, extracted with CHCl ₃ and dried over anhydrous MgSO ₄ . Insoluble matter was removed by filtration, the concentrate was concentrated, and the residue was purified by gel filtration column chromatography (chloroform:methanol=1:1) to obtain a mixture of 208a and 208b (754.4 mg, α 67%, β 13%).

(x) Synthesis of Methyl α-D-xylopyranosyl-(1→3)-β-(4-methoxyphenyl 2,4-di-O-acetyl-β-D-glucopyranosid)uronate (202aa) 90% trifluoroacetic acid (10.0 mL) was added to a mixture of compounds 208a and 208b (754.4 mg, α:β = 67:13) and stirred at 0°C. After 2.5 hours, the mixture was concentrated and purified by silica gel column chromatography (n-hexane: ethyl acetate = 1:1 to ethyl acetate: methanol = 10:1) to obtain a mixture of 202aa and 202ab (474.5 mg, yield 91%). This was suspended in chloroform and filtered. Insoluble matter was collected to obtain 202aa (427.8 mg) (yield from the mixture of 208a and 208b 82%). ¹ H-NMR δ _H (CD ₃ OD):6.98-6.92(m,2H,Ar-H),6.84-6.82(m,2H,Ar-H),5.19-5.11(m,4H,Glc A-1,2,4),4.95(d,1H,J _1,2 =3.84Hz,Xyl-1),4.27(d,1H,J _4,5 =9.96Hz,Glc A-5),4.12(t,1H,J _2,3 =J _3,4 =9.24Hz,Glc A-3),3.74,3.69(2s,3H×2,2O CH ₃ ),3.53-3.39(m,4H,Xyl-3,4,5ab),2.12,2.07(2s,3H×2,2A c).

Example 2 Synthesis of tetrasaccharide derivative (compound of formula (5) where n = 1) (1) Synthesis of methyl [3-O-levulinoyl-2,4-di-O-(4-methyl)benzoyl-α-D-xylopyranosyl]-(1→3)-(methyl 2,4-di-O-benzoyl-β-D-glucopyranosyluronate)-(1→3)-[2,4-di-O-(4-methyl)benzoyl-α-D-xylopyranosyl]-(1→3)-(4-methoxyphenyl 2,4-di-O-benzoyl-β-D-glucopyranosid)uronate (206b)
The disaccharide donor (205b, 109.7 mg, 106.7 μmol) and the disaccharide acceptor (203b1, 90.2 mg, 101 μmol) were dissolved in dichloromethane (3.5 mL), MSAW300 (0.31 g) was added, and the mixture was stirred for 30 minutes. The mixture was cooled to -20°C, TMSOTf (10 μL, 53.5 μmol) was added, and the mixture was stirred for 3 hours. Saturated sodium bicarbonate water was added to the reaction solution, and the mixture was filtered through Celite. The product was post-treated by the usual method, and purified by gel filtration column chromatography (CHCl ₃ :MeOH=1:1), to obtain 206b (56.3 mg, 32.1 μmol) in a yield of 32%. [α] _D +21.0°(c 0.66,CHCl ₃ ). ¹ H-NMR δ _H (CDCl ₃ ): 7.92-7.899 (m, 4H, Ar), 7.66-7.62 (m, 5H, Ar), 7.59 (d, 2H, J=8.16 Hz, Ar), 7.55-7.50 (m, 7H, Ar), 7.41 (d, 2H, J=7.44 Hz, Ar), 7.33-7.27 (m, 5H, Ar), 7.18 (m, 3H, Ar), 7.15 (d, 2H, J=8.04 Hz, Ar), 7.11 (d, 2H, J=7.75 Hz, Ar), 7.06 (d, 2H, J=7.92 Hz, Ar), 6.91-6.89 (m, 2H, Ar), 6.88-6.86 (m, 2H, Ar), 6.71-6.70 (m, 2H, Ar), 5.55 (dd, 1H, J _1,2 =7.26 Hz, J _2,3 =8.64 Hz, H-2 ¹ ), 5.50 (brt, 1H, J=8.94 Hz, H-4 ¹ ), 5.45 (t, 1H, J _2,3 = J _3,4 =9.78 Hz, H-3 ⁴ ), 5.42 (brt, 1H, J=9.45 Hz, H-4 ³ ), 5.34 (d, 1H, J _1,2 =3.60 Hz, H-1 ² ), 5.28 (d, 1H, J _1,2 =3.96 Hz, H-1 ⁴ ), 5.25 (brt, 1H, J=8.52 Hz, H-2 ³ ), 5.09 (d, 1H, H-1 ¹ ), 5.00 (dd, 1H, J _1,2 =7.98 Hz, H-1 ³ ), 4.81-4.76 (m, 3H, H-4 ² , 2 ⁴ , 4 ⁴ ), 4.71 (dd, 1H, J _2,3 =9.60 Hz, H-2 ³ ), 4.43 (brt, 1H, J=9.27 Hz, H-3 ² ), 4.33 (brt, 1H, J=8.73 Hz, H-3 ¹ ), 4.17 (brt, 1H, J=9.08 Hz, H-3 ³ ), 4.12 (d, 1H, J _4,5 =9.84 Hz, H-5 ³ ), 4.11 (d, 1H, J _4,5 =9.19 Hz, H-5 ¹ ), 3.70, 3.53, 3.44 (3s, 3Hx3, 3OMe), 3.66 (dd, 1H, J _4,5e =5.93 Hz, J _5a,5e =11.53 Hz, H-5 ² e), 3.49 (brt, 1H, J=10.78 Hz, H-5 ² a), 3.31 (brt, 1H, J=11.05 Hz, H-5 ⁴ a), 3.21 (dd, 1H, J _4,5e =6.00 Hz, J _5a,5e =11.33 Hz, H-5 ⁴ e), 2.50, 2.45, 2.39, 2.37 (4s, 12H, 4Ph Me ), 2.44-2.10 (m, 4H, 2CH ₂ ), 1.83 (s, 3H, Lev). ESI-HRMS m/z [(M+Na) ⁺ ]: calcd. for C ₉₆ H ₉₀ NaO ₃₂ : 1777.5307; found, 1777.5330.

(2) Synthesis of α-D-xylopyranosyl-(1→3)-β-D-glucopyranosyluronic acid-(1→3)-α-D-xylopyranosyl-(1→3)-4-methoxyphenyl β-D-glucopyranosyluronic acid, disodium salt (207b) Compound (206b, 15.1 mg, 8.60 μmol) was dissolved in a mixture of THF (1.4 ml) and H ₂ O (0.1 ml), 1.25 M LiOH (70 μL, 86 μmol) was added, and the mixture was stirred for 2.5 hours. After distillation under reduced pressure, the mixture was cooled to 0°C, 1.0 mL of MeOH was added, and 0.5 M NaOH (50 μL) was added, and the mixture was stirred at room temperature for 16 days. 50% acetic acid (2 drops) was added and evaporated under reduced pressure, and the resulting residue was purified by gel filtration column chromatography (LH-20, 1% acetic acid) and Bond Elut (C8) to obtain 4.1 mg of 207b (yield 64%). [α] _D +18.3°(c 0.41,H ₂ O). ¹ H-NMR δ _H (H ₂ O) (DHO=4.70 ppm): 7.00-6.99 (m, 2H, Ar), 6.87-6.85 (m, 4H, Ar), 5.26 (d, 1H, J _1,2 =3.78 Hz, H-1 ^4or2 ), 5.22 (d, 1H, J _1,2 =3.84 Hz, H-1 ^2or4 ), 4.87 (d, 1H, J _1,2 =7.80 Hz, H-1 ¹ ), 4.63 (d, 1H, J _1,2 =8.00 Hz, H-1 ³ ), 3.68 (s, 3H, OMe), 3.61 (m, 1H, H-2 ^4or2 ), 3.56 (m, 2H, H-3 ³ , 3 ^2or4 ), 3.55(m, 1H, H-2 ¹ ), 3.41 (dd, 1H, J _2,3 =9.72 Hz, H-2 ^2or4 ), 3.38 (dd, 1H, J _2,3 =9.12 Hz, H-2 ³ ). ESI-HRMS m/z[(M+Na) ⁺ ]: calcd. for C ₂₉ H ₄₀ NaO ₂₂ : 763.1903; found, 763.1884.

Example 3 Synthesis of tetrasaccharide derivative represented by formula (9) (1) Synthesis of compound 402 Compound 401 (273.6 mg, 0.2516 mmol) of formula 10 dissolved in a mixed solvent of toluene (6.0 mL) and ethanol (3.0 mL) was added with H ₂ NNH ₂ · AcOH (112.1 mg) and stirred at room temperature overnight. The reaction solution was then concentrated and purified by gel filtration column chromatography (CHCl ₃ : MeOH = 1:1) and silica gel column chromatography (ethyl acetate: methanol = 100:1) to obtain 137.0 mg of compound 402 in a yield of 75%.

(2) Synthesis of Compound 403 (Formula 8) 10-Camphorsulfonic acid (3.9 mg) was added to Compound 402 (97.4 mg, 0.1340 mmol) dissolved in DMF, and the mixture was stirred at room temperature. 2-Methoxypropene (14 μL) was added thereto. After 8 hours, 2-methoxypropene (14 μL) was further added and stirred overnight. Then, 2-methoxypropene (28 μL) was further added, and after 2 hours, diisopropylethylamine was added to stop the reaction, extracted with EtOAc, and dried over anhydrous MgSO _4. Insoluble matter was filtered, concentrated, and purified by silica gel column chromatography (toluene:ethyl acetate=6:1 to 4:1), and 70.2 mg (yield 68%) of Compound 403 (Formula 8) was obtained.

(3) Synthesis of Compound 404 Formula 7, Compound 205a (135.4 mg, 0.1948 mmol) and Formula 8, Compound 403 (110.0 mg, 0.1405 mmol) dissolved in CH ₂ Cl ₂ were added to MSAW300 and stirred at −78° C. TMSOTf (4.0 μL) was added thereto. After 1.5 hours, the reaction was stopped by adding saturated NaHCO ₃ , extracted with CHCl ₃ , and dried with anhydrous MgSO _4. Insoluble matter was filtered, concentrated, and purified by gel filtration column chromatography (CHCl ₃ :MeOH=1:1) to obtain a product (75.8 mg). Ac ₂ O (3.0 mL) and pyridine (3.0 mL) were added to the obtained product and stirred at room temperature overnight. Thereafter, the reaction solution was concentrated and purified by silica gel column chromatography (toluene:ethyl acetate=3:1) to obtain 67.3 mg of compound 404 (two-step yield: 35%).

(4) Synthesis of Compound 405
(1,5-cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexafluorophosphate was added with THF (1.0 mL) and activated with H ₂ , and then placed in an Ar atmosphere. Compound 404 (67.3 mg, 0.04950 mmol) dissolved in THF (5.0 mL) was added thereto and stirred at room temperature for 1.5 hours. Water was then added, the temperature was lowered to 0°C, sodium bicarbonate (8.9 mg) and iodine (26.2 mg) were added, and the mixture was stirred for 2 hours. 1M hypo was then added to stop the reaction, and the mixture was extracted with CHCl ₃ and dried with anhydrous MgSO _4. Insoluble matter was filtered, concentrated, and purified by silica gel column chromatography (toluene:ethyl acetate=3:1 to ethyl acetate:methanol=10:1) to obtain the product (60.0 mg). The obtained product was dissolved in THF (4.0 mL), AcOH (25 μL) and 1M t-butylammonium fluoride (220 μL) were added, and the mixture was stirred at room temperature for 2 days. Then, the mixture was extracted with CHCl ₃ and dried over anhydrous MgSO _4. Insoluble matter was filtered, concentrated, and purified by silica gel column chromatography (toluene:ethyl acetate=1:1), to obtain 29.1 mg of compound 405 (two-step yield: 54%).

(5) Synthesis of Compound 406 (Formula 9) Compound 405 (29.1 mg, 0.0270 mmol) dissolved in a mixed solvent of THF (4.0 mL) and water (0.2 mL) was added with 1.25 M LiOH (210 μL) at 0° C. and stirred for 1 hour. The reaction solution was concentrated, methanol (4.0 mL) was added, and 0.1 M NaOH (5 drops) was added and stirred at room temperature overnight. The reaction was stopped by adding AcOH, the reaction solution was concentrated, and the product (20.0 mg) was obtained by purifying it by gel filtration column chromatography (LH-20, 1% acetic acid). The obtained product was dissolved in a mixed solvent of methanol (2.0 mL) and water (2.0 mL), and palladium-carbon was added. Then, the flask was filled with H ₂ atmosphere and stirred at room temperature overnight. The reaction solution was filtered through Celite, concentrated and dried to obtain 14.6 mg of Compound 406 (2 steps yield 92%) of Formula 9.
¹ H-NMR δ _H (D ₂ O): 5.17(d, 1H, J _1,2 =3.84 Hz, Xyl ² -1), 4.44(d, 1H, J _1,2 =7.86 Hz, Xyl ¹ -1), 4.42(d, 1H, J _1,2 =8.22 Hz, GlcA-1), 3.94(dd, 1H, J=5.34, 11.88 Hz, Xyl-5a), 3.86-3.84(m, 1H, Rbo-4), 3.74-3.72(m, 4H, Xyl ² -4, Rbo-3, 5a, 1a), 3.69-3.60(m, 5H, Xyl ¹ -4, GlcA-5, Rbo-2, 5b, Xyl ² -5a), 3.54-3.43(m, 6H, Xyl ² -3, 5b, Xyl ¹ -3, GlcA-3,4, Rbo- ^1b ), 3.38(dd, 1H, J _2,3 = _9.66 Hz ¹³ C NMR δ _C (D ₂ O): 105.46( ^{Xyl 1 -1} ), 103.84(GlcA-1), 101.52(Xyl ² -1), 83.51(GlcA- ⁴ ), 83.33(Rbo-4), 79.48(Xyl ¹ -4), 77.56(Rbo-3), 76.37(Xyl ¹ -3), 75.67(Xyl ¹ -2), 75.60(Xyl ² -3), 74.60, 74.40(GlcA-5, Rbo-2), 74.20(Xyl ² -2), 74.12(Xyl ² -4), 73.85(GlcA-2), 71.95(GlcA-3), 65.53(Xyl ¹ -5), 65.22(Xyl ² -5), 64.03(Rbo-1), 62.77(Rbo-5). ESI-HRMS m/z[(M+Na) ⁺ ]: calcd. for C ₂₁ H ₃₆ NaO ₁₉ : 615.1748; found, 615.1744.

The present invention can be used in the fields of pharmaceutical manufacturing, glycoengineering, etc.

Claims (7)

Formula (2'):
[In the formula, X ² ' is a leaving group, Y' is a group that fixes the conformation of the xylose residue, and R ⁶ ' is a protecting group for a hydroxyl group that does not have neighboring group participation ability]
and a xylose derivative represented by the formula:
Formula (3'):
[wherein X ³ ' is a leaving group or a protected hydroxyl group, R ⁷ ' is a hydroxyl-protecting group capable of neighboring group participation, R ⁸ ' is a hydroxyl-protecting group, Y ⁴ ' is a group which is a precursor of a carboxyl group or a hydroxymethyl group, and R ⁷ ' and R ⁸ ' are not benzoyl groups ]
From a glucose derivative or a glucuronic acid derivative represented by
Formula (4'):
[In the formula, X ³ ', R ⁷ ', R ⁸ ' and Y ⁴ ' are as defined above.]
The method for producing an α-glycoside represented by the following steps 1) to 3):
Step 1) directly condensing a xylose derivative represented by formula (2') with a glucose derivative or a glucuronic acid derivative represented by formula (3') to obtain a mixture of α1→3 glycosides and β1→3 glycosides;
Step 2) The mixture obtained in step 1) is deprotected, and then
Step 3) separating the α-glycosides from the mixture deprotected in step 2), wherein the separation in step 3) is carried out by dissolving the β-glycosides in chloroform to remove the β-glycosides . 2. The method of claim 1 , wherein the condensation in step 1) is carried out using NIS-AgOTf, NIS-TfOH, MeOTf, CuBr ₂ -AgOTf-nBu ₄ NI, or NBS-AgOTf as a condensing agent. 3. The method according to claim 1 or 2 , wherein the deprotection in step 2) is carried out by hydrolysis with trifluoroacetic acid (TFA) to form a triol having hydroxyl groups at the 2-, 3- and 4-positions of the xylose residue. R ⁷ ' and R ⁸ 4. The method according to claim 1, wherein ' is an acetyl group. Formula (9):
or a derivative thereof. Formula (7):
[In the formula, X ¹ is a leaving group or a protected hydroxyl group, X ² to X ⁶ are protecting groups for the hydroxyl groups, and X ⁷ is an alkyl group.]
With a disaccharide donor represented by formula (8):
[In the formula, X ⁸ to X ¹³ are protecting groups for hydroxyl groups, and X ¹² and X ¹³ may combine together to form an acetal]
and then the tetrasaccharide derivative is deprotected to give the disaccharide acceptor of formula (9):
A method for producing a tetrasaccharide represented by formula (9), comprising obtaining a tetrasaccharide represented by formula (9): 7. The method of claim 6, wherein ^X1 is trichloroacetimidoyloxy, ^X2 to ^X6 are acetyl, ^X7 is methyl, ^X8 is allyl, ^X9 and ^X10 are benzyl, ^X11 is TBDPS , and ^X12 and ^X13 together form an isopropylidene acetal.