Stereoselective reaction of xyloside stereoisomer mixture

Abstract

The present invention provides a method for stereoselectively reacting a xyloside stereoisomer mixture. The method includes a step for bringing a reaction mixture that contains a cyclic esterified intermediate compound, in which a cyclic ester of boronic acid or boric acid is formed at the 2-position and the 4-position of the xylose moiety of each of α-xyloside and β-xyloside, into contact with silica gel. The step includes returning the cyclic esterified intermediate compound corresponding to α-xyloside to the α-xyloside form before cyclic esterification, and maintaining the cyclic esterified form of the cyclic esterified intermediate compound corresponding to β-xyloside.

Description

Stereoselective reactions of xyloside stereoisomeric mixtures.

[0001] The present disclosure relates to sugar-related compounds, including oligosaccharides, glycosides, etc., and techniques for producing them.

[0002] Glycosylation-associated muscular dystrophy, a type of muscular dystrophy, is caused by defects in the biosynthesis of the glycoprotein dystroglycan, and is associated with muscle cell damage as well as central nervous system disorders such as brain malformations and mental retardation. Recent research has revealed that abnormalities in the glycosylation of dystroglycan present in muscle cells are the cause of glycosylation-associated muscular dystrophy. The glycosylation moiety of dystroglycan that has affinity for laminin is called matriglycan. Matriglycan connects muscle cells to the basement membrane via laminin, and abnormalities in the biosynthesis of this glycosylation moiety cause glycosylation-associated muscular dystrophy.

[0003] The overall structure of the glycan modifying the Ser / Thr residues of dystroglycan has been investigated, and it has been found to be an O-mannosylglycan, which is an extension of matriglycan via ribitol phosphate. Matriglycan contains a glycan with repeating disaccharides consisting of α-xylose (Xyl) and β-glucuronic acid (GlcA) at its non-reducing end (Non-Patent Document 1), and it has been found that this repeating disaccharide glycan is necessary for binding to laminin (Non-Patent Document 2).

[0004] In the artificial synthesis of biologically significant sugar chains or their constituent oligosaccharides or glycosides, it is important to select specific conformations and configurations of sugars. In Patent Document 1 and Non-Patent Document 3, the present inventors synthesized a mixture of α- and β-stereoisomers of Xyl-GlcA disaccharides and discovered that the former (α1→3) isomer, related to matriglycan, could be selectively isolated by a specific solvent separation method. Specifically, the β1→3 isomer is soluble in chloroform, whereas the α1→3 isomer is insoluble in chloroform. Therefore, the α1→3 isomer could be selectively isolated by dissolving the mixture in chloroform and separating the insoluble matter.

[0005] The specific solvent (chloroform) separation method described above was significantly simpler and produced a superior yield (45% vs. 24%) than the selective synthesis of the α1→3 isomer by the Intramolecular Aglycon Delivery (IAD) method, also disclosed in Patent Document 1 and Non-Patent Document 3. Generally, separation of different stereoisomers can be achieved by distillation, recrystallization, or various chromatography techniques that utilize the affinity of the isomer to the carrier. However, some isomers have too high boiling points, do not crystallize easily, and cannot be separated at all by chromatography. The Xyl-GlcA disaccharide isomers described above are such an example. The unexpected discovery that a pair of stereoisomers (diastereomers) differing only in the configuration at one of the multiple asymmetric carbons can be separated using a common solvent, chloroform. Furthermore, in Patent Document 2, the inventors discovered that a mixture of similar disaccharide stereoisomers containing benzoyl protecting groups could be separated using dichloromethane as a solvent.

[0006] Dystroglycanopathies are caused by over 18 genes, but regardless of the causative gene, matriglycan deficiency is the underlying cause of all disease types. Gene therapy targeting individual genes has not been fully developed due to limited patient numbers and safety concerns regarding viral vectors. Replenishing deficient matriglycan with artificially synthesized matriglycan could open up a therapeutic strategy that is independent of the causative gene and resolve the aforementioned issues. Furthermore, artificial glycosylation-related compounds are not limited to muscular dystrophy. For example, Lassa fever virus infection is closely related to the binding of the virus to matriglycan, and the potential for therapeutic agents utilizing this binding has been described (Non-Patent Documents 4 and 5).

[0007] As described in Non-Patent Documents 2 and 5, there are promising methods for extending sugar chains such as matriglycan using enzymes, but chemical synthesis methods may be more advantageous in terms of industrial productivity and flexibility in chemical modification.

[0008] Therefore, particularly from the standpoint of therapeutic drug development, there is a need to develop methods for chemically producing and separating isomeric compounds having specific stereoconfigurations related to sugar chains, oligosaccharides, glycosides, etc. As part of such development, there is a need for methods for stereoselectively reacting mixtures of stereoisomers.

[0009] International Publication No. 2021 / 054474 Patent Publication No. 2023-041438

[0010] Kanagawa, M. et al., Cell Rep., 14, 2209-2223 (2016)Goddeeris, M. et al., Nature 503, 136-140 (2013)Tamura, T. et al., J. Org. Chem. 2020, 85, 20, 12935-12946Katz, M. et al., Nature 603, 174-179 (2022)Sheikh, MO. Et al., Nat Commun. 13, 3617 (2022)

[0011] The present disclosure provides a method for stereoselectively reacting a mixture of xyloside stereoisomers. In one aspect, the method includes a step of contacting a reaction mixture containing a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moieties of each of the α-xylosides and the β-xylosides with silica gel, the step including converting the cyclic esterified intermediate compound corresponding to the α-xyloside back to the α-xyloside form before cyclic esterification and maintaining the cyclic esterified form of the cyclic esterified intermediate compound corresponding to the β-xyloside. In some embodiments, the method further includes a step of obtaining the reaction mixture, which includes reacting the mixture containing the α-xylosides and the β-xylosides in which the hydroxyl groups at the 2- and 4-positions of the xylose moieties are unprotected with boronic acid or boric acid to obtain a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moieties of each of the α- and β-xylosides contained in the mixture. In another aspect, a composition is provided comprising an α-xyloside in which the hydroxyl groups at the 2- and 4-positions of the xylose moiety are not protected, and an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moiety of the β-xyloside corresponding to the α-xyloside.

[0012] In one aspect, the present disclosure provides a method for producing a cyclic esterified intermediate compound, comprising an α-xyloside and a β-xyloside, each containing a cyclic ester of boronic acid or boric acid at the 2- and 4-positions of the xylose moiety of the xylose moiety, by contacting the reaction mixture with silica gel. This method includes converting the cyclic esterified intermediate compound corresponding to the α-xyloside back to the α-xyloside form before cyclic esterification and maintaining the cyclic esterified form of the cyclic esterified intermediate compound corresponding to the β-xyloside. This method allows selective separation or at least a significant increase in the proportion of the α-stereoisomer in the non-cyclic esterified form, leaving only the β-stereoisomer in the cyclic esterified form. The method according to some embodiments may further comprise a step of obtaining the reaction mixture, which comprises reacting the mixture containing α-xylosides and β-xylosides, in which the hydroxyl groups at the 2- and 4-positions of the xylose moieties are unprotected, with boronic acid or boric acid to obtain a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moieties of each of the α- and β-xylosides contained in the mixture. In some embodiments, the method may be used, for example, to selectively separate α-xylosides or β-xylosides, or to selectively remove β-xylosides, from a mixture containing α-xylosides and β-xylosides.

[0013] Some embodiments of the method utilize as a starting material a mixture containing α-xylosides and β-xylosides, in which the hydroxyl groups at positions 2 and 4 of each xylose moiety are unprotected. Preferably, the hydroxyl group at position 3 of the xylose moiety is also unprotected. The molar ratio of α-xylosides to β-xylosides in this mixture is typically close to 1:1, but other ratios are also possible, such as 1:10 to 10:1. As will be understood by those skilled in the art, xylosides are glycosides in which the glycocone is xylose (including xylose with a protecting group), and α-xylosides and β-xylosides exist depending on whether the aglycon is glycosidicly bonded to the anomeric carbon of xylose in a downward or upward orientation relative to the xylose ring. Chemical synthesis often produces a mixture of the corresponding diastereomers, α-xylosides and β-xylosides, as a synthetic product, but these are not easily separated from each other. Therefore, a novel method for selectively separating α-xylosides or β-xylosides from a mixture of the corresponding diastereomers, α-xylosides and β-xylosides, or for increasing the ratio of α-xylosides to β-xylosides, or for selectively removing β-xylosides, would be useful.

[0014] The present disclosure discloses a method involving an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moiety of the xyloside. In this disclosure, "selectively separating" an α-xyloside or β-xyloside from a mixture containing an α-xyloside and a β-xyloside means obtaining the α-xyloside as a fraction substantially free of β-xyloside, or obtaining the β-xyloside as a fraction substantially free of α-xyloside, from the α-xyloside and β-xyloside derived from the α-xyloside and β-xyloside, respectively, in the starting mixture but not subjected to the cyclic esterification described above; or both may be achieved. The separated xyloside may differ from the xyloside present in the starting mixture in terms of the presence or type of protecting group. In the present disclosure, "selectively removing β-xylosides" from a mixture containing α-xylosides and β-xylosides means completely or partially removing (i.e., eliminating) β-xylosides derived from β-xylosides in the starting mixture but not cyclically esterified, thereby increasing the proportion of α-xylosides derived from α-xylosides in the starting mixture but not cyclically esterified, in other words, increasing the α / β ratio. In some embodiments, a fraction containing an increased proportion of α-xylosides derived from α-xylosides in the mixture but not cyclically esterified is obtained (e.g., as an elution fraction from silica gel chromatography). However, in such a fraction, the proportion of the corresponding β-xylosides, i.e., β-xylosides derived from β-xylosides in the mixture but not cyclically esterified, is at least reduced, and the fraction may be obtained as a fraction containing no β-xylosides. Selective removal of β-xylosides can be achieved by creating a state in which only β-xylosides are converted to cyclic esterified products, as described below.

[0015] The method according to embodiments of the present disclosure can also be described as a method for selectively modifying β-xylosides in a mixture containing α-xylosides and β-xylosides.

[0016] In some embodiments, the method includes reacting a mixture containing α-xylosides and β-xylosides in which the hydroxyl groups at the 2- and 4-positions of the xylose moieties are unprotected with boronic acid or boric acid to obtain an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moieties of the α- and β-xylosides contained in the mixture, thereby obtaining a reaction mixture. This reaction can be carried out at room temperature. This reaction can be carried out in an organic solvent, preferably a nonpolar solvent, an aprotic polar solvent, or a mixture thereof. More specific examples of organic solvents include, but are not limited to, toluene, hexane, ethyl acetate, or any combination thereof. The reaction time can be adjusted appropriately by those skilled in the art based on the progress of the reaction, and can be, for example, 5 hours or more, or 8 hours or more, or 48 hours or less, or 24 hours or less. As a result, the hydroxyl groups at the 2- and 4-positions of the xylose moiety of each of the α- and β-xylosides are esterified with the two acid groups of boronic acid or boric acid to form cyclic ester compounds corresponding to the α-xylosides and β-xylosides, respectively.

[0017] As will be appreciated by those skilled in the art, boronic acids and boric acids have the general formula R—B(OH) ２where R is OH in the case of boric acid and R is a substituent in the case of boronic acid. The R group in the boronic acid can be, for example, an alkyl, alkenyl, aryl, cycloalkyl, or organic heterocyclic group, or a combination thereof. The alkyl group can be, for example, 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl, or butyl). The alkenyl group can be, for example, 2 to 6 carbon atoms (e.g., propenyl). The aryl group can be, for example, a phenyl group, and may be a substituted aryl group. Examples of cycloalkyl or organic heterocyclic groups include, but are not limited to, cyclopentyl, cyclohexyl, thiophene, furan, and pyridine. Boronic acids or boric acids often form cyclic esters or complexes with diols. Embodiments of the present disclosure utilize this coupling reaction involving the two hydroxyl groups of the xylose moiety. In the case of boric acid, when one boron atom bonds with the oxygen atoms of two hydroxyl groups, a molecular complex is formed, as in the case of an ester of boronic acid. In the present disclosure, the bond between boric acid and the two hydroxyl groups of the xylose moiety that forms such a complex is also referred to as a "cyclic ester." Phenylboronic acid and ethylboronic acid are non-limiting examples of suitable boronic acids.

[0018] In this disclosure, the term "intermediate compound" or "intermediate" refers to a cyclically esterified compound that is distinct from and corresponds to (i.e., is a derivative or precursor of) the non-cyclically esterified α-xylosides and β-xylosides. In this disclosure, these intermediate compounds are sometimes referred to as cyclically esterified intermediate compounds. Furthermore, for example, a cyclically esterified intermediate compound corresponding to an α-xyloside (also referred to as an "α-intermediate" in this disclosure) can subsequently be hydrolyzed to regenerate the non-cyclically esterified α-xyloside. For example, a cyclically esterified intermediate compound corresponding to a β-xyloside (also referred to as a "β-intermediate" in this disclosure) can be separated from the α-xyloside and then hydrolyzed to regenerate the non-cyclically esterified β-xyloside, or the cyclically esterified intermediate can be used or discarded as is (in this sense, the "intermediate" is not necessarily converted into another compound). In this disclosure, the term "reaction mixture" refers to a mixture containing an α-intermediate and a β-intermediate that is contacted with silica gel to cause a hydrolysis reaction of the cyclic esters.

[0019] The method of this embodiment includes a step of contacting a reaction mixture containing the cyclic esterified boronic acid (or boric acid) intermediate compounds corresponding to the α-xylosides and β-xylosides, respectively, with silica gel. Surprisingly, it was discovered that the cyclic esterified intermediate compounds corresponding to the α-xylosides are relatively quickly hydrolyzed back to the triol form, i.e., the α-xyloside form before cyclic esterification, upon contact with silica gel, whereas the cyclic esterified intermediate compounds corresponding to the β-xylosides contained in the same reaction mixture maintain their cyclic esterified form even upon contact with silica gel. That is, silica gel reacts selectively or preferentially with the α-intermediate over the β-intermediate in a mixture of α-intermediates and β-intermediates, hydrolyzing it to the non-cyclic esterified α-xyloside form. Under the same conditions, the non-cyclic esterified β-xyloside form hydrolyzed from the β-intermediate is not recovered, or at least recovered at a significantly reduced rate compared to the α-xyloside form. After contact with silica gel, typically, the cyclic esterified intermediate compound corresponding to the β-xyloside is recovered, but the cyclic esterified intermediate compound corresponding to the α-xyloside is not recovered. In other words, by contacting a mixture of the α-intermediate and the β-intermediate with silica gel, only the α-intermediate can be hydrolyzed to a xyloside form that is not completely cyclic esterified.

[0020] Silica gel has acidic properties based on silicic acid and has the ability to adsorb moisture from the environment, as evidenced by its well-known use as a desiccant. Based on the difference in the angle at which the xylose glycocone is presented between the α and β isomers, silica gel appears to provide a "just right" hydrolysis environment for the α isomer to be selectively hydrolyzed. Selective hydrolysis of the α isomer can be expressed as preferential hydrolysis of the α isomer over the β isomer. While not wishing to be bound by any particular theory, the following schematic diagram explains a possible reaction mechanism. The α isomer (i.e., the α intermediate) and the β isomer (i.e., the β intermediate) have different coordination structures with the silanol on the silica gel surface, and the α isomer can form a more stable complex supported by hydrogen bonds between three oxygen atoms, as shown in the diagram below as α2. In the α isomer, complex formation activates the oxygen atoms of the coordinated cyclic ester, which in turn makes the boron atom electron-deficient, making it more susceptible to water attack and promoting hydrolysis. In contrast, in the β isomer, the silanol coordination becomes relatively unstable because there are two support points, making it difficult to convert from β1 to β2. Therefore, it can be thought that the oxygen atom of the cyclic ester is difficult to activate, making it relatively difficult to increase the hydrolysis rate.

[0021] The particle size of particulate silica gel is hundreds of times larger than the interatomic distances involved in the reaction shown in the diagram above, so the silica gel particles do not penetrate into the compound. Therefore, it is unlikely that the specific effect on the cyclic esterified intermediate compound depends on, for example, the specific particle size of the silica gel. It is understood that the atomic-level environment involving the silanol on the silica gel surface and the cyclic esterified xylose moiety is what is being considered. Even when using different types of silica gel obtained from different suppliers, it was consistently the β-intermediate rather than the α-intermediate that remained unhydrolyzed. This suggests that the acceleration of hydrolysis of the α-intermediate due to silanol coordination is a general tendency for silica gel.

[0022] Additionally, known reactions for acetalizing diols (e.g., benzylidene acetalization) were attempted to achieve the same 2,4-position acetalization instead of boronic acid or boric acid esterification, but the reaction conditions were too harsh, resulting in decomposition of the glycone. Therefore, the "just right" hydrolysis relies not only on the use of silica gel, but also on the coupling of silica gel with a boronic acid or cyclic ester of boric acid, which is formed under milder conditions than acetals and can be hydrolyzed under milder conditions.

[0023] In certain embodiments, the step of contacting the reaction mixture with silica gel may include separating the reaction mixture by silica gel chromatography. This embodiment is preferred because contacting the mixture with silica gel, which serves as the stationary phase for chromatography, selectively hydrolyzes the cyclic ester of the α-isomer, while efficiently separating the regenerated α-xyloside in triol (or diol, if the 3-position is protected) form from the unreacted cyclic esterified intermediate of the β-isomer in the same procedure. Therefore, the α-xyloside can be selectively separated by silica gel chromatography. In a preferred embodiment, the elution fraction containing the hydrolyzed non-cyclic esterified α-xyloside does not contain non-cyclic esterified β-xyloside, or if it does contain non-cyclic esterified β-xyloside, the proportion of β-xyloside is significantly reduced compared to the starting mixture. For example, if a small amount of non-cyclic esterified β-xyloside is present, the elution fraction can be contacted with silica gel again to further remove the β-xyloside. The cyclic esterified intermediate of the β-isomer may be hydrolyzed after separation from the non-cyclic esterified xyloside to regenerate the β-xyloside. This allows selective separation of β-xylosides from a mixture containing α- and β-xylosides. Because α / β selectivity is not required during the hydrolysis treatment, stronger hydrolysis conditions can be used than with silica gel (silicic acid).

[0024] Silica gel chromatography can be, for example, silica gel column chromatography or silica gel thin-layer chromatography. Alternatively, the silica gel used in the step of contacting the reaction mixture with silica gel can be a silica gel reagent provided without being a chromatographic stationary phase. For example, by mixing and contacting a reaction mixture containing the cyclic ester compounds corresponding to the α-xylosides and β-xylosides, respectively, with a silica gel reagent in a container, selective hydrolysis of the cyclic ester of the α-isomer can be caused, yielding a mixture containing the α-xyloside compound regenerated in triol (or diol) form and an intermediate compound in which the cyclic ester of boronic acid or boric acid remains formed at the 2- and 4-positions of the β-xyloside corresponding to the α-xyloside. While this mixture may contain a small amount of non-cyclic esterified β-xylosides, it can be said that the non-cyclic esterified β-xylosides have been selectively removed compared to the mixture containing the starting α-xylosides and β-xylosides.

[0025] Particulate silica gel typically has a particle size in the micrometer range, for example, but not limited to, within the range of 5 μm to 600 μm, 10 μm to 300 μm, or 30 μm to 100 μm. Particulate silica gel may be spherical or irregularly shaped silica gel. Irregularly shaped silica gel is sometimes called crushed silica gel. Silica gel is typically porous, and its pore size may typically be 3 to 15 nm, for example, 5 to 10 nm, or 6 to 7 nm, although the pore size of silica gel used in embodiments is not particularly limited. Methods for determining the particle size and pore size of silica gel are well known to those skilled in the art and are typically listed in silica gel product catalogs. Specific examples of silica gel products that can be used in the present embodiment include, but are not limited to, Wakogel® C-300 available from Fujifilm Wako Pure Chemical Industries, Ltd., Silica gel 60 (0.063-0.200 mm) available from Merck, and Silica gel 60N (spherical, neutral) available from Kanto Chemical Co., Inc. The particularly preferred silica gel product, C-300, is a crushed silica gel with a particle size of 45-75 μm. Silica gels chemically modified with functional groups are known to those skilled in the art and can be used in the present embodiment, but unmodified silica gel is more preferred.

[0026] The reaction mixture can be contacted with silica gel in the same organic solvent as described above. The specific type of solvent can be appropriately selected by those skilled in the art. For example, it is preferable that the mobile phase of silica gel chromatography is a mixed organic solvent based on toluene and containing ethyl acetate in an amount of less than half by volume.

[0027] In one aspect, a composition is provided that includes (i) an α-xyloside in which the hydroxyl groups at the 2- and 4-positions of the xylose moiety are unprotected, and (ii) an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moiety of the β-xyloside corresponding to the α-xyloside (i.e., diastereomeric to the α-xyloside). This composition can be used to selectively separate the α-xyloside or to selectively separate a cyclic esterified intermediate compound that serves as a precursor to the acyclic esterified β-xyloside. In some embodiments, the composition further includes silica gel. For example, a mixture of the reaction mixture and silica gel, or a silica gel chromatography column or TLC plate loaded with the reaction mixture, may correspond to this composition. In another embodiment, the composition does not include silica gel. For example, a sample obtained after removing silica gel from the mixture of the reaction mixture and silica gel may correspond to this composition. Compositions containing the acyclic esterified β-xyloside corresponding to the acyclic esterified α-xyloside in an amount (molar or mass) smaller than the acyclic esterified α-xyloside are also contemplated.

[0028] In various embodiments of the present disclosure, the α-xylosides and β-xylosides can be, for example, O-xylosides, S-xylosides, N-xylosides, or C-xylosides. More preferably, the α-xylosides and β-xylosides are O-xylosides or N-xylosides, and especially O-xylosides.

[0029] The glycone, i.e., xylose moiety, of α-xylosides and β-xylosides is preferably the first monosaccharide moiety, and the aglycone is also preferably a monosaccharide moiety. In this case, the aglycone is referred to as the second monosaccharide moiety, and it is understood that α-xylosides and β-xylosides are disaccharides. In the present disclosure, the monosaccharide moiety is preferably a D-isomer. The monosaccharide moiety is preferably a six-membered ring monosaccharide moiety. The disaccharide is preferably a disaccharide with a 1→3 glycosidic bond. Non-limiting examples of sugars that can serve as the second monosaccharide moiety include glucuronic acid, glucose, glucosamine, mannose, galactose, galactosamine, galacturonic acid, xylose, etc. Prior to reacting α-xylosides and β-xylosides with boronic acid or boric acid, these second monosaccharide moieties are preferably protected at one or more or all of their hydroxyl, carboxyl, and amino groups with protecting groups known to those skilled in the art. In particularly preferred embodiments, the second monosaccharide moiety is a glucuronic acid moiety (including a glucuronic acid moiety having a protecting group). Disaccharides consisting of a xylose moiety and a glucuronic acid moiety are highly useful because they are building blocks of matriglycan. In certain embodiments, the glucuronic acid moiety, which is the second monosaccharide moiety, is a glucuronic acid moiety in which functional groups such as hydroxyl groups (e.g., hydroxyl and carboxyl groups) have been protected prior to reaction with boronic acid or boric acid. For example, the hydroxyl groups at the 2- and 4-positions of the glucuronic acid moiety can be protected with acyl groups such as benzoyl, acetyl, substituted acetyl, substituted benzoyl, and levulinoyl, or substituted alkyl groups (i.e., RCH2- bonded to the O-derived hydroxyl group) such as benzyl, substituted benzyl, naphthylmethyl, substituted naphthylmethyl, and allyl. In some preferred embodiments, the hydroxyl groups at the 2- and 4-positions of the glucuronic acid moiety are protected with benzoyl groups. The functional group bonded to the 1-carbon atom of the glucuronic acid moiety may be, for example, a phenoxy group such as phenoxy, substituted phenoxy, naphthoxy, or substituted naphthoxy; an alkoxy group such as methoxy, ethoxy, or propyloxy; or a substituent such as a thiophenoxy group or a thioalkoxy group in which the oxygen atom of a phenoxy group or alkoxy group is replaced with a sulfur atom.The carboxy group at the 6-position of the glucuronic acid moiety can be protected, for example, as an ester with an alcohol or phenol, or as a thioester with a thiol or thiophenol. These are non-limiting examples of protecting groups (or protected functional groups). In a particularly preferred embodiment, the hydroxyl groups at the 2- and 4-positions of the glucuronic acid moiety are each protected with a benzoyl group, the hydroxyl group at the 1-position is protected with a methoxyphenyl group, and the carboxyl group at the 6-position is protected with a methyl group. A second monosaccharide moiety other than glucuronic acid can also have a protecting group similar to those exemplified herein. Examples of protecting groups that can be carried by the monosaccharide moiety in embodiments of the present disclosure are described in Greene's Protective Groups in Organic Synthesis, PGM Wuts & TW Greene, WILEY (2007).

[0030] When the aglycone is not a sugar, for example, the aglycone may be a cyclic or acyclic chain group or combinations thereof having 6 to 30 carbon atoms, such as an aryl, cyclic alkyl, heterocyclic, or linear or branched alkyl or alkenyl group, or combinations thereof. These functional groups may be linked to the first carbon of the first monosaccharide via an oxygen atom (O-xyloside) or via a non-oxygen atom such as a sulfur atom, nitrogen atom, or carbon atom (S-xyloside, N-xyloside, and C-xyloside, respectively).

[0031] The reaction and separation schemes according to preferred embodiments of the present disclosure are shown below, with the yields indicated for specific examples. In the schemes below, compounds are labeled with numbers 1 to 5, and the same labels are used for corresponding compounds in the examples described below.

[0032] The present disclosure includes the following non-limiting embodiments: Embodiment 1: A method comprising the step of contacting a reaction mixture containing a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid has been formed at the 2- and 4-positions of the xylose moieties of each of the α-xylosides and the β-xylosides with silica gel, the step comprising returning the cyclic esterified intermediate compound corresponding to the α-xyloside to the α-xyloside form before cyclic esterification and maintaining the cyclic esterified form of the cyclic esterified intermediate compound corresponding to the β-xyloside. Embodiment 2: The method of embodiment 1, further comprising the step of obtaining the reaction mixture, which includes an α-xyloside and a β-xyloside in which the hydroxyl groups at the 2- and 4-positions of the xylose moieties are unprotected, with boronic acid or boric acid to obtain a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid has been formed at the 2- and 4-positions of the xylose moieties of each of the α- and β-xylosides contained in the mixture. Embodiment 3: The method of embodiment 1 or 2, wherein the ratio of non-cyclically esterified α-xylosides to non-cyclically esterified β-xylosides is increased relative to the starting mixture. Embodiment 4: The method of embodiment 1-3, further comprising selectively separating cyclically esterified intermediate compounds corresponding to the β-xylosides. Embodiment 5: The method of embodiment 1-4, wherein the step of contacting the reaction mixture with silica gel comprises separating the reaction mixture by silica gel chromatography. Embodiment 6: The method of embodiment 5, wherein the silica gel chromatography is silica gel column chromatography. Embodiment 7: The method of embodiment 1-6, wherein the α-xylosides and β-xylosides are O-xylosides. Embodiment 8: The method of embodiment 1-7, wherein the aglycones of the α-xylosides and β-xylosides are second monosaccharide moieties, and the α-xylosides and β-xylosides are disaccharides. Embodiment 9: The method of embodiment 8, wherein the second monosaccharide moiety is a glucuronic acid moiety. Embodiment 10: The method of embodiment 9, wherein the glucuronic acid moiety has the hydroxyl groups at positions 2 and 4 protected with benzoyl groups.Embodiment 11 A composition comprising an α-xyloside in which the hydroxyl groups at the 2- and 4-positions of the xylose moiety are not protected, and an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moiety of a β-xyloside corresponding to the α-xyloside.

[0033] Embodiment 21: A method for selectively separating an α-xyloside or a β-xyloside or selectively removing a β-xyloside from a mixture containing α-xylosides and β-xylosides in which the hydroxyl groups at the 2- and 4-positions of the xylose moieties are unprotected, the method comprising the steps of reacting the mixture with boronic acid or boric acid to obtain a reaction mixture containing an intermediate compound in which cyclic esters of boronic acid or boric acid are formed at the 2- and 4-positions of the xylose moieties of the α- and β-xylosides contained in the mixture, and contacting the reaction mixture with silica gel. Embodiment 22: The method of embodiment 21, wherein the step of contacting the reaction mixture with silica gel comprises separating the reaction mixture by silica gel chromatography, and the α-xyloside is selectively separated in the silica gel chromatography. Embodiment 23: The method of embodiment 22, wherein the silica gel chromatography is silica gel column chromatography. Embodiment 24: The method of any one of embodiments 21 to 23, wherein the α-xyloside and β-xyloside are O-xylosides. Embodiment 25. The method of any one of Embodiments 21 to 24, wherein the aglycone of the α-xyloside and β-xyloside is a second monosaccharide moiety, and the α-xyloside and β-xyloside are disaccharides.Embodiment 26. The method of Embodiment 25, wherein the second monosaccharide moiety is a glucuronic acid moiety.Embodiment 27. A composition comprising an α-xyloside in which the hydroxyl groups at the 2- and 4-positions of the xylose moiety are not protected, and an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moiety of the β-xyloside corresponding to the α-xyloside.

[0034] The following examples are provided to further describe the embodiments in detail, but are for illustrative purposes only and are not intended to limit the scope of the present disclosure. In particular, aglycones other than the specific glucuronic acid exemplified below can also be used in further embodiments.

[0035] Example 1 Separation of α-disaccharide isomer (1) and synthesis of β-disaccharide isomer (5) via cyclic phenylboronic acid ester (3',3) A mixture of methyl[α-d-xylopyranosyl-(1→3)-β-(4-metoxyphenyl 2,4-di-O-benzoyl-β-d-glucopyranosid)]uronate (1) (318.6 mg, 486.6 μmol) and methyl[β-d-xylopyranosyl-(1→3)-β-(4-metoxyphenyl 2,4-di-O-benzoyl-β-d-glucopyranosid)]uronate (2) (245.0 mg, 374.3 μmol) was dissolved in toluene (5.7 ml), and phenylboronic acid (525.0 mg, 4.306 mmol) was added. The mixture was stirred at room temperature for 12 hours to allow the reaction to proceed. At this point, NMR confirmed that 1 (α-xyloside) and 2 (β-xyloside) had reacted substantially completely with phenylboronic acid, and the reaction mixture contained the cyclic phenylboronic acid esterified products of 1 and 2 (i.e., cyclic esterified intermediates; designated 3' and 3, respectively). The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (toluene:ethyl acetate = 5:1 to 1:1, followed by ethyl acetate:methanol = 100:1 to 30:1). The separated α-isomer, 1 (255.1 mg, 389.7 μmol), was recovered in 80% yield. This fraction did not contain the β-isomer (2). In other words, the α-xyloside was selectively separated from the starting mixture containing α-xyloside and β-xyloside. The crude β-isomer, 3 (756.2 mg), which was cyclic phenylboronic acid esterified at the 2- and 4-positions of the xylose moiety, was also obtained. This fraction did not contain 3', the α-isomer converted to cyclic phenylboronic acid ester. Therefore, by contacting the reaction mixture containing the α-intermediate and the β-intermediate with silica gel, selective hydrolysis of the α-intermediate occurred. The silica gel used in the silica gel column chromatography in this example was Wakogel® C-300 (particle size 45-75 μm, crushed particles) available from Fujifilm Wako Pure Chemical Industries, Ltd., and the weight of the silica gel used was 30 times that of the substrate.

[0036] 3 was dissolved in pyridine (13 ml), 1.0 M levulinic anhydride (2 ml) and 4,4-dimethylaminopyridine (DMAP) were added, and the mixture was stirred at room temperature for 1 day. Further addition of 0.1 M HCl (1.3 ml) resulted in levulinoylation at the 3-position and acid hydrolysis of the cyclic boronate ester. After extraction with ethyl acetate and washing with saturated brine, the organic layer was dried over anhydrous MgSO4. The insoluble matter was filtered and concentrated, and the residue was purified by gel filtration column chromatography (chloroform:methanol = 1:1) to quantitatively obtain 5 (283.3 mg, 376.4 μmol) from 2 (i.e., derived from 2 in the mixture of 1 and 2). Consequently, the β-xyloside was selectively separated from the starting mixture containing both α- and β-xylosides.

[0037] In another example, the experiment was carried out essentially in the same manner as above, except that Silica gel 60 (particle size 0.063-0.200 mm, crushed particles) available from Merck was used as the silica gel. The same mixture of α-isomer 1 and β-isomer 2 (0.1694 mmol: 0.1303 mmol) was used as the starting material. After cyclic esterification with boronic acid and separation by silica gel column chromatography, all of the α-isomer eluted as a non-cyclic esterified triol form, with a yield of 88%. Of the β-isomer, 44% eluted as the cyclic esterified intermediate (3) (yield after levunoylation (5)) and 24% eluted as the triol form (2). The α / β ratio of the mixture of α- and β-isomer triols recovered from silica gel column chromatography was 1 / 0.21. In yet another example, silica gel 60N (spherical, neutral, particle size 40-100 μm) available from Kanto Chemical Co., Inc. was used as the silica gel, and the reaction mixture was applied to the column by the dusting column technique, but essentially the same results were obtained, with only the β isomer remaining as a cyclic esterified intermediate and eluting.

[0038] Example 2: Separation of α-disaccharide isomer (1) and synthesis of β-disaccharide isomer (5) via cyclic ethylboronic acid ester (4', 4). The same experiment as in Example 1 was carried out, except that ethylboronic acid was used instead of phenylboronic acid. Specifically, a mixture of 1 (629.1 mg, 961.0 μmol) and 2 (483.9 mg, 739.2 μmol) was dissolved in toluene (12 ml), and ethylboronic acid (638.4 mg, 8.640 mmol) was added and stirred for 12 hours. The reaction mixture was purified by silica gel column chromatography using Wakogel® C-300 silica gel (toluene:ethyl acetate = 5:1 to 1:1, ethyl acetate:methanol = 100:1 to 30:1), and 1 (456.6 mg, 696.0 μmol) was recovered in 72% yield. This fraction did not contain β-isomer (2). In addition, 4 (762.4 mg) was obtained in crude form. This fraction did not contain 4', the α-isomer converted to cyclic ethylboronic acid ester. Therefore, selective hydrolysis of the α-intermediate occurred when the reaction mixture containing the α-intermediate and β-intermediate was contacted with silica gel. 4 was dissolved in pyridine (12 ml), 1.0 M levulinic anhydride (6 ml) and DMAP were added, and the mixture was stirred at room temperature for 30 minutes. 1.0 M HCl (1.3 ml) was added, and the mixture was extracted with ethyl acetate. After washing with saturated brine, the organic layer was dried over anhydrous MgSO4. The insoluble matter was filtered and concentrated, and the residue was purified by gel filtration column chromatography (chloroform:methanol = 1:1). Subsequently, the product was further purified by column chromatography (n-hexane:ethyl acetate = 50:1 to 1:3), and 5 (575.8 mg, 765.0 μmol) was quantitatively obtained from 2.

[0039] [Example 3] Synthesis of Methyl[{3-O-levulynoyl-2,4-di-O-(4-methyl)benzoyl-β-d-xylopyranosyl}-(1→3)-β-(4-methoxyphenyl 2,4-di-O-benzoyl-β-d-glucopyranosid)]uronate (6) 5 (575.8 mg, 765.0 μmol) obtained in Example 2 was dissolved in pyridine (12 mL), p-toluoyl chloride (0.60 mL, 4.5 mmol) and DMAP (9.1 mg, 75 μmol) were added, and the mixture was stirred at room temperature for 5.5 hours. 1.0 M HCl (1.0 mL) was added, and the mixture was extracted with chloroform, washed with saturated brine, and the organic layer was dried over anhydrous MgSO. Insoluble matter was filtered and concentrated, and the residue was purified by gel filtration column chromatography (chloroform:methanol = 1:1) to give 6 (670.7 mg, 678.2 μmol) in 89% yield.

[0040] [Reference Example] Below, we outline the experiments in the Reference Example that investigated the possibility of separating stereoisomers by protection using benzylidene acetalization rather than boronate esterification. In each experiment, either the xylose moiety was decomposed to give monosaccharides, or a mixture of disaccharides without isomer separation was obtained.

[0041] Reference Example 1: A mixture of 1 and 2 (282.5 mg, 431.5 μmol) (1:2 = 0.06:1) was dissolved in benzaldehyde (10 ml), zinc chloride (289.7 mg, 2.126 μmol) was added, and the mixture was stirred at 45°C for 1 day. Saturated sodium bicarbonate was added to terminate the reaction, and the mixture was extracted with ethyl acetate and washed with saturated sodium bicarbonate and saturated brine. The organic layer was dried over MgSO4, the insoluble matter was filtered, and the residue was concentrated. The residue was purified by silica gel chromatography (toluene:ethyl acetate = 50:1 to ethyl acetate:methanol = 50:1) to give methyl(4-methoxyphenyl 2,4-di-O-benzoyl-β-d-glucopyranosid)uronate (7) (269.2 mg). It appeared that the xylose moiety was decomposed under the reaction conditions for acetalization.

[0042] Reference Example 2: A mixture of 1 and 2 (227.2 mg, 347.0 μmol) (1:2 = 0.07:1) was dissolved in benzaldehyde (10 ml), zinc chloride (240.1 mg, 1.762 μmol) was added, and the mixture was stirred at room temperature for 1 day. Saturated sodium bicarbonate was added to terminate the reaction, and the mixture was extracted with ethyl acetate and then washed with saturated sodium bicarbonate and saturated brine. The organic layer was dried over MgSO4, insoluble matter was filtered, and the mixture was concentrated. The residue was purified by silica gel chromatography (toluene:ethyl acetate = 50:1 to ethyl acetate:methanol = 50:1) to give 7 (89.5 mg).

[0043] Reference Example 3: A mixture of 1 and 2 (112.2 mg, 171.3 μmol) (1:2 = 1:5) was dissolved in acetonitrile (3 mL), and benzaldehyde dimethyl acetal (38 μL, 254.6 μmol) and p-toluenesulfonic acid monohydrate (3.4 mg, 17.80 μmol) were added, followed by stirring at room temperature for 1 day. The mixture was neutralized with triethylamine, and the concentrated residue was purified by column chromatography (toluene:ethyl acetate = 50:1 to ethyl acetate:methanol = 50:1) to give 7 (29.4 mg).

[0044] Reference Example 4: A mixture of 1 and 2 (16.3 mg, 24.80 μmol) (1:2 = 1:5) was dissolved in DMF (500 μl) under reduced pressure, and benzaldehyde dimethyl acetal (5.6 μl, 37.52 μmol) and a catalytic amount of camphorsulfonic acid were added, followed by stirring at room temperature for 5 hours. The mixture was neutralized with triethylamine, and the concentrated residue was purified by gel filtration chromatography (chloroform:methanol = 1:1), recovering a mixture of 1 and 2 (34.2 mg). In Reference Example 4, acetalization was attempted under milder reaction conditions than in Reference Examples 1 to 3, but it appeared that acetalization did not proceed.

Claims

1. A method comprising a step of contacting a reaction mixture containing a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moieties of each of the α-xyloside and the β-xyloside with silica gel, wherein the step includes returning the cyclic esterified intermediate compound corresponding to the α-xyloside to the α-xyloside form before being cyclic esterified, and maintaining the cyclic esterified form of the cyclic esterified intermediate compound corresponding to the β-xyloside.

2. The method of claim 1, further comprising a step of obtaining the reaction mixture, which step comprises reacting the mixture containing α-xylosides and β-xylosides in which the hydroxyl groups at the 2- and 4-positions of the xylose moieties are not protected with boronic acid or boric acid to obtain a cyclic esterified intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2- and 4-positions of the xylose moieties of each of the α- and β-xylosides contained in the mixture.

3. The method of claim 2, wherein the ratio of non-cyclically esterified alpha xylosides to non-cyclically esterified beta xylosides is increased compared to the starting mixture.

4. The method of claim 1, further comprising selectively isolating a cyclic esterified intermediate compound corresponding to said β-xyloside.

5. The method of claim 1, wherein the step of contacting the reaction mixture with silica gel comprises separating the reaction mixture by silica gel chromatography.

6. The method according to claim 5, wherein the silica gel chromatography is silica gel column chromatography.

7. The method of any one of claims 1 to 6, wherein the α-xylosides and β-xylosides are O-xylosides.

8. The method of any one of claims 1 to 6, wherein the aglycone of the alpha xyloside and beta xyloside is a second monosaccharide moiety, and the alpha xyloside and beta xyloside are disaccharides.

9. The method of claim 8, wherein the second monosaccharide moiety is a glucuronic acid moiety.

10. The method according to claim 9, wherein the hydroxyl groups at the 2- and 4-positions of the glucuronic acid moiety are protected with benzoyl groups, respectively.

11. A composition comprising an α-xyloside in which the hydroxyl groups at the 2nd and 4th positions of the xylose moiety are not protected, and an intermediate compound in which a cyclic ester of boronic acid or boric acid is formed at the 2nd and 4th positions of the xylose moiety of the β-xyloside corresponding to the α-xyloside.

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