Novel oligosaccharide, intermediate for production of said oligosaccharide, and method for producing same

Abstract

The present invention addresses the problem of providing: a novel oligosaccharide that is a double-antenna glycan having an [alpha] 2, 6-sialic acid structure at a non-reducing terminal; a method for producing the oligosaccharide; an intermediate thereof; and a method for producing the intermediate thereof. The present invention provides a novel oligosaccharide represented by the following formula A-22, a method for producing the oligosaccharide represented by Figure 1, an intermediate thereof, and a method for producing the intermediate thereof. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a novel oligosaccharide as a biantennary glycan having an α2,6-sialic acid structure at a non-reducing end, a method for producing the oligosaccharide, and an intermediate thereof and a method for producing the intermediate thereof. Background Art

[0002] The addition of sugar chains (glycosylation) to proteins is known to significantly influence their function and structure. N-linked sugar chains are closely associated with the physiological activity of proteins, and biantennary N-glycans with an α2,6-sialic acid structure at their non-reducing termini have been reported to be the optimal structure for enhancing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity (Non-Patent Document 1).

[0003] The development and commercialization of pharmaceuticals utilizing sugar chains requires the stable, large-scale production of high-purity sugar chains at an industrially viable price. The reported synthesis methods for α2,6-sialylated sugar chains (α2,6-sialylglycan) to date have been broadly categorized into two types: 1) semi-chemical synthesis, which involves the isolation and purification of natural extracts or a combination of chemical synthesis of the main backbone precursor and enzymatic chemical conversion; and 2) pure chemical synthesis.

[0004] For example, as a semi-chemical synthesis method, a combination of enzymes and chemical methods has been reported to produce N-linked sugar chains from egg yolks (Non-Patent Document 2). While these methods can synthesize the target sugar chains with fewer steps than pure chemical synthesis, they require a large supply of egg yolks, and the subsequent isolation and purification of the water-soluble, unprotected sugar chains from the egg yolks and subsequent chemical conversion often require specialized techniques and purification equipment (Patent Documents 1-4).

[0005] On the other hand, the following examples have been reported as purely chemical synthesis methods for α2,6-sialylated sugar chains.

[0006] (1) Total synthesis of a complex 11-glycosylated polysaccharide having an α2,6-sialic acid moiety (Non-Patent Document 3).

[0007] (2) Total synthesis of immunoglobulin G13 glycopeptide having an α2,6-sialic acid site (Non-Patent Document 4).

[0008] (3) Total synthesis of an α2,6-sialylated 12-sugar N-linked sugar chain containing core fucose (Non-Patent Document 5).

[0009] (4) Total synthesis of α2,6-sialylated 10-saccharide oligosaccharide chains fluorinated at position 3 (Non-Patent Document 6).

[0010] (5) Total synthesis of asymmetrically deuterated α2,6-sialylated biantennary 11-glycan oligosaccharide chains and tetraantennary 17-glycan oligosaccharide chains (Non-Patent Document 7).

[0011] It is believed that the purely chemical synthesis of sugar chains, provided reliable production methods are established, offers a high degree of flexibility in production quantities, similar to conventional low-molecular-weight compounds, through derivatization from monosaccharides. Furthermore, since sugars modified with protective groups are converted, most purification operations involve handling water-insoluble compounds, significantly reducing operational complexity and man-hours compared to semi-chemical synthesis. Furthermore, by applying established chemical synthesis methods, a wide variety of non-natural sugar chains can be readily synthesized.

[0012] On the other hand, in the above-mentioned past reports, the following two points were cited as major technical challenges in the synthesis: 1) the conversion of sugar moieties, such as the construction of the β-mannoside moiety and the α-sialic acid moiety, which are difficult, and the presence of low selectivity and low yield in their ligation steps; and 2) in both the sugar moiety conversion and ligation steps, silica gel column chromatography purification, which is not suitable for scale-up, was often used in each step. In order to remove isomers and impurities produced as by-products of the reaction, precise chromatographic separation and purification operations had to be performed in many steps.

[0013] As described above, although purely chemical synthesis methods for sugar chains have potential advantages over semi-chemical synthesis methods in terms of large-scale synthesis, from the perspectives of yield, selectivity, efficiency, and cost, it is difficult to say that the technology has been fully developed, and there are very few examples of its use as a practical large-scale synthesis method.

[0014] Phthaloyl-protected sugar derivatives sometimes require deacylation. However, in the presence of trace amounts of water, the phthalimide group readily undergoes ring-opening reactions under alkaline conditions, requiring strict control of the water content. Even ppm-level water levels are difficult to completely suppress ring-opening. Therefore, methods are needed that suppress the ring-opening of the phthalimide group while enabling high-yield deacylation.

[0015] Furthermore, in the chemical synthesis of oligosaccharide chains containing (possibly multiple) hydroxyl groups, efficient production of target compounds requires the rational utilization of selective deprotection and deprotection methods for these hydroxyl groups. In particular, the 2-naphthylmethyl group has been widely used as a common protecting group for hydroxyl groups. Meanwhile, a known method for deprotecting 2-naphthylmethyl groups is the use of 2,3-dichloro-5,6-dicyano-p-benzoquinone in dichloromethane-water. This method provides the target deprotected product in moderate to high yields for a wide range of substrates. However, due to the general immiscibility of dichloromethane and water, and the fact that 2,3-dichloro-5,6-dicyano-p-benzoquinone and its byproduct, 2,3-dichloro-5,6-dicyano-p-benzohydroquinone, are virtually insoluble in dichloromethane-water, this affects stirring properties, thus presenting challenges for large-scale synthesis. Furthermore, there are reports of cases where the target deprotected product may not always be obtained in satisfactory yields depending on the substrate used (Non-Patent Document 8). Furthermore, the increase in benzyl groups in the substrate tends to reduce reaction yields (Non-Patent Document 9), leading to a desire for a milder and more efficient method applicable to complex substrates. As a method for achieving this, improved conditions using β-pinene as an additive have been reported (Non-Patent Document 10), but even with this method, the yield remains moderate for complex substrates containing multiple benzyl groups (Non-Patent Document 11). Given this background, there is a desire to develop a de-2-naphthylmethylation reaction that can remove the protecting group of the 2-naphthylmethyl group under milder conditions and obtain the deprotected product in high yield.

[0016] Furthermore, as chemical synthesis methods for oligosaccharide chains, liquid phase synthesis and solid phase synthesis are known. However, liquid phase synthesis can use conventional organic synthesis methods, so it is easier to track reactions and scale up. However, since post-treatment and purification are performed at each step, there are disadvantages of being time-consuming and labor-intensive. In addition, solid phase synthesis is advantageous in that it can be automatically synthesized and can be manufactured quickly, but there are limitations on scale up due to equipment limitations. In addition, due to its low reactivity, it is necessary to use an excessive amount of sugar donors used in the sugar extension reaction, which is not suitable for industrial large-scale synthesis. In addition, there is a disadvantage of being difficult to confirm the progress of the reaction in the middle stage (patent document 5). In order to solve these problems, several methods for producing oligosaccharides using a matrix with a molecular structure (tag) have been developed. The molecular structure (tag) is precipitated, distributed, adsorbed, etc. specifically for a certain environment (patent document 6, non-patent documents 12 to 16). These methods have the advantages of both liquid and solid phase synthesis methods, that is, they are easy to analyze because they can react in a homogeneous system, and they can be separated from reagent residues, etc. by utilizing the characteristics of the tag. For example, a method is known in which a branched long-chain alkane is used as a hydrophobic tag and the post-reaction solution is adsorbed onto octadecyl-modified silica gel, thereby separating untagged compounds (Non-Patent Document 16). However, both methods require the use of a tag, which has the following disadvantages: a desorption step is required; and as oligomerization progresses, the matrix becomes larger than the tag, gradually becoming more dominant due to the physical properties of the matrix, weakening the functionality of the tag. Therefore, in the production of oligosaccharide chains, a method for more efficiently purifying oligosaccharides is desired.

[0017] During glycosylation reactions, the presence of -NHAc groups within the reaction substrate significantly reduces the reactivity of the desired glycosylation reaction due to interaction with Lewis acids, often requiring an excess of sugar donor for completion. Consequently, in the synthesis of oligosaccharide chains containing acetylglucosamine, a method has been employed: a Troc group (Non-Patent Documents 5, 6, and 7), a phthaloyl group (Non-Patent Document 15), or a sulfonyl group (Non-Patent Document 4) is used as a temporary protecting group on the glucosamine nitrogen during the glycosylation reaction, followed by deprotection and N-Ac conversion. However, the Troc group requires deprotection reaction conditions using zinc / AcOH or a long reaction time with excess lithium hydroxide, and complex sugar chains are accompanied by substrate decomposition under these conditions. Furthermore, the use of excess ethylenediamine in deprotection of the phthaloyl group poses the problem of amidation of the sialic acid ester site as a side reaction. To avoid this, a two-step process is required: selective hydrolysis of the ester site prior to deprotection. Furthermore, in the case of a sulfonyl group, deprotection is performed under reaction conditions using metallic sodium, which makes scalability difficult. Therefore, in the production of oligosaccharide chains containing acetylglucosamine, there is a demand for the development of a protecting group that does not reduce the reactivity of the glycosylation reaction and can be easily replaced with an acetyl group.

[0018] Prior art literature

[0019] Patent Literature

[0020] Patent Document 1: International Publication No. 2011 / 027868

[0021] Patent Document 2: International Publication No. 96 / 02255

[0022] Patent Document 3: International Publication No. 2014 / 208742

[0023] Patent Document 4: International Publication No. 2017 / 110984

[0024] Patent Document 5: International Publication No. 2002 / 16384

[0025] Patent Document 6: Japanese Patent No. 6001267

[0026] Non-patent literature

[0027] Non-patent literature 1: Proc. Natl. Acad. Sci. USA 2015, 112, 10611

[0028] Non-patent document 2: Beilstein J. Org. Chem. 2018, 14, 416

[0029] Non-patent document 3: Tetrahedron Lett. 1986, 27, 5739

[0030] Non-patent document 4: J. Am. Chem. Soc. 2009, 131, 16669-16671

[0031] Non-patent literature 5: J.Org.Chem.2016, 81, 10600-10616

[0032] Non-patent literature 6: J. Am. Chem. Soc. 2019, 141, 6484-6488

[0033] Non-patent literature 7: Angew. Chem. Int. Ed. 2021, 60, 24686-24693

[0034] Non-patent document 8: Org. Lett. 2002, 4, 4551

[0035] Non-patent literature 9: J. Am. Chem. Soc. 2018, 140, 4632

[0036] Non-patent literature 10: J.Org.Chem., 2017, 82, 3926

[0037] Non-patent literature 11: Angew. Chem. Int. Ed. 2021, 60, 19287

[0038] Non-patent document 12: Tetrahedron Lett., 40, 7479 (1999)

[0039] Non-Patent Document 13: Synlett, 2001, 590

[0040] Non-Patent Document 14: Synlett, 2001, 1693

[0041] Non-patent literature 15: Org. Biomol. Chem. 2018, 16, 4720

[0042] Non-patent document 16: J. AM. CHEM. SOC. 2005, 127, 7296 Summary of the Invention

[0043] Problems to be solved by the invention

[0044] The present invention aims to provide a novel oligosaccharide as a biantennary glycan having an α2,6-sialic acid structure at a non-reducing terminal, a method for producing the oligosaccharide, and an intermediate thereof and a method for producing the intermediate thereof.

[0045] Solutions for solving problems

[0046] The present inventors conducted intensive research to solve the above-mentioned problems and, as a result, discovered a novel oligosaccharide represented by the following formula A-22 as a biantennary glycan having an α2,6-sialic acid structure at the non-reducing end, a novel method for efficiently producing the oligosaccharide, and an intermediate thereof and a method for producing the intermediate thereof, thereby completing the present invention.

[0047] That is, the present invention relates to the following, but is not limited thereto. [1]

[0049] A method for producing the oligosaccharide represented by the following formula A-22.

[0050]

[0051] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0052] The method includes step I-1, step I-2, step I-3, step I-4, and step I-5.

[0053] (Step I-1) is a step of producing a compound represented by the following formula A-8, and includes the step of producing a compound represented by the following formula A-6 by forming an α-1,3-glycosidic bond and an α-1,6-glycosidic bond between the compound represented by the following formula A-3 and the compound represented by the following formula A-5.

[0054]

[0055]

[0056] (Step I-2) is a step of producing a compound represented by the following formula A-11, and includes the step of forming a β-1,4-glycosidic bond between the compound represented by the above formula A-8 and the compound represented by the following formula A-9 to produce a compound represented by the following formula A-10.

[0057]

[0058] (wherein, is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0059]

[0060] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0061]

[0062] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0063] (Step I-3) is a step of producing a compound represented by the following formula A-15, wherein the compound represented by the above formula A-11 and the compound represented by the following formula A-12 form a β-1,2-glycosidic bond to produce the compound represented by the following formula A-13, and then the phthaloyl group as a protecting group for the amino group and the acetyl group on the hydroxyl group on the compound represented by the above formula A-13 are removed to produce the compound represented by the following formula A-14, and then a phthaloyl group selected from an aryloxycarbonyl group (COOAr), an acetyl group (Ac), a 2,2,2-trichloroethoxycarbonyl group (Troc), and a phthaloyl group (Ph The protecting group in the th) protects the amino group in the compound represented by the formula A-14 to generate the compound represented by the following formula A-15 (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group); or, the acetyl group (Ac) on the compound represented by the following formula A-13 is removed to generate the compound represented by the following formula A-15 (wherein, R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0064]

[0065] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0066]

[0067] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0068]

[0069] (Step I-4) is a step for generating a compound represented by the following formula A-20, and includes: generating a compound represented by the following formula A-17 by forming a β-1,4-glycosidic bond between the compound represented by the following formula A-15 and the compound represented by the following formula A-16, and then generating a compound represented by the following formula A-18 by removing the amino protecting group and the acyl protecting group of the hydroxyl group in the compound represented by the following formula A-17.

[0070]

[0071] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0072]

[0073] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0074]

[0075] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0076] (Step I-5) is a step of producing the oligosaccharide represented by the above-mentioned formula A-22 by reacting the compound represented by the above-mentioned formula A-20 with the compound represented by the following formula A-21.

[0077] [2]

[0079] The method according to [1], comprising: reacting a compound represented by the following formula A-4 with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in a mixed solvent of a fluorous alcohol and water to remove the 2-naphthylmethyl group in the compound represented by the formula A-4, thereby generating the compound represented by the formula A-5.

[0080] [3]

[0082] The method according to [2], wherein the fluorine-containing alcohol is selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butanol, and combinations thereof. [4]

[0084] The method according to [2] or [3], wherein the reaction is carried out at -35°C to 70°C. [5]

[0086] The method according to [2] or [3], wherein the reaction is carried out at -30°C to -10°C. [6]

[0088] The method according to any one of [1] to [5], wherein the step I-2 includes reacting the compound represented by the formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid to generate the compound represented by the formula A-11. [7]

[0090] The method according to [6], wherein the alkyl ester of the perfluorocarboxylic acid is methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, or butyl undecanoate. [8]

[0092] The method according to [6] or [7], wherein the strong base is selected from sodium salt, lithium salt, and potassium salt of metal amide; C1-C 20 Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyllithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene (DBU), diazabicyclononene (DBN), and 1,1,3,3-tetramethylguanidine (TMG); and combinations thereof. [9]

[0094] The method according to [6] or [7], wherein the strong base is potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, or lithium hexamethyldisilazide (LHMDS).

[10]

[0096] The method according to any one of [6] to [9], wherein the step of reacting the compound represented by the formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid to produce the compound represented by the formula A-11 is performed in a single step. 10 In alcohol solvent, or in C1~C 10 The reaction is carried out in a mixed solvent of an alcohol solvent and an amide solvent, an ether solvent, an ester solvent, an aromatic solvent, a halogen solvent, a hydrocarbon solvent, or a nitrile solvent.

[11]

[0098] The method according to any one of [1] to

[10] , wherein in the step I-3, the compound represented by the formula A-15 is produced by protecting the amino group in the compound represented by the formula A-14 with an aryloxycarbonyl group (COOAr).

[12]

[0100] The method according to any one of [1] to

[11] , wherein the step of generating the compound represented by Formula A-15 from the compound represented by Formula A-14 in Step I-3 is carried out in an aqueous solution of sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate.

[13]

[0102] The method according to any one of [1] to

[12] , wherein the R 1 is isopropyl or 4-methoxyphenyl.

[14]

[0104] A method according to any one of [1] to

[13] , comprising a step for generating the compound represented by the formula A-16, wherein the step is as follows: a solution containing the compound represented by the following formula G-11 and carbonylchlorohydrotris(triphenylphosphine)ruthenium (II) is stirred, and then water and 1,3-dibromo-5,5-dimethylhydantoin, iodine, or N-bromosuccinimide are added to the solution and stirred, thereby causing the allyl group bonded to the carbon 1 position of D-galactopyranoside in the compound represented by the formula G-11 to be detached via oxygen to generate the compound represented by the following formula G-12, and further, the hydroxyl group on the carbon 1 position of D-galactopyranoside in the compound represented by the formula G-12 is converted into a 2,2,2-trifluoro-N-phenylacetimido group, thereby generating the compound represented by the formula A-16.

[0105]

[15]

[0107] A method for producing the compound represented by the following formula A-5.

[0108]

[0109] The method includes reacting a compound represented by the following formula A-4 with DDQ (2,3-dichloro-5,6-dicyano-p-benzoquinone) in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group in the compound represented by the formula A-4, thereby generating a compound represented by the formula A-5.

[0110]

[16]

[0112] The method according to

[15] , wherein the fluorine-containing alcohol is selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butanol, and combinations thereof.

[17]

[0114] The method according to

[15] or

[16] , wherein the reaction is carried out at -35°C to 70°C.

[18]

[0116] The method according to

[15] or

[16] , wherein the reaction is carried out at -30°C to -10°C.

[19]

[0118] A method for producing a compound represented by the following formula A-11.

[0119]

[0120] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0121] The method includes the step of reacting a compound represented by the following formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid.

[0122]

[0123] (R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[20]

[0125] The method according to

[19] , wherein the alkyl ester of the perfluorocarboxylic acid is methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, or butyl undecanoate. [twenty one]

[0127] The method according to

[19] or

[20] , wherein the strong base is selected from sodium salt, lithium salt, and potassium salt of metal amide; C1-C 20Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyllithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene (DBU), diazabicyclononene (DBN), and 1,1,3,3-tetramethylguanidine (TMG); and combinations thereof. [twenty two]

[0129] The method according to

[19] or

[20] , wherein the strong base is potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, or lithium hexamethyldisilazide (LHMDS). [twenty three]

[0131] The method according to any one of

[19] to

[22] , wherein the reaction is carried out in a single C1 to C 10 In alcohol solvent, or in C1~C 10 The reaction is carried out in a mixed solvent of an alcohol solvent and an amide solvent, an ether solvent, an ester solvent, an aromatic solvent, a halogen solvent, a hydrocarbon solvent, or a nitrile solvent. [twenty four]

[0133] A method for producing a compound represented by the following formula A-17.

[0134]

[0135] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0136] The method includes the steps of generating a compound represented by the following formula A-15, and then forming a β-1,4-glycosidic bond with a compound represented by the following formula A-16 to generate a compound represented by the above formula A-17.

[0137]

[0138] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0139]

[25]

[0141] The method according to

[24] , wherein R5 is an aryloxycarbonyl group (COOAr).

[26]

[0143] A method for producing a compound represented by the following formula A-18.

[0144]

[0145] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0146] The method includes removing the amino protecting group and the acyl protecting group of the hydroxyl group in the compound represented by the following formula A-17.

[0147]

[0148] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[27]

[0150] An oligosaccharide represented by the following formula A-22.

[0151]

[0152] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[28]

[0154] An oligosaccharide represented by the following formula A'-22.

[0155]

[29]

[0157] A compound represented by the following formula A-8.

[0158]

[30]

[0160] A compound represented by the following formula A-10.

[0161]

[0162] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[31]

[0164] A compound represented by the following formula A-11.

[0165]

[0166] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[32]

[0168] A compound represented by the following formula A-13.

[0169]

[0170] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[33]

[0172] A compound represented by the following A-14.

[0173]

[0174] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[34]

[0176] A compound represented by the following formula A-14-FMA.

[0177]

[0178] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[35]

[0180] A compound represented by the following formula A-15.

[0181]

[0182] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[36]

[0184] A compound represented by the following formula A-17.

[0185]

[0186] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[37]

[0188] A compound represented by the following formula A-18-FF or a salt thereof.

[0189]

[0190] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[38]

[0192] A compound represented by the following formula A-19-FF or a salt thereof.

[0193]

[0194] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[39]

[0196] A compound represented by the following formula A-20-FF or a salt thereof.

[0197]

[0198] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[40]

[0200] A compound represented by the following formula A'-20-FF or a salt thereof.

[0201]

[41]

[0203] A method for producing a sugar chain remodeled antibody or a molecule comprising the Fc region thereof, comprising: a step of obtaining a compound represented by formula A-22 using the method described in any one of [1] to

[14] ; and a step of reacting the obtained compound represented by formula A-22 with a receptor molecule to obtain the sugar chain remodeled antibody or a molecule comprising the Fc region thereof, wherein the receptor molecule is an antibody or a molecule comprising the Fc region thereof having a core GlcNAc optionally with fucose added as an N297-linked sugar chain.

[42]

[0205] The production method according to

[41] further comprises a step of reacting an azide group (N3-) with a molecule having an alkyne structure.

[43]

[0207] The production method according to

[42] , wherein the molecule having an alkyne structure is selected from chemotherapeutic agents, molecular targeted drugs, immunostimulants, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins and hormones.

[44]

[0209] A method for producing an antibody-drug conjugate, comprising the method described in any one of

[41] to

[43] .

[45]

[0211] The method according to any one of [1] to

[14] , wherein the step I-1 includes: after stopping the reaction of the compound represented by the formula A-3 and the compound represented by the formula A-5, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A-6 and impurities, so that the compound represented by the formula A-6 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A-6 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-6.

[46]

[0213] A method according to any one of [1] to

[14] and

[45] , wherein step I-2 includes: after stopping the reaction of the compound represented by the formula A-8 and the compound represented by the formula A-9, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A-10 and impurities, so that the compound represented by the formula A-10 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A-10 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-10.

[47]

[0215] A method according to any one of [1] to

[14] ,

[45] and

[46] , wherein the step I-3 includes: after stopping the reaction of the compound represented by the formula A-11 and the compound represented by the formula A-12, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A-13 and impurities, so that the compound represented by the formula A-13 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A-13 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-13.

[48]

[0217] The method according to any one of [1] to

[14] and

[45] to

[47] , wherein the step I-3 includes: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A-14 and impurities, so that the compound represented by the formula A-14 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then using an organic solvent to elute the compound represented by the formula A-14 from the hydrophobic carrier, thereby purifying the compound represented by the formula A-14.

[49]

[0219] A method according to any one of [1] to

[14] and

[45] to

[48] , wherein step I-4 includes: after stopping the reaction of the compound represented by the formula A-15 and the compound represented by the formula A-16, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A-17 and impurities, so that the compound represented by the formula A-17 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A-17 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-17.

[50]

[0221] The method according to

[45] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-6, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[51]

[0223] The method according to

[46] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-10, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[52]

[0225] The method according to

[47] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-13, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[53]

[0227] The method according to

[48] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-14, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[54]

[0229] The method according to

[49] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-17, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[55]

[0231] The method according to any one of

[45] to

[54] , wherein the hydrophobic carrier is a resin for packing in reverse phase partition chromatography.

[56]

[0233] The method according to

[55] , wherein the reverse phase partition chromatography filling resin is selected from the group consisting of poly(styrene / divinylbenzene) polymer gel resin, polystyrene-divinylbenzene resin, polyhydroxymethyl acrylate resin, styrene-vinylbenzene copolymer resin, polyvinyl alcohol resin, polystyrene resin, polymethacrylate resin, chemically bonded silica gel resin, and combinations thereof.

[57]

[0235] The method according to

[56] , wherein the chemically bonded silicone resin is selected from the group consisting of the following resins: (1) a resin obtained by reacting a silane coupling agent with silicone; (2) a resin obtained by chemically bonding a dimethyloctadecyl group, an octadecyl group, a trimethyloctadecyl group, a dimethyloctyl group, an octyl group, a butyl group, an ethyl group, a methyl group, a phenyl group, a cyanopropyl group, or an aminopropyl group to silicone; (3) a resin obtained by chemically bonding a docosyl group or a triacontyl group to silicone; and (4) a combination of (1) to (3).

[58]

[0237] The method according to

[56] , wherein the chemically bonded silica gel resin is octadecyl bonded silica gel resin (ODS resin).

[59]

[0239] The method according to any one of

[45] to

[56] , wherein the water-soluble organic solvent is a water-soluble alcohol solvent, a water-soluble nitrile solvent, a water-soluble ether solvent, a water-soluble ketone solvent, a water-soluble amide solvent, or a water-soluble sulfoxide solvent, or a mixed solvent containing at least one of the above water-soluble organic solvents.

[60]

[0241] The method according to

[59] , wherein the water-soluble nitrile solvent is acetonitrile.

[61]

[0243] The method according to any one of

[45] to

[60] , wherein the organic solvent used in the step of eluting the target substance from the hydrophobic carrier is a nitrile solvent, an ether solvent, an ester solvent, a ketone solvent, a halogen solvent, an aromatic solvent, or a mixed solvent containing at least one of the above solvent systems.

[62]

[0245] A method for purifying the compound represented by the following formula A-6.

[0246]

[0247] The method comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by formula A-6 and impurities, so that the compound represented by formula A-6 is adsorbed in the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; and then, eluting the compound represented by formula A-6 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by formula A-6.

[63]

[0249] A method for purifying the compound represented by the following formula A-10.

[0250]

[0251] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0252] The method comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by formula A-10 and impurities, so that the compound represented by formula A-10 is adsorbed on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water to remove the impurities; and then, eluting the compound represented by formula A-10 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by formula A-10.

[64]

[0254] A method for purifying the compound represented by the following formula A-13.

[0255]

[0256] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0257] The method comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A-13 and impurities, so that the compound represented by the formula A-13 is adsorbed on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; and then, eluting the compound represented by the formula A-13 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-13.

[65]

[0259] A method for purifying the compound represented by the following formula A-14.

[0260]

[0261] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0262] The method comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A-14 and impurities, so that the compound represented by the formula A-14 is adsorbed on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; and then, eluting the compound represented by the formula A-14 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-14.

[66]

[0264] A method for purifying the compound represented by the following formula A-17.

[0265]

[0266] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0267] The method comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A-17 and impurities, so that the compound represented by the formula A-17 is adsorbed on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; and then, eluting the compound represented by the formula A-17 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-17.

[67]

[0269] The method according to

[62] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-6, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[68]

[0271] The method according to

[63] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-10, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[69]

[0273] The method according to

[64] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-13, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[70]

[0275] The method according to

[65] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-14, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[71]

[0277] The method according to

[66] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-17, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[72]

[0279] The method according to any one of

[62] to

[71] , wherein the hydrophobic carrier is a resin for packing in reverse phase partition chromatography.

[73]

[0281] The method according to

[72] , wherein the reverse phase partition chromatography filling resin is selected from the group consisting of poly(styrene / divinylbenzene) polymer gel resin, polystyrene-divinylbenzene resin, polyhydroxymethyl acrylate resin, styrene-vinylbenzene copolymer resin, polyvinyl alcohol resin, polystyrene resin, polymethacrylate resin, chemically bonded silica gel resin, and combinations thereof.

[74]

[0283] The method according to

[73] , wherein the chemically bonded silicone resin is selected from the group consisting of the following resins: (1) a resin obtained by reacting a silane coupling agent with silicone; (2) a resin obtained by chemically bonding a dimethyloctadecyl group, an octadecyl group, a trimethyloctadecyl group, a dimethyloctyl group, an octyl group, a butyl group, an ethyl group, a methyl group, a phenyl group, a cyanopropyl group, or an aminopropyl group to silicone; (3) a resin obtained by chemically bonding a docosyl group or a triacontyl group to silicone; and (4) a combination of (1) to (3).

[75]

[0285] The method according to

[73] , wherein the chemically bonded silica gel resin is octadecyl bonded silica gel resin (ODS resin).

[76]

[0287] The method according to any one of

[62] to

[75] , wherein the water-soluble organic solvent is a water-soluble alcohol solvent, a water-soluble nitrile solvent, a water-soluble ether solvent, a water-soluble ketone solvent, a water-soluble amide solvent, a water-soluble sulfoxide solvent, or a mixed solvent containing at least one of the above water-soluble organic solvents.

[77]

[0289] The method according to

[76] , wherein the water-soluble nitrile solvent is acetonitrile.

[78]

[0291] The method according to any one of

[62] to

[77] , wherein the organic solvent used in the step of eluting the target substance from the hydrophobic carrier is a nitrile solvent, an ether solvent, an ester solvent, a ketone solvent, a halogen solvent, an aromatic solvent, or a mixed solvent containing at least one of the above solvent systems.

[0292] Furthermore, in one aspect, the present invention relates to the following, but is not limited thereto. [1]

[0294] A method for producing the oligosaccharide represented by the following formula A'-22.

[0295]

[0296] The method includes step I-1, step I-2, step I-3, step I-4, and step I-5.

[0297] (Step I-1) is a step of producing a compound represented by the following formula A-8, and includes the step of producing a compound represented by the following formula A-6 by forming an α-1,3-glycosidic bond and an α-1,6-glycosidic bond between the compound represented by the following formula A-3 and the compound represented by the following formula A-5.

[0298]

[0299] (Step I-2) is a step of producing a compound represented by the following formula A'-11, and includes the step of forming a β-1,4-glycosidic bond between the compound represented by the above formula A-8 and the compound represented by the following formula A'-9 to produce a compound represented by the following formula A'-10.

[0300]

[0301] (Step I-3) is a step of producing a compound represented by the following formula A'-15, wherein the compound represented by the above formula A'-11 and the compound represented by the following formula A-12 form a β-1,2-glycosidic bond to produce the compound represented by the following formula A'-13, and then the phthaloyl group as a protecting group for the amino group and the acetyl group on the hydroxyl group on the compound represented by the above formula A'-13 are removed to produce the compound represented by the following formula A'-14, and then a phthaloyl group selected from an aryloxycarbonyl group (COOAr), an acetyl group (Ac), a 2,2,2-trichloroethoxycarbonyl group (Troc), and a phthaloyl group (P hth) protects the amino group in the compound represented by the formula A'-14 to generate the compound represented by the following formula A'-15 (wherein R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group); or, by removing the acetyl group (Ac) on the compound represented by the following formula A'-13 to generate the compound represented by the following formula A'-15 (wherein, R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0302]

[0303]

[0304] (Step I-4) is a step of producing a compound represented by the following formula A'-20, and comprises: forming a β-1,4-glycosidic bond between the compound represented by the above formula A'-15 and the compound represented by the following formula A-16 to produce a compound represented by the following formula A'-17 (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group), and then removing the amino protecting group and the acyl protecting group of the hydroxyl group in the compound represented by the above formula A'-17 to produce a compound represented by the following formula A'-18 (wherein, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0305]

[0306]

[0307] (Step I-5) is a step of producing the oligosaccharide represented by the above formula A'-22 by reacting the compound represented by the above formula A'-20 with the compound represented by the following formula A-21.

[0308] [2]

[0310] The method according to [1], comprising: reacting a compound represented by the following formula A-4 with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group in the compound represented by the formula A-4, thereby generating the compound represented by the formula A-5.

[0311] [3]

[0313] The method according to [2], wherein the fluorine-containing alcohol is selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butanol, and combinations thereof. [4]

[0315] The method according to [2] or [3], wherein the reaction is carried out at -35°C to 70°C. [5]

[0317] The method according to [2] or [3], wherein the reaction is carried out at -30°C to -10°C. [6]

[0319] The method according to any one of [1] to [5], wherein the step I-1 includes: after stopping the reaction of the compound represented by the formula A-3 and the compound represented by the formula A-5, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A-6 and impurities, so that the compound represented by the formula A-6 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A-6 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A-6. [7]

[0321] The method according to any one of [1] to [6], wherein the step I-2 includes: after stopping the reaction of the compound represented by the formula A-8 and the compound represented by the formula A'-9, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A'-10 and impurities, so that the compound represented by the formula A'-10 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A'-10 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A'-10. [8]

[0323] The method according to any one of [1] to [7], wherein the step I-3 includes: after stopping the reaction of the compound represented by the formula A'-11 and the compound represented by the formula A-12, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A'-13 and impurities, so that the compound represented by the formula A'-13 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A'-13 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A'-13. [9]

[0325] The method according to any one of [1] to [8], wherein the step I-3 includes: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A'-14 and impurities, so that the compound represented by the formula A'-14 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A'-14 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A'-14.

[10]

[0327] The method according to any one of [1] to [9], wherein the step I-4 includes: after stopping the reaction of the compound represented by the formula A'-15 and the compound represented by the formula A-16, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by the formula A'-17 and impurities, so that the compound represented by the formula A'-17 is adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities, and then eluting the compound represented by the formula A'-17 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the formula A'-17.

[11]

[0329] The method according to [6], wherein the inclusions contain sugar compounds other than the compound represented by Formula A-6, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[12]

[0331] The method according to [7], wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-10, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[13]

[0333] The method according to [8], wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-13, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[14]

[0335] The method according to [9], wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-14, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[15]

[0337] The method according to

[10] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-17, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[16]

[0339] The method according to any one of [6] to

[15] , wherein the hydrophobic carrier is a packing resin for reverse phase partition chromatography.

[17]

[0341] The method according to

[16] , wherein the reverse phase partition chromatography filling resin is selected from the group consisting of poly(styrene / divinylbenzene) polymer gel resin, polystyrene-divinylbenzene resin, polyhydroxymethyl acrylate resin, styrene-vinylbenzene copolymer resin, polyvinyl alcohol resin, polystyrene resin, polymethacrylate resin, chemically bonded silica gel resin, and combinations thereof.

[18]

[0343] The method according to

[17] , wherein the chemically bonded silicone resin is selected from the group consisting of the following resins: (1) a resin obtained by reacting a silane coupling agent with silicone; (2) a resin obtained by chemically bonding a dimethyloctadecyl group, an octadecyl group, a trimethyloctadecyl group, a dimethyloctyl group, an octyl group, a butyl group, an ethyl group, a methyl group, a phenyl group, a cyanopropyl group, or an aminopropyl group to silicone; (3) a resin obtained by chemically bonding a docosyl group or a triacontyl group to silicone; and (4) a combination of (1) to (3).

[19]

[0345] The method according to

[17] , wherein the chemically bonded silica gel resin is octadecyl bonded silica gel resin (ODS resin).

[20]

[0347] The method according to any one of [6] to

[19] , wherein the water-soluble organic solvent is a water-soluble alcohol solvent, a water-soluble nitrile solvent, a water-soluble ether solvent, a water-soluble ketone solvent, a water-soluble amide solvent, or a water-soluble sulfoxide solvent, or a mixed solvent containing at least one of the above water-soluble organic solvents. [twenty one]

[0349] The method according to

[20] , wherein the water-soluble nitrile solvent is acetonitrile. [twenty two]

[0351] The method according to any one of [6] to

[21] , wherein the organic solvent used in the step of eluting the target substance from the hydrophobic carrier is a nitrile solvent, an ether solvent, an ester solvent, a ketone solvent, a halogen solvent, an aromatic solvent, or a mixed solvent containing at least one of the above solvent systems. [twenty three]

[0353] The method according to any one of [1] to

[22] , wherein the step I-2 includes reacting the compound represented by the formula A'-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid to generate the compound represented by the formula A'-11. [twenty four]

[0355] The method according to

[23] , wherein the alkyl ester of the perfluorocarboxylic acid is methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, or butyl undecanoate.

[25]

[0357] The method according to

[23] or

[24] , wherein the strong base is selected from sodium salt, lithium salt, and potassium salt of metal amide; C1-C 20 Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyllithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene (DBU), diazabicyclononene (DBN), and 1,1,3,3-tetramethylguanidine (TMG); and combinations thereof.

[26]

[0359] The method according to

[23] or

[24] , wherein the strong base is potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, or lithium hexamethyldisilazide (LHMDS).

[27]

[0361] The method according to any one of

[23] to

[26] , wherein the step of generating the compound represented by the formula A'-11 by reacting the compound represented by the formula A'-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid is carried out in a single step of C1 to C 10 In alcohol solvent, or in C1~C 10 The reaction is carried out in a mixed solvent of an alcohol solvent and an amide solvent, an ether solvent, an ester solvent, an aromatic solvent, a halogen solvent, a hydrocarbon solvent, or a nitrile solvent.

[28]

[0363] The method according to any one of [1] to

[27] , wherein in the step I-3, the compound represented by the formula A'-15 is produced by protecting the amino group in the compound represented by the formula A'-14 with an aryloxycarbonyl group (COOAr).

[29]

[0365] The method according to any one of [1] to

[28] , wherein the step of generating the compound represented by the formula A'-15 from the compound represented by the formula A'-14 in the step I-3 is carried out in an aqueous solution of sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate.

[30]

[0367] A method for producing the compound represented by the following formula A-5.

[0368]

[0369] The method comprises: reacting a compound represented by the following formula A-4 with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group in the compound represented by the formula A-4, thereby generating a compound represented by the formula A-5.

[0370]

[31]

[0372] The method according to

[30] , wherein the fluorine-containing alcohol is selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butanol, and combinations thereof.

[32]

[0374] The method according to

[30] or

[31] , wherein the reaction is carried out at -35°C to 70°C.

[33]

[0376] The method according to

[30] or

[31] , wherein the reaction is carried out at -30°C to -10°C.

[34]

[0378] A method for purifying the compound represented by the following formula A-6.

[0379]

[0380] The method comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by formula A-6 and impurities, so that the compound represented by formula A-6 is adsorbed in the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; and then, eluting the compound represented by formula A-6 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by formula A-6.

[35]

[0382] A method for purifying the compound represented by the following formula A'-10.

[0383]

[0384] The method includes: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A'-10 and impurities, so that the compound represented by the formula A'-10 is adsorbed on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; then, using an organic solvent to elute the compound represented by the formula A'-10 from the hydrophobic carrier, thereby purifying the compound represented by the formula A'-10.

[36]

[0386] A method for purifying the compound represented by the following formula A'-13.

[0387]

[0388] The method includes: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A'-13 and impurities, so that the compound represented by the formula A'-13 is adsorbed in the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; then, using an organic solvent to elute the compound represented by the formula A'-13 from the hydrophobic carrier, thereby purifying the compound represented by the formula A'-13.

[37]

[0390] A method for purifying the compound represented by the following formula A'-14.

[0391]

[0392] The method includes: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A'-14 and impurities, so that the compound represented by the formula A'-14 is adsorbed in the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water to remove the impurities; then, using an organic solvent to elute the compound represented by the formula A'-14 from the hydrophobic carrier, thereby purifying the compound represented by the formula A'-14.

[38]

[0394] A method for purifying the compound represented by the following formula A'-17.

[0395]

[0396] (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0397] The method includes: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the formula A'-17 and impurities, so that the compound represented by the formula A'-17 is adsorbed in the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and the water, thereby removing the impurities; then, using an organic solvent to elute the compound represented by the formula A'-17 from the hydrophobic carrier, thereby purifying the compound represented by the formula A'-17.

[39]

[0399] The method according to

[34] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A-6, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[40]

[0401] The method according to

[35] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-10, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[41]

[0403] The method according to

[36] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-13, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[42]

[0405] The method according to

[37] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-14, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[43]

[0407] The method according to

[38] , wherein the inclusions contain sugar compounds other than the compound represented by the formula A'-17, and / or compounds derived from a reaction reagent for obtaining the compound to be purified.

[44]

[0409] The method according to any one of

[34] to

[43] , wherein the hydrophobic carrier is a resin for packing in reverse phase partition chromatography.

[45]

[0411] The method according to

[44] , wherein the reverse phase partition chromatography filling resin is selected from the group consisting of poly(styrene / divinylbenzene) polymer gel resin, polystyrene-divinylbenzene resin, polyhydroxymethyl acrylate resin, styrene-vinylbenzene copolymer resin, polyvinyl alcohol resin, polystyrene resin, polymethacrylate resin, chemically bonded silica gel resin, and combinations thereof.

[46]

[0413] The method according to

[45] , wherein the chemically bonded silicone resin is selected from the group consisting of the following resins: (1) a resin obtained by reacting a silane coupling agent with silicone; (2) a resin obtained by chemically bonding a dimethyloctadecyl group, an octadecyl group, a trimethyloctadecyl group, a dimethyloctyl group, an octyl group, a butyl group, an ethyl group, a methyl group, a phenyl group, a cyanopropyl group, or an aminopropyl group to silicone; (3) a resin obtained by chemically bonding a docosyl group or a triacontyl group to silicone; and (4) a combination of (1) to (3).

[47]

[0415] The method according to

[45] , wherein the chemically bonded silica gel resin is octadecyl bonded silica gel resin (ODS resin).

[48]

[0417] The method according to any one of

[34] to

[47] , wherein the water-soluble organic solvent is a water-soluble alcohol solvent, a water-soluble nitrile solvent, a water-soluble ether solvent, a water-soluble ketone solvent, a water-soluble amide solvent, a water-soluble sulfoxide solvent, or a mixed solvent containing at least one of the above water-soluble organic solvents.

[49]

[0419] The method according to

[48] , wherein the water-soluble nitrile solvent is acetonitrile.

[50]

[0421] The method according to any one of

[34] to

[49] , wherein the organic solvent used in the step of eluting the target substance from the hydrophobic carrier is a nitrile solvent, an ether solvent, an ester solvent, a ketone solvent, a halogen solvent, an aromatic solvent, or a mixed solvent containing at least one of the above solvent systems.

[51]

[0423] A method for producing a compound represented by the following formula A'-11.

[0424]

[0425] The method includes the step of reacting a compound represented by the following formula A'-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid.

[0426]

[52]

[0428] The method according to

[51] , wherein the alkyl ester of the perfluorocarboxylic acid is methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, or butyl undecanoate.

[53]

[0430] The method according to

[51] or

[52] , wherein the strong base is selected from sodium salt, lithium salt, and potassium salt of metal amide; C1-C 20 Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyllithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene (DBU), diazabicyclononene (DBN), and 1,1,3,3-tetramethylguanidine (TMG); and combinations thereof.

[54]

[0432] The method according to

[51] or

[52] , wherein the strong base is potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, or lithium hexamethyldisilazide (LHMDS).

[55]

[0434] The method according to any one of

[51] to

[54] , wherein the reaction is carried out in a single C1 to C 10 In alcohol solvent, or in C1~C 10 The reaction is carried out in a mixed solvent of an alcohol solvent and an amide solvent, an ether solvent, an ester solvent, an aromatic solvent, a halogen solvent, a hydrocarbon solvent, or a nitrile solvent.

[56]

[0436] A method for producing a compound represented by the following formula A'-17.

[0437]

[0438] (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0439] The method includes the steps of generating a compound represented by the following formula A'-15, and then forming a β-1,4-glycosidic bond with a compound represented by the following formula A-16 to generate a compound represented by the formula A'-17.

[0440]

[0441] (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0442]

[57]

[0444] The method according to

[56] , wherein R5 is an aryloxycarbonyl group (COOAr).

[58]

[0446] A method for producing a compound represented by the following formula A'-18.

[0447]

[0448] (Where M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation. The method comprises: removing the amino protecting group and the acyl protecting group of the hydroxyl group in the compound represented by the following formula A'-17.

[0449]

[0450] (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[59]

[0452] An oligosaccharide represented by the following formula A'-22.

[0453]

[60]

[0455] A compound represented by the following formula A'-8.

[0456]

[61]

[0458] A compound represented by the following formula A'-10.

[0459]

[62]

[0461] A compound represented by the following formula A'-11.

[0462]

[63]

[0464] A compound represented by the following formula A'-13.

[0465]

[64]

[0467] A compound represented by the following A'-14.

[0468]

[65]

[0470] A compound represented by the following formula A'-15.

[0471]

[0472] (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[66]

[0474] A compound represented by the following formula A'-17.

[0475]

[0476] (wherein, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[67]

[0478] A compound represented by the following formula A'-18.

[0479]

[0480] (Where M +is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[68]

[0482] A compound represented by the following formula A'-19.

[0483]

[0484] (Where M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[69]

[0486] A compound represented by the following formula A'-20.

[0487]

[0488] (Where M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0489] Effects of the Invention

[0490] According to the present invention, there are provided an oligosaccharide represented by the above formula A-22 and a novel method for producing the same, as well as a production intermediate of the oligosaccharide and a method for producing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0491] Figure 1A This is a diagram partially and schematically illustrating an example of the novel method for producing the oligosaccharide represented by formula A-22 provided by the present invention. More specifically, it schematically illustrates a method for producing compounds represented by formulas A-2 to A-14 using the compound represented by formula A-1 as a starting material.

[0492] Figure 1B This is a partial schematic illustration of an example of the novel method for producing the oligosaccharide represented by formula A-22 provided by the present invention. More specifically, it schematically illustrates methods for producing compounds represented by formulas A-15 to A-22 using the compound represented by formula A-14 as a starting material. DETAILED DESCRIPTION

[0493] Hereinafter, preferred embodiments for carrying out the present invention will be described. It should be noted that the embodiments described below are examples of representative embodiments of the present invention and are not intended to narrowly interpret the scope of the present invention.

[0494] <1. Method for producing oligosaccharide represented by formula A-22>

[0495] In one embodiment of the present invention, a novel oligosaccharide represented by formula A-22 and a novel method for producing the same are provided.

[0496] In the present invention, the oligosaccharide represented by formula A-22 refers to the following oligosaccharide.

[0497]

[0498] The novel synthesis route of the oligosaccharide represented by the above formula A-22 includes the following steps I-1 to I-5.

[0499] Throughout this specification, the substituent "R1" in oligosaccharides and compounds is a C1-C6 alkyl group or a phenyl group, which may be substituted with 1 to 3 (i.e., 1, 2, or 3) C1-C6 alkoxy groups. The phenyl group may be unsubstituted.

[0500] As used in this specification, the term "C x ~C y "Alkyl" refers to a straight-chain, branched, or cyclic saturated aliphatic hydrocarbon group containing x to y carbon atoms. Examples of straight-chain saturated aliphatic hydrocarbon groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Examples of branched saturated aliphatic hydrocarbon groups include isopropyl, isobutyl, tert-butyl, sec-butyl, isopentyl, tert-pentyl, sec-pentyl, isohexyl, tert-hexyl, and sec-hexyl. In addition, as described above, "alkyl" also includes cyclic saturated aliphatic hydrocarbon groups, i.e., "cycloalkyl", which may include a condensed or cross-linked ring system. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0501] As used herein, the term "alkoxy" refers to an alkyl group bonded via an oxygen group (i.e., alkyl-O-), and the alkyl group is as defined above. Examples of alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[0502] In one embodiment of the present invention, the substituent "R1" in the oligosaccharide and the compound is preferably n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, tert-butyl, isopentyl, tert-pentyl, sec-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl, 3,4-dimethoxyphenyl, 2,4-dimethoxyphenyl, 2,3-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, or phenyl, more preferably isopropyl, isobutyl, tert-butyl, 4-methoxyphenyl, 3-methoxyphenyl, or 2-methoxyphenyl, and even more preferably isopropyl or 4-methoxyphenyl.

[0503] Throughout this specification, the present invention, in one embodiment, also encompasses "salts" of the oligosaccharide represented by Formula A-22 and its novel production intermediates (e.g., compounds represented by Formulas A-2 to A-20). For example, salts can be formed at carboxyl groups, amino groups, and the like in the oligosaccharide represented by Formula A-22 and the compounds used to produce the oligosaccharide. Examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, and phosphoric acid; or organic acids such as acetic acid, propionic acid, trimethylacetic acid, tert-butylacetic acid, hexanoic acid, succinic acid, malic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, methanesulfonic acid, and ethanesulfonic acid. Furthermore, salts can also be formed when the acidic protons present react with inorganic or organic bases. Inorganic bases include, but are not limited to, sodium hydroxide, potassium hydroxide, sodium carbonate, calcium hydroxide, and lithium hydroxide, and organic bases include, but are not limited to, ethanolamine, diethanolamine, triethanolamine, and triethylamine. In one embodiment, a "salt" is preferably a pharmaceutically acceptable salt.

[0504] <Process I-1>

[0505] Step I-1 is a step for producing the compound represented by the following formula A-8, and includes the step of forming an α-1,3-glycosidic bond and an α-1,6-glycosidic bond between the compound represented by the following formula A-3 and the compound represented by the following formula A-5 to produce the compound represented by the following formula A-6. Step I-1 includes the following steps I-1-1 to I-1-3.

[0506]

[0507]

[0508] <Process I-1-1>

[0509] Step I-1-1 is a step for producing a compound represented by Formula A-6 by forming an α-1,3-glycosidic bond and an α-1,6-glycosidic bond between the compound represented by Formula A-3 and the compound represented by Formula A-5. This step can be performed using or applying known methods, but is preferably performed using the method described in Examples 32 or 55. For example, molecular sieve 4A powder and trimethylsilyl trifluoromethanesulfonate (TMSOTf) can be added sequentially to an organic solvent (such as toluene) to form an α-1,3-glycosidic bond and an α-1,6-glycosidic bond between the compound represented by Formula A-3 and the compound represented by Formula A-5, thereby producing the compound represented by Formula A-6. Alternatively, the compound represented by Formula A-3 and the compound represented by Formula A-5, which serve as starting materials, can be produced as follows.

[0510] <Production of the compound represented by formula A-3>

[0511] In one embodiment of the present invention, the compound represented by formula A-3 can be produced by the following steps, but is not limited to this production method.

[0512] First, water and p-TsOH·H 2 O are added to the compound represented by the following formula A-1 (3,4,6-tri-O-benzyl-1,2-O-(1-methoxyethylidene)-β-D-mannopyranose), for example, and then the compound is reacted with triethylamine to produce the compound represented by the following formula A-2.

[0513]

[0514] This step can be preferably performed by the method shown in Example 29 or 52, for example.

[0515] Next, the compound represented by Formula A-3 is produced by adding, for example, trichloroacetonitrile and diazabicycloundecene (DBU) to the compound represented by Formula A-2. This step can be preferably performed by the method described in Example 30 or 53, for example.

[0516] <Production of the compound represented by formula A-5>

[0517] Regarding the compound represented by formula A-5, first, the compound represented by the following formula A-4 is produced, and then the compound represented by formula A-5 is produced from this compound.

[0518] In one embodiment of the present invention, the compound represented by formula A-4 is produced by the following steps X-1 to X-14, or the following steps X-1 to X-8 + X-15 to X-16. The details of each step are exemplified below, and each step can be carried out using or applying a conventional method for producing monosaccharides or oligosaccharides.

[0519]

[0520] <Process X-1>

[0521] Step X-1 is a step of producing a compound represented by the following formula C-2 by protecting the hydroxyl group bonded to the 3-carbon of the compound represented by the following formula C-1 with a 2-naphthylmethyl group (NAP).

[0522]

[0523] The compound represented by Formula C-1, which serves as the starting material for this step, can be produced by known methods or a commercially available product. Commercially available products of the compound represented by Formula C-1 include, for example, 1,2:5,6-di-O-isopropylidene-α-D-furanose produced by Sigma-Aldrich. This step can be carried out using or applying known methods, preferably, the method described in Example 1.

[0524] <Process X-2>

[0525] Step X-2 is a step of producing a compound represented by the following formula C-3 by subjecting the compound represented by formula C-2 to acid hydrolysis of two isopropylidene groups and formation of a pyranose ring.

[0526]

[0527] This step can be performed by utilizing or applying a known method, and preferably, for example, can be performed by the method described in Example 2.

[0528] <Process X-3>

[0529] Step X-3 is a step of producing a compound represented by the following formula C-4 by protecting the hydroxyl group on the compound represented by formula C-3 with an acetyl group.

[0530]

[0531] This step can be performed by utilizing or applying a known method, and preferably, for example, can be performed by the method described in Example 3.

[0532] <Process X-4>

[0533] Step X-4 is a step of producing a compound represented by the following formula C-5 by selectively removing only the acetyl group in the acetyloxy group bonded to the carbon 1 position of the compound represented by formula C-4.

[0534]

[0535] This step can be performed by utilizing or applying a known method, and preferably, for example, can be performed by the method described in Example 3.

[0536] The above-mentioned step of producing the compound represented by Formula C-5 from the compound represented by Formula C-3 can be carried out by a one-pot method as shown in Example 3, for example.

[0537] <Process X-5>

[0538] Step X-5 is a step of reacting the compound represented by Formula C-5 with trichloroacetonitrile to produce a compound represented by the following Formula C-6.

[0539]

[0540] This step can be performed by utilizing or applying a known method, and preferably, for example, can be performed by the method described in Example 4.

[0541] <Process X-6>

[0542] Step X-6 is a step of producing a compound represented by the following formula C-8 by reacting the compound represented by the following formula C-6 with the compound represented by the following formula C-7.

[0543]

[0544] The compound represented by Formula C-7 can be produced by known methods, or a commercially available product can be used. Commercially available products of the compound represented by Formula C-7 include, for example, 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-pyranoside manufactured by Tokyo Chemical Industry Co., Ltd. This step can be carried out by utilizing or applying known methods, preferably, by the method described in Example 5.

[0545] <Process X-7>

[0546] Step X-7 is a step of producing a compound represented by the following formula C-9 by removing an acetyl group from the compound represented by formula C-8.

[0547]

[0548] This step can be performed by utilizing or applying a known method, and preferably, for example, can be performed by the method described in Example 6.

[0549] In one embodiment of the present invention, step X-7 is a step in which the acetyl group of the compound represented by formula C-8 is removed by reacting it with a strong base in the presence of trifluoroacetate, thereby producing the compound represented by formula C-9. The use of sodium methoxide in methanol for this acetyl group elimination reaction has been reported (Org. Biomol. Chem., 2018, 16, 4720-4727), but this may also cause an undesirable side reaction, such as the ring-opening of the phthalimide group. On the other hand, by reacting the compound with a strong alkoxide base in the presence of an alkyl ester of a perfluorocarboxylic acid, the acetyl group can be removed while suppressing the ring-opening of the phthalimide group.

[0550] The "alkyl ester of perfluorocarboxylic acid" used in the above step is not limited as long as the reaction proceeds, and includes methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, and butyl undecanoate, and methyl trifluoroacetate is preferably used.

[0551] The "strong base" mentioned above is not limited as long as it allows the reaction to proceed, and can be selected from sodium salts, lithium salts, and potassium salts of metal amides; C1-C 20 Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyllithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene (DBU), diazabicyclononene (DBN), and 1,1,3,3-tetramethylguanidine (TMG); and combinations thereof, for example, as C1-C 20 Examples of the sodium, lithium, and potassium salts of alkoxides include lithium methoxide, sodium methoxide, potassium methoxide, lithium ethoxide, sodium ethoxide, potassium ethoxide, lithium isopropoxide, sodium isopropoxide, potassium isopropoxide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-amylate, sodium tert-amylate, and potassium tert-amylate. Particularly preferred examples include sodium tert-butoxide, lithium tert-butoxide, potassium tert-butoxide, and lithium hexamethyldisilazide (LHMDS).

[0552] The solvent in this step is not limited as long as it allows the reaction to proceed. For example, a single C1 to C 10 Alcohol solvent, or use C1~C 10 Mixed solvents of alcohol solvents and amide solvents (dimethylformamide, dimethylacetamide, etc.), ether solvents (tetrahydrofuran, dimethoxyethane, cyclopentyl methyl ether, etc.), ester solvents (ethyl acetate, etc.), aromatic solvents (toluene, etc.), halogen solvents (dichloromethane, etc.), hydrocarbon solvents (hexane, etc.) or nitrile solvents (acetonitrile, etc.), preferably methanol, or a mixed solvent system of methanol and tetrahydrofuran can be used, but it is not limited thereto. It should be noted that the above-mentioned "C1 to C 10 The "alcohol solvent" may be replaced by an alcohol having a larger carbon number. On the other hand, from the perspective of availability and convenience, C1 to C5 alcohols (methanol, ethanol, propanol, butanol, etc.) can be preferably used.

[0553] The reaction temperature in this step is not limited as long as the reaction proceeds, and is, for example, -20°C to 80°C, preferably 0°C to 70°C, more preferably 20°C to 65°C, and particularly preferably 40°C to 60°C.

[0554] <Process X-8>

[0555] Step X-8 is a step of producing a compound represented by the following formula C-10 by selectively protecting the hydroxyl groups bonded to the 4- and 6-carbons of D-glucopyranoside in the compound represented by formula C-9 with benzaldehyde dimethyl acetal.

[0556]

[0557] This step can be performed by utilizing or applying a known method, and preferably, for example, by the method described in Example 7.

[0558] <Process X-9>

[0559] Step X-9 is a step of reacting the compound represented by formula C-10 with a compound that provides a leaving group selected from the group consisting of trifluoromethanesulfonyloxy, nonafluorobutanesulfonyloxy, 2-nitrobenzenesulfonyloxy, and 4-nitrobenzenesulfonyloxy to produce the compound represented by the following formula C-11.

[0560]

[0561] (wherein X1 represents a substituent selected from the group consisting of trifluoromethanesulfonyl, nonafluorobutanesulfonyl, 2-nitrobenzenesulfonyl, and 4-nitrobenzenesulfonyl.) This step can be performed by utilizing or applying a known leaving group-imparting method, preferably, for example, the method described in Example 8.

[0562] In this step, examples of the “compound that imparts a leaving group selected from the group consisting of a trifluoromethanesulfonyloxy group, a nonafluorobutanesulfonyloxy group, a 2-nitrobenzenesulfonyloxy group, and a 4-nitrobenzenesulfonyloxy group” include trifluoromethanesulfonic anhydride, nonafluoro-1-butanesulfonyl fluoride, bis(nonafluoro-1-butanesulfonic acid) anhydride, 2-nitrobenzenesulfonyl chloride, and 4-nitrobenzenesulfonyl chloride, with trifluoromethanesulfonic anhydride being preferred.

[0563] The solvent in this step is not limited as long as it allows the reaction to proceed, and examples thereof include ethyl acetate, toluene, dichloromethane, acetonitrile, cyclopentyl methyl ether, and tert-butyl methyl ether. Preferred examples include ethyl acetate, toluene, and dichloromethane.

[0564] The reaction temperature in this step is not limited as long as the reaction proceeds, and is, for example, -40°C to 60°C, preferably -30°C to 40°C, and more preferably -20°C to 10°C.

[0565] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as it allows the reaction to proceed. Examples thereof include 1-methylimidazole, pyridine, 4-dimethylaminopyridine, picoline, lutidine, and collidine. 1-methylimidazole is preferred.

[0566] <Process X-10>

[0567] Step X-10 is a step of reacting a compound represented by formula C-11 with cesium acetate or tetrabutylammonium acetate to produce a compound represented by formula C-12 (wherein X2 is an acetyl group); or reacting a compound represented by formula C-11 with tetrabutylammonium benzoate to produce a compound represented by formula C-12 (wherein X2 is a benzoyl group).

[0568]

[0569] The conversion of glucose to mannose is a well-known reaction, but there have been no reports on the conversion of glucose-glucosamine disaccharides linked by a β-glycosidic bond, where the protecting group for the hydroxyl group bonded to the 3-carbon of the D-glucopyranoside is 2-naphthylmethyl (Nap). This method enables the conversion of glucose to mannose, allowing the construction of a mannose-glucosamine disaccharide backbone linked by a β-glycosidic bond to be achieved with high yield and selectivity.

[0570] The solvent in this step is not limited as long as it allows the reaction to proceed, and examples thereof include dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, N,N-dimethylimidazolidinone, sulfolane, tetrahydrofuran, and acetonitrile. Dimethyl sulfoxide is preferred.

[0571] The reaction temperature in this step is not limited as long as the reaction proceeds, and is, for example, 20°C to 80°C, preferably 23°C to 70°C, more preferably 26°C to 60°C, and particularly preferably 30°C to 50°C.

[0572] <Process X-11>

[0573] Step X-11 is a step of producing a compound represented by the following formula C-13 by eliminating the X2 group in the compound represented by formula C-12 and ring-opening the phthalimide group.

[0574]

[0575] This step can be performed by utilizing or applying a known hydrolysis method, and preferably, for example, by the method shown in Example 9.

[0576] In this step, the compound represented by Formula C-13 can be used directly in the next step after dissolving in a solvent, or it can be isolated and purified by recrystallization. A major advantage of the compound represented by Formula C-13 is that it can be isolated and purified by crystallization. This allows for the near-complete removal of impurities with similar structures that are difficult to remove during column purification. In this case, the compound represented by Formula C-13 can be obtained with an HPLC purity of 99% or higher.

[0577] Isolation and purification by recrystallization in this step include, for example, a method of completely removing the solvent from the solvent-dissolved state by drying under reduced pressure, or a method of using tetrahydrofuran as a good solvent and adding isopropyl alcohol dropwise as a poor solvent in the presence of a small amount of water.

[0578] Recrystallization in this step can also be performed using seed crystals of the compound represented by Formula C-13. When using seed crystals, crystallization can be performed, for example, by using tetrahydrofuran as a good solvent and, in the presence of a trace amount of water, adding dropwise a portion of isopropyl alcohol as a poor solvent. After adding the seed crystals, confirming the precipitation of crystals, the remaining isopropyl alcohol is then added dropwise.

[0579] The above-mentioned step of producing the compound represented by Formula C-13 from the compound represented by Formula C-11 can be carried out by a one-pot method as shown in Example 9, for example.

[0580] <Process X-12>

[0581] Step X-12 is a step of producing a compound represented by the following formula C-14 by ring-closing the ring-opened phthalimide group in the compound represented by formula C-13 by dehydration condensation.

[0582]

[0583] This step can be performed by utilizing or applying a known method, and preferably, for example, by the method described in Example 10.

[0584] <Process X-13>

[0585] Step X-13 is a step of producing a compound represented by the following formula C-15 by protecting the hydroxyl group bonded to the carbon 2 of D-mannopyranoside in the compound represented by formula C-14 with a benzyl group.

[0586]

[0587] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method described in Example 11.

[0588] In one embodiment of the present invention, Step X-13 comprises the step of protecting the hydroxyl group bonded to the carbon-2 position of the D-mannopyranoside in the compound represented by Formula C-14 with a benzyl group in the presence of lithium tert-butoxide or lithium tert-amylate to produce the compound represented by Formula C-15. Carrying out Step X-15 in the presence of lithium tert-butoxide or lithium tert-amylate suppresses the ring-opening of the phthalimide. Furthermore, compared to conventional conditions using sodium hydride, the process can be performed safely and is easier to scale up.

[0589] The solvent in this step is not limited as long as the reaction proceeds, and examples thereof include dimethylacetamide, dimethylformamide, N-methylpyrrolidone, and N,N-dimethylimidazolidinone. Preferably, dimethylacetamide is used.

[0590] The reaction temperature in this step is not limited as long as the reaction proceeds, and is, for example, -20°C to 100°C, preferably -15°C to 70°C, and particularly preferably -10°C to 50°C.

[0591] <Process X-14>

[0592] Step X-14 is a step of selectively reducing the benzylidene protecting group in the compound represented by formula C-15 (for more details, refer to Angew. Chem. Int. Ed. 2005, 44, 1665-1668) to produce a compound represented by the following formula A-4 in which only the hydroxyl group bonded to the carbon 6 of D-mannopyranoside is deprotected.

[0593]

[0594] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method described in Example 12.

[0595] In this step, the compound represented by formula A-4 produced may be used in the next step as it is in the solvent, or may be isolated and purified by column purification or the like.

[0596] In one embodiment of the present invention, steps X-15 and X-16 shown below are included for performing the stereoreversal of glucose to mannose by redox reaction, instead of steps X-16 for performing the stereoreversal of glucose to mannose by S N The second reaction is steps X-9 to X-12, which are steps of performing the stereoreversal of glucose to mannose.

[0597]

[0598] <Process X-15>

[0599] Step X-15 is a step of producing a compound represented by the following formula C-16 by oxidizing the 2-position of D-glucopyranoside in the compound represented by formula C-10.

[0600]

[0601] This step can be performed by utilizing or applying a known method.

[0602] <Process X-16>

[0603] Step X-16 is a step of producing a compound represented by the following formula C-14 by reducing the keto group bonded to the 2-carbon of 2-keto-D-glucopyranoside in the compound represented by formula C-16.

[0604]

[0605] This step can be performed by utilizing or applying a known method.

[0606] The solvent in this step is not limited as long as the reaction proceeds, and examples thereof include diethyl ether, cyclopentyl methyl ether, tert-butyl methyl ether, diisopropyl ether, dipropyl ether, dibutyl ether, and 1,4-dioxane, with tetrahydrofuran being preferred.

[0607] The reaction temperature of this step is not limited as long as the reaction proceeds, and examples thereof include -80° C. to 20° C. As described below, the optimal reaction temperature varies depending on the reducing agent used.

[0608] In one embodiment of the present invention, the oxo group bonded to the carbon 2-position of 2-keto-D-glucopyranoside in the compound represented by formula C-16 is reduced in the presence of a reducing agent selected from the group consisting of lithium tri-sec-butylborohydride, lithium tripentylborohydride, lithium diisobutyl-tert-butoxyaluminum hydride (LDBBA), the compound represented by the following formula W, and combinations thereof.

[0609]

[0610] (wherein, R3 is a di-tert-butylmethylphenoxylate or hydride represented by the following formula, wherein at least two R3 are di-tert-butylmethylphenoxylate.

[0611] )

[0612] For example, when using NaBH4, the reduction step has a low stereoselectivity (around 7:3), making it difficult to efficiently obtain the desired Glc→Man stereoinversion (Org. Biomol. Chem., 2018, 16, 4720-4727). On the other hand, when using the reducing agents listed above, the selectivity of the Glc→Man stereoinversion is significantly improved (93.6:6.4 to 98.1:1.9) compared to when using NaBH4.

[0613] A compound represented by Formula W in which three R3 are di-tert-butylmethylphenoxide can be obtained, for example, by adding dibutylhydroxytoluene (885.41 mg, 4.02 mmol) to a suspension (2 mL) of lithium aluminum hydride (50.0 mg, 1.32 mmol) in tetrahydrofuran at 0°C, followed by stirring at 25°C. A compound represented by Formula W in which two R3 are di-tert-butylmethylphenoxide can be obtained by the same method using 2 molar equivalents of dibutylhydroxytoluene per 1 molar equivalent of lithium aluminum hydride.

[0614] As described above, the reaction temperature of this step is not limited as long as the reaction proceeds. When lithium tri-sec-butylborohydride, lithium tripentylborohydride, or LDBBA is used as the reducing agent, the reaction temperature is preferably -80°C to -20°C, more preferably -80°C to -30°C, even more preferably -80°C to -40°C, and particularly preferably -80°C to -50°C. When the compound represented by formula W is used as the reducing agent, the reaction temperature is preferably -20°C to 20°C, more preferably -15°C to 15°C, and particularly preferably -10°C to 10°C. Therefore, the compound represented by formula W is particularly preferred as the reducing agent used in this step because the reaction proceeds at a temperature that is easier to handle.

[0615] Next, the compound represented by formula A-4 is used to produce the compound represented by formula A-5. In one embodiment of the present invention, this step includes reacting the compound represented by formula A-4 with DDQ (2,3-dichloro-5,6-dicyano-p-benzoquinone) in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group in the compound represented by formula A-4 (de-2-naphthylmethylation reaction), thereby producing the oligosaccharide represented by formula A-5. This can be preferably carried out, for example, by the method described in Example 31 or 54.

[0616] Regarding the aforementioned de-2-naphthylmethylation reaction, the inventors of the present application have discovered that by reacting 2,3-dichloro-5,6-dicyano-p-benzoquinone with a substrate having a 2-naphthylmethyl group bonded via an oxygen atom in a fluorinated alcohol and water, the reaction can be conducted under mild conditions with good stirring properties, and the de-2-naphthylmethylated product can be obtained in high yield. The advantages of this de-2-naphthylmethylation reaction are described in further detail below. In the aforementioned de-2-naphthylmethylation reaction of the present invention, the de-2-naphthylmethylated product can be obtained in high yield from a substrate such as a sugar having a 2-naphthylmethyl group bonded via an oxygen atom under mild conditions. Furthermore, there is no evidence of deterioration in stirring properties or adhesion to the vessel wall caused by the by-product 2,3-dichloro-5,6-dicyano-p-benzohydroquinone, allowing the reaction to be conducted with good reproducibility, making it suitable for large-scale synthesis of the aforementioned product. Furthermore, due to the abnormal freezing point depression of HFIP-H2O, no solidification of the solvent was observed even when the reaction temperature was lowered to -30°C, making it applicable over a wide temperature range (melting point HFIP: -3.3°C, H2O: 0°C) depending on the reactivity of the reaction substrate. Furthermore, it was discovered that when performing the denaphthylmethylation reaction of the compound represented by Formula A-5 having multiple benzyl groups, using DDQ as the oxidant and HFIP-H2O as the solvent, the reaction proceeds with superior selectivity compared to conventional conditions. For this conversion reaction, a large number of reported examples employ a two-layer reaction system using dichloromethane and water. In this case, debenzylation of multiple benzyl groups proceeds at a moderate rate, with yields remaining at a moderate level. While there are reports of improved conditions using β-pinene as an additive (see, for example, J. Org. Chem., 2017, 82, 3926), the yields for compounds with multiple Bn groups remain moderate (see, for example, Angew. Chem. Int. Ed., 2021, 60, 19287). Furthermore, the selectivity for substrates with four or more benzyl groups, such as the compound represented by Formula A-5, is not fully understood. Furthermore, the dichloromethane-water system presents the problem of poor stirring properties due to DDQ and DDQ-derived byproducts, making it difficult to define suitable reaction conditions for large-scale synthesis. On the other hand, the DDQ / HFIP-H2O system of this method achieves high selectivity of over 95% for the compound represented by Formula A-5, which has four benzyl groups. This deterioration in stirring properties due to DDQ, as described above, has not been observed in this method. Furthermore, due to the abnormal freezing point depression of HFIP-H2O, even when the reaction temperature is lowered to -30°C, no solidification of the solvent is observed. This allows for a wide temperature range (melting point HFIP: -3.3°C, H2O: 0°C) depending on the reactivity of the reaction substrate.

[0617] It should be noted that the aforementioned 2-naphthylmethylation reaction is not limited to the reaction in step I-1-1. Therefore, one embodiment of the present invention further provides a method for producing the oligosaccharide represented by formula A-5, comprising the steps of reacting the compound represented by formula A-4 with DDQ (2,3-dichloro-5,6-dicyano-p-benzoquinone) in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group from the compound represented by formula A-5.

[0618] The "fluorinated alcohol" is not limited as long as it allows the reaction to proceed, but is preferably selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butanol, and combinations thereof.

[0619] The above-mentioned de-2-naphthylmethylation reaction is not limited as long as the reaction proceeds, but is preferably carried out at -35°C to 70°C, more preferably at -30°C to -10°C.

[0620] <Purification of the compound represented by Formula A-6>

[0621] In this step I-1-1, the compound represented by Formula A-6 can be obtained in a purified form by the following purification method. This purification method comprises: after terminating the reaction between the compound represented by Formula A-3 and the compound represented by Formula A-5, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by Formula A-6 and impurities, allowing the compound represented by Formula A-6 to adsorb onto the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water to remove impurities; and then eluting the compound represented by Formula A-6 from the hydrophobic carrier using an organic solvent to purify the compound represented by Formula A-6. According to this purification method, in the liquid-phase synthesis of oligosaccharide chains, by using a small amount of hydrophobic carrier for washing, residual reagent residues and impurities derived from sugar donors and sugar acceptors after the glycosylation reaction can be easily removed. It also suppresses reaction inhibition and side reactions caused by these impurities, thereby enabling the efficient and large-scale production of high-quality oligosaccharides. Furthermore, compared to existing methods, the present invention utilizes the inherent hydrophobicity of the matrix to reduce the number of tag desorption steps and prevent degradation of tag functionality during oligomerization, enabling more efficient oligosaccharide production. In particular, in this step, the aforementioned purification method facilitates separation and purification of the compound represented by Formula A-6 from degradation products derived from the compound represented by Formula A-4.

[0622] It should be noted that the purification of the compound represented by Formula A-6 is not limited to the purification in this step I-1-1. Therefore, one embodiment of the present invention also provides a method comprising: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by Formula A-6 and impurities, allowing the compound represented by Formula A-6 to adsorb on the hydrophobic carrier; then filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water to remove impurities; and then eluting the compound represented by Formula A-6 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-6. Furthermore, the above purification method can also be applied to the purification of organic compounds other than sugar compounds. Furthermore, when the organic compound to be purified is a sugar compound, it is preferred to purify an oligosaccharide having a sugar chain structure consisting of 3 to 15 sugar residues (protected oligosaccharide) in which one or all of the hydroxyl groups in the sugar are protected. Protective groups for the sugar chain in this case include, but are not limited to, alkyl ethers, benzyl ethers, silyl ethers, esters, and carbonates.

[0623] The above-mentioned "inclusions" refer to compounds and reagents other than the protected oligosaccharide (in this step, the compound represented by the above formula A-6), mainly referring to the reagents used in the synthesis reaction of the protected oligosaccharide, their residues, sugars other than the protected oligosaccharide such as monosaccharide or disaccharide compounds used in the extension reaction of the protected oligosaccharide, or by-products generated by the deprotection reaction of the protected oligosaccharide.

[0624] The above-mentioned "hydrophobic carrier" refers to a hydrophobic adsorption material that adsorbs specific compounds including sugar compounds. For example, a resin for reversed-phase partition chromatography filling can be listed. The "resin for reversed-phase partition chromatography filling" is selected from the group consisting of poly(styrene / divinylbenzene) polymer gel resin, polystyrene-divinylbenzene resin, polyhydroxymethylacrylate resin, styrene-vinylbenzene copolymer resin, polyvinyl alcohol resin, polystyrene resin, polymethacrylate resin, chemically bonded silica gel resin, and combinations thereof, but is not limited thereto.

[0625] The above-mentioned "chemically bonded silicone resin" is selected from the group consisting of (1) a resin obtained by reacting a silane coupling agent with silicone, (2) a resin obtained by chemically bonding a dimethyloctadecyl group, an octadecyl group, a trimethyloctadecyl group, a dimethyloctyl group, an octyl group, a butyl group, an ethyl group, a methyl group, a phenyl group, a cyanopropyl group, or an aminopropyl group to silicone, (3) a resin obtained by chemically bonding a docosyl group or a triacontyl group to silicone; and (4) a combination of the above (1) to (3). Octadecyl bonded silicone resin (ODS resin) is preferably used, but is not limited thereto.

[0626] The above-mentioned "water-soluble organic solvent" is not particularly limited, and water-soluble alcohol solvents (preferably C1 to C4), water-soluble nitrile solvents (acetonitrile, etc.), water-soluble ether solvents (tetrahydrofuran, etc.), water-soluble ketone solvents (acetone, etc.), water-soluble amide solvents (dimethylformamide, etc.), or water-soluble sulfoxide solvents (dimethyl sulfoxide, etc.) can be used, and acetonitrile can be preferably used.

[0627] The "organic solvent" used in the process of eluting the target substance from the above-mentioned hydrophobic carrier is not particularly limited. Nitrile solvents (acetonitrile, etc.), ether solvents (tetrahydrofuran, etc.), ester solvents (ethyl acetate, etc.), ketone solvents (acetone, etc.), halogen solvents (dichloromethane, etc.), or aromatic solvents (toluene, etc.), or mixed solvents containing at least one of the above-mentioned solvent systems can be used. Acetonitrile, ethyl acetate, tetrahydrofuran, and toluene can be preferably used.

[0628] The above-mentioned purification step is not particularly limited and can be performed at a temperature of 0°C to 50°C.

[0629] After the above step I-1-1, the compound represented by the above formula A-8 can be produced from the compound represented by the above formula A-6 by the following steps I-1-2 to I-1-3, but the production process is not limited to these steps.

[0630] <Process I-1-2>

[0631] Step I-1-2 is a step of producing a compound represented by Formula A-7 by deprotecting the 4-methoxyphenyl group from the compound represented by Formula A-6.

[0632]

[0633] In one embodiment of the present invention, this step involves reacting the compound represented by Formula A-6 with λ3-iodide in a fluorinated alcohol and water to deprotect the 4-methoxyphenyl group, thereby producing the compound represented by Formula A-7. This step can preferably be performed, for example, by the method described in Examples 33 or 56.

[0634] The above-mentioned "λ3-iodide" refers to a trivalent hypervalent iodine compound. In one embodiment, it is a compound of formula R 4a -I(OR 4b )2 shown in the compound (wherein, R 4a is unsubstituted or substituted phenyl, R 4b is selected from the group consisting of H, acetoxy, trifluoroacetoxy, toluenesulfonyloxy, methanesulfonyloxy and combinations thereof). As shown in the definition of the above formula, R 4aThe phenyl group may be a "substituted phenyl group." Examples of such substituents include linear or branched saturated or unsaturated hydrocarbon groups, oxygen-containing groups (such as alkoxy and ester groups), nitrogen-containing groups (such as cyano and azide groups), and halogen atoms (such as fluorine, chlorine, bromine, and iodine atoms). Hydrocarbon groups, oxygen-containing substituents, and halogen atoms are more preferred. When these substituents contain carbon, for example, substituents having 1 to 5 carbon atoms or 1 to 3 carbon atoms are preferably used. Specific examples of λ3-iodides include, but are not limited to, [bis(trifluoroacetoxy)iodo]benzene (PIFA), [hydroxy(toluenesulfonyloxy)iodo]benzene (HTIB), (diacetoxyiodo)benzene (PIDA), [bis(trifluoroacetoxy)iodo]pentafluorobenzene, and [hydroxy(methanesulfonyloxy)iodo]benzene.

[0635] The "fluorinated alcohol" used in the above process refers to a fluorinated alcohol compound in which all carbons except the carbon bonded to the hydroxyl group have fluorine. As long as fluorine substitution is allowed, the fluorinated alcohol preferably has more fluorine. Fluorinated alcohols include fluorinated aliphatic alcohols, but are not limited to them. The hydrocarbon part in the fluorinated aliphatic alcohol can be saturated or unsaturated, can be linear or branched, or can be cyclic. Fluorinated aliphatic alcohols are, for example, fluorinated C2 to C8 aliphatic alcohols, preferably fluorinated C2 to C5 aliphatic alcohols, and more preferably fluorinated C2 to C3 aliphatic alcohols. Specific examples of fluorinated alcohols include hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butanol, and a group consisting of combinations thereof, but are not limited to them. Hexafluoro-2-propanol (HFIP) is preferably used.

[0636] This step is carried out in the presence of the aforementioned fluorinated alcohol and water. The amount of water can be appropriately adjusted to achieve a high yield of the product. For example, the amount of water can be set to approximately 1.0 equivalents or more, approximately 1.5 equivalents or more, approximately 2.0 equivalents or more, or approximately 2.5 equivalents or more relative to the compound represented by Formula A-6, on a molar basis. Furthermore, the amount of water can be set to approximately 10 or less, approximately 8 or less, approximately 5 or less, or approximately 3 or less relative to the compound represented by Formula A-6, on a volume basis.

[0637] In this step, an "additive" may be further added to the fluorinated alcohol and water. The additive is preferably selected from the group consisting of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, trifluoroacetic acid, and combinations thereof. The amount of the additive can be appropriately set, for example, to about 0.5 to 8 equivalents, about 1 to 6 equivalents, or about 1.5 to 5 equivalents relative to the compound represented by Formula A-6.

[0638] <Process I-1-3>

[0639] Step I-1-3 is a step of producing the compound represented by the above formula A-8 from the compound represented by the above formula A-7. This step can be performed by utilizing or applying a known method, preferably by the method described in Example 34 or 57, for example.

[0640] In one embodiment of the present invention, this step involves reacting the compound represented by Formula A-7 with 2,2,2-trifluoro-N-phenylacetimidazole (TFPC) in the presence of N-methylimidazole to produce the compound represented by Formula A-8. DBU can be used in place of N-methylimidazole. By using N-methylimidazole or DBU as the base, for example, the amount of TFPC can be reduced compared to using potassium carbonate, resulting in a higher yield of the target product. Since TFPC is an expensive reagent, improving the yield in this step is highly beneficial in commercial production.

[0641] The solvent in this step is not limited as long as it allows the reaction to proceed, and examples thereof include dichloromethane, toluene, ethyl acetate, acetonitrile, and tetrahydrofuran, with dichloromethane being preferred.

[0642] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably -20°C to 40°C, more preferably -10°C to 35°C, and particularly preferably 0°C to 30°C.

[0643] This step is preferably carried out in the presence of a dehydrating agent. The dehydrating agent in this step is not limited as long as it allows the reaction to proceed, and examples thereof include molecular sieves, preferably molecular sieve 4A powder having a particle size of 10 μm or less.

[0644] In this step, the compound represented by Formula A-8 produced can be used directly in the next step after removing the base used in the reaction, or it can be isolated / purified by column purification or the like. Examples of isolation / purification using a chromatographic column include those using silica gel as the stationary phase and dichloromethane or a toluene-ethyl acetate mixed solvent system as the mobile phase.

[0645] <Process I-2>

[0646] Step I-2 is a step of producing a compound represented by the following formula A-11, and includes forming a β-1,4-glycosidic bond between the compound represented by the above formula A-8 and the compound represented by the following formula A-9 to produce a compound represented by the following formula A-10.

[0647]

[0648] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0649]

[0650] (In the formula, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted with 1 to 3 C1-C6 alkoxy groups.) Step I-2 includes the following steps I-2-1 to I-2-2.

[0651] <Process I-2-1>

[0652] Step I-2-1 is a step in which a compound represented by Formula A-8 and a compound represented by Formula A-9 form a β-1,4-glycosidic bond to produce a compound represented by Formula A-10. The compound represented by Formula A-9 can be produced by known methods, or a commercially available product can be used. Commercially available products of the compound represented by Formula A'-9 (a compound represented by Formula A-9 in which R1 is a 4-methoxyphenyl group) include, for example, 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-pyranoside manufactured by Tokyo Chemical Industry Co., Ltd. Alternatively, with respect to the compound represented by formula A"-9, the compound represented by formula A-12 produced as described later is subjected to the treatment shown in Example 59, for example, to obtain isopropyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-pyranoside (the compound represented by formula F"-1). Furthermore, the compound represented by formula F"-1 is subjected to the treatment shown in Example 60, for example, to obtain the compound represented by formula A"-9. This step, which is a step for producing the compound represented by formula A-10, can be carried out by utilizing or applying a known method, preferably by the method shown in Example 35 or 61, for example.

[0653] <Purification of the compound represented by Formula A-10>

[0654] In this step I-2-1, the compound represented by Formula A-10 can be obtained in a purified form by the following purification method. This purification method comprises: after terminating the reaction between the compound represented by Formula A-8 and the compound represented by Formula A-9, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by Formula A-10 and impurities, allowing the compound represented by Formula A-10 to adsorb onto the hydrophobic carrier. The impurities are then removed by filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water. Subsequently, the compound represented by Formula A-10 is eluted from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-10. Similar to the purification method for the compound represented by Formula A-6, this purification method utilizes a small amount of hydrophobic carrier, enabling the efficient production of high-quality oligosaccharides in large quantities during liquid-phase synthesis of oligosaccharide chains. In particular, the compound represented by formula A-9, which is a monosaccharide, and the compound represented by formula A-10, which is a pentasaccharide, have extremely similar polarities in normal-phase silica gel column chromatography, for example, having the same Rf value under the conditions of a typical column solvent system of hexane-ethyl acetate, making separation difficult. However, by utilizing the purification method of the present invention, monosaccharides and pentasaccharides having extremely similar polarities can be easily separated.

[0655] It should be noted that the purification of the compound represented by Formula A-10 is not limited to the purification in step I-2-1. Therefore, one embodiment of the present invention also provides a method comprising: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by Formula A-10 and impurities, allowing the compound represented by Formula A-10 to adsorb on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water to remove impurities; and then, eluting the compound represented by Formula A-10 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-10.

[0656] The "impurities" mentioned above refer to compounds and reagents other than the protected oligosaccharide (the compound represented by Formula A-10 in this step). These primarily include reagents used in the synthesis reaction of the protected oligosaccharide, their residues, sugars other than the protected oligosaccharide, such as monosaccharide or disaccharide compounds used in the elongation reaction of the protected oligosaccharide, and byproducts resulting from the deprotection reaction of the protected oligosaccharide. The "hydrophobic support" (such as a resin for reversed-phase partition chromatography packing), "water-soluble organic solvent," "organic solvent," and purification temperature used in this step are the same as those described in the purification method for the compound represented by Formula A-6 above.

[0657] <Process I-2-2>

[0658] Step I-2-2 is a step of producing a compound represented by Formula A-11 from a compound represented by Formula A-10.

[0659] In one embodiment of the present invention, step I-2-2 is a step of reacting the compound represented by formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid in a solvent to remove the acetyl group (deacetylation reaction), thereby producing a compound represented by formula A-11. This deacetylation reaction can be preferably carried out, for example, by the method described in Example 36 or 62. This deacetylation reaction can be carried out in the same manner as the deacetylation reaction described in step X-7, except that the substrate is different. By using a method of reacting with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid, the deacetylation reaction can be carried out while suppressing the ring-opening of the phthalimide group.

[0660] It should be noted that the above-mentioned deacetylation reaction is not limited to use in this step I-2-2. Therefore, in one embodiment of the present invention, there is also provided a method for producing the compound represented by the above-mentioned formula A-11, which includes reacting the compound represented by the above-mentioned formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid.

[0661] The perfluorocarboxylic acid alkyl ester, strong base, solvent, and reaction temperature used in this step are the same as those described in the above step X-7.

[0662] <Process I-3>

[0663] Step I-3 includes a step of generating a compound represented by the following formula A-15, wherein the compound represented by the above formula A-11 and the compound represented by the following formula A-12 form a β-1,2-glycosidic bond to generate a compound represented by the following formula A-13, and then, removing the phthaloyl group as a protecting group for the amino group and the acetyl group on the hydroxyl group on the compound represented by the above formula A-13 to generate a compound represented by the following formula A-14, and then protecting the compound represented by the formula A-14 with a protecting group selected from the group consisting of an aryloxycarbonyl group (COOAr), an acetyl group (Ac), a 2,2,2-trichloroethoxycarbonyl group (Troc), and a phthaloyl group (Phth). The amino group in the compound of the following formula A-13 is removed to generate a compound of the following formula A-15 (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is hydrogen, or, R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group); or, the acetyl group (Ac) on the compound of the following formula A-13 is removed to generate a compound of the following formula A-15 (wherein, R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0664]

[0665]

[0666] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0667]

[0668] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0669]

[0670] In one embodiment of the present invention, step I-3 includes the following steps I-3-1 to I-3-3.

[0671] <Process I-3-1>

[0672] Step I-3-1 is a step in which the compound represented by Formula A-11 and the compound represented by Formula A-12 form a β-1,2-glycosidic bond to produce the compound represented by Formula A-13. The glycosidic bonding step can be performed using or applying known methods, preferably, for example, the method described in Examples 37 or 63. Alternatively, the compound represented by Formula A-12 can be produced as follows. Furthermore, the compound represented by Formula A-13 can be purified as described below.

[0673] <Production of the compound represented by formula A-12>

[0674] The compound represented by the above formula A-12 can be produced by the following sub-steps Z-1 to Z-3.

[0675] <Small process Z-1>

[0676] First, the hydroxyl group of the compound represented by the formula A-9 used in the above step I-2 is protected with an acetyl group to produce a compound represented by the following formula F-1.

[0677]

[0678] This step can be performed by utilizing or applying a known method, preferably by the method shown in Example 13, which can be performed by adding triethylamine, dimethylaminopyridine and acetic anhydride to an ethyl acetate solution of the compound represented by Formula A-9, but is not limited to this method.

[0679] <Small process Z-2>

[0680] Next, by removing the 4-methoxyphenyl group from the compound represented by Formula F-1, the compound represented by the following Formula F-2 is produced.

[0681]

[0682] In one embodiment of the present invention, sub-step Z-2 is a step of reacting the compound represented by formula F-1 with λ3-iodide in a fluorinated alcohol and water to remove the 4-methoxyphenyl group, thereby producing the compound represented by formula F-2. This step can be preferably carried out, for example, by the method described in Example 14. This step can be carried out according to step I-1-2 described above, and the fluorinated alcohol and λ3-iodide used in this step can be the same as those used in step I-1-2 described above.

[0683] <Small process Z-3>

[0684] Sub-step Z-3 is a step of subsequently reacting the compound represented by formula F-2 with 2,2,2-trifluoro-N-phenylacetimidochloride (TFPC) to produce the compound represented by formula A-12.

[0685]

[0686] This step can be performed by utilizing or applying a known method.

[0687] In one embodiment of the present invention, sub-step Z-3 is a step in which the compound represented by formula A-12 is produced by reacting with 2,2,2-trifluoro-N-phenylacetimidochloride (TFPC) in the presence of N-methylimidazole. This can be preferably carried out, for example, by the method described in Example 15 or 58. As described for the similar reaction in step I-1-3, the use of N-methylimidazole as the base reduces the equivalent amount of TFPC, for example, compared to the case of using potassium carbonate, and the target product can be obtained in high yield. The solvent and reaction temperature used, as well as the preferred presence of a dehydrating agent and the possibility of isolation and purification by column purification, are similar to those in step I-1-3.

[0688] <Purification of the compound represented by Formula A-13>

[0689] In step I-3-1, the compound represented by Formula A-13 can be obtained in a purified form by the following purification method. This purification method comprises: after terminating the reaction between the compound represented by Formula A-11 and the compound represented by Formula A-12, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by Formula A-13 and impurities, allowing the compound represented by Formula A-13 to adsorb onto the hydrophobic carrier. The impurities are then removed by filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water. Subsequently, the compound represented by Formula A-13 is eluted from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-13. Similar to the purification method for the compound represented by Formula A-6, this purification method, using a small amount of hydrophobic carrier, enables the efficient production of high-quality oligosaccharides in large quantities during liquid-phase synthesis of oligosaccharide chains.

[0690] It should be noted that the purification of the compound represented by Formula A-13 is not limited to the purification in this step I-3-1. Therefore, in one embodiment of the present invention, a method is also provided, comprising: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by Formula A-13 and impurities, allowing the compound represented by Formula A-13 to adsorb on the hydrophobic carrier; then, filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water to remove impurities; and then, eluting the compound represented by Formula A-13 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-13.

[0691] The above-mentioned "inclusions" refer to compounds and reagents other than protected oligosaccharides (compounds represented by formula A-13 in this step), mainly referring to reagents used in the synthesis reaction of protected oligosaccharides, their residues, sugars other than protected oligosaccharides such as monosaccharide or disaccharide compounds used in the extension reaction of protected oligosaccharides, or by-products generated by the deprotection reaction of protected oligosaccharides.

[0692] The "hydrophobic support" (such as a resin for reverse phase partition chromatography packing), "water-soluble organic solvent," "organic solvent," and purification temperature used in the above steps are the same as those described in the purification method for the compound represented by formula A-5.

[0693] <Process I-3-2>

[0694] Step I-3-2 is the step of removing the phthaloyl protecting group on the amino group and the acetyl protecting group on the hydroxyl group of the compound represented by Formula A-13, thereby producing the compound represented by Formula A-14. This step can preferably be carried out, for example, by the method described in Examples 38 or 64. For example, n-butanol and ethylenediamine can be added to a solution containing the compound represented by Formula A-13, but the method is not limited thereto. Alternatively, an acid (e.g., fumaric acid) can be added after the addition of n-butanol and ethylenediamine, followed by stirring, and seeding to obtain crystals of an acid salt (e.g., a fumarate salt) of the compound represented by Formula A-14 (see, for example, Example 64). By isolating the acid salt and removing analogs (e.g., isomers) of the compound represented by Formula A-14 by filtration, the compound can be highly purified. It should be noted that fumaric acid is preferably used as the "acid" to be added, but the method is not limited thereto.

[0695] <Purification of the compound represented by Formula A-14>

[0696] In this step I-3-2, the compound represented by Formula A-14 can be obtained in a purified form by the following purification method. This purification method comprises adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by Formula A-14 produced as described above and impurities, allowing the compound represented by Formula A-14 to adsorb onto the hydrophobic carrier. The impurities are then removed by filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water. Subsequently, the compound represented by Formula A-14 is eluted from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-14. Similar to the purification method for the compound represented by Formula A-6 in step I-1-1 above, this purification method utilizes a small amount of hydrophobic carrier, enabling the efficient production of high-quality oligosaccharides in large quantities during liquid-phase synthesis of oligosaccharide chains.

[0697] It should be noted that the purification of the compound represented by the above-mentioned formula A-14 is not limited to the purification in this step. Therefore, in one embodiment of the present invention, the following method is also provided, which comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the above-mentioned formula A-14 and impurities, allowing the compound represented by the above-mentioned formula A-14 to be adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the above-mentioned water-soluble organic solvent and water to remove the impurities, and then eluting the compound represented by the above-mentioned formula A-14 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the above-mentioned formula A-14.

[0698] The "impurities" mentioned above refer to compounds and reagents other than the protected oligosaccharide (the compound represented by Formula A-14 in this step), primarily including reagents used in the synthesis reaction of the protected oligosaccharide, their residues, sugars other than the protected oligosaccharide, such as monosaccharide or disaccharide compounds used in the elongation reaction of the protected oligosaccharide, and byproducts generated by the deprotection reaction of the protected oligosaccharide. It should be noted that the "hydrophobic support" (such as a resin for reversed-phase partition chromatography packing), "water-soluble organic solvent," "organic solvent," and purification temperature used in this step are the same as those described in the purification method for the compound represented by Formula A-6 in Step I-1-1 above.

[0699] <Process I-3-3>

[0700] Step I-3-3 is a step in which the amino group in the compound represented by the above formula A-14 is protected with a protecting group selected from the group consisting of an aryloxycarbonyl group (COOAr), an acetyl group (Ac), a 2,2,2-trichloroethoxycarbonyl group (Troc), and a phthaloyl group (Phth) to produce a compound represented by the above formula A-15 (wherein R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc) and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group). The purpose of introducing the amino-protecting group described above is to more efficiently produce the target compound (the compound represented by Formula A-22) by using an acetyl group as the linear pathway for the amino-protecting group. However, in the glycosylation reaction between the compound represented by Formula A-15 and the compound represented by Formula A-16 in the next step I-4, if the acetyl-protected amino group (-NHAc group) is present in the reaction matrix, the reactivity of the target glycosylation reaction is significantly reduced due to interaction with the Lewis acid, and an excess of sugar donor is required to complete the reaction. Therefore, during the glycosylation reaction, the nitrogen of the glucosamine is temporarily protected with a protecting group selected from the group consisting of an aryloxycarbonyl group (COOAr), a 2,2,2-trichloroethoxycarbonyl group (Troc), and a phthaloyl group (Phth). After the glycosylation reaction, the protection is removed to form a -NHAc group, thereby avoiding the above disadvantages. Furthermore, the aryloxycarbonyl group (COOAr) is most preferably used as the protecting group. The "aryl (Ar)" in the aryloxycarbonyl group refers to a group derived from an aromatic hydrocarbon by removing a hydrogen atom from an aromatic ring. Examples include, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl, 2-pyridyl, 3-pyridyl, nitrophenyl, chlorophenyl, fluorophenyl, bromophenyl, iodophenyl, methoxyphenyl, and C1-C4 alkylphenyl groups, with phenyl being preferred. Aryloxycarbonyl (COOAr) has been found to facilitate glycosylation reactions compared to other protecting groups, and subsequent deprotection reactions can be performed under conventional hydrolysis conditions, preferably at room temperature within one hour.

[0701] The above step can preferably be carried out, for example, by the method shown in Example 39 or 65, for example, by adding tetrahydrofuran and an aqueous solution of sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate (preferably sodium bicarbonate) dissolved in water to a solution of the compound represented by formula A-14 in tetrahydrofuran, but is not limited thereto.

[0702] Instead of steps I-3-2 and I-3-3, the compound represented by formula A-15 (wherein R5 and R6, together with the nitrogen atom to which they are bound, form a phthalimide group) can be produced by selectively removing the acetyl group (Ac) from the compound represented by formula A-13. The selective removal of the acetyl group can be performed under methyl trifluoroacetate conditions, but is not limited thereto. This step produces the same result as the case where a phthaloyl group (Phth) is selected as the protecting group for the amino group in the compound represented by formula A-14 in steps I-3-2 and I-3-3.

[0703] <Process I-4>

[0704] Step I-4 is a step for producing a compound represented by the following formula A-20, and includes the following steps: forming a β-1,4-glycosidic bond between the compound represented by the above-mentioned formula A-15 and the compound represented by the following formula A-16 to produce a compound represented by the following formula A-17, and then removing the protecting group of the amino group in the compound represented by the following formula A-17 to produce a compound represented by the following formula A-18.

[0705]

[0706] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0707]

[0708] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0709]

[0710] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + (wherein the cation is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation). In one embodiment of the present invention, step I-4 includes the following steps I-4-1 to I-4-4.

[0711] <Process I-4-1>

[0712] This step involves forming a β-1,4-glycosidic bond between the compound represented by Formula A-15 and the compound represented by Formula A-16 to produce the compound represented by Formula A-17. The glycosidic bonding step can be performed using or applying known methods, preferably, the method described in Examples 40 or 66.

[0713] <Production of the compound represented by formula A-16>

[0714] In one embodiment of the present invention, the compound represented by formula A-16 can be produced by the following sub-steps V-1 to V-11. This step includes, as an essential sub-step, the later-described sub-step V-7 of synthesizing a disaccharide block by forming an α-2,6-glycosidic bond between two monosaccharide molecules. Otherwise, the step can be carried out by using or applying conventional methods for producing monosaccharides or oligosaccharides.

[0715] In one embodiment of the present invention, step V includes the following sub-steps.

[0716]

[0717] <Small process V-1>

[0718] Sub-step V-1 is a step of producing a compound represented by the following formula G-2 by protecting the hydroxyl group on the compound represented by the following formula G-1 with a benzoyl group.

[0719]

[0720]

[0721] The compound represented by formula G-1, which is the starting material of this step, is specified as CAS No. 100759-10-2 and can be produced by known methods, for example, by the methods described in Examples 16 and 17. This step can be carried out by utilizing or applying known methods, preferably, by the method described in Example 18.

[0722] <Small Process V-2>

[0723] Sub-step V-2 is a step of producing a compound represented by the following formula G-3 by removing the benzylidene protecting group from the compound represented by formula G-2.

[0724]

[0725] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method shown in Example 19.

[0726] In one embodiment of the present invention, this step includes the step of subjecting the generated compound of Formula G-3 to solid-phase extraction by contacting a solvent containing the compound of Formula G-3 with silica gel. Unreacted compound of Formula G-2 and dissociated benzaldehyde are not adsorbed on the silica gel, allowing efficient purification of the compound of Formula G-3 through this step.

[0727] Examples of the solvent for dissolving the compound represented by Formula G-3 include toluene, heptane, dichloromethane, chloroform, or a combination thereof. Preferred examples include toluene, dichloromethane, chloroform, or a combination thereof, and particularly preferred is toluene, but the solvent is not limited thereto.

[0728] Examples of the silica gel used in this step include 2 to 5 times the amount of the raw material, preferably 2 to 4 times the amount of the raw material, and more preferably about 3 times the amount of the raw material.

[0729] In this step, the solvent used to elute the compound represented by formula G-3 adsorbed on silica gel is not particularly limited as long as it does not dissolve silica gel and can elute the target substance. Examples include cyclopentyl methyl ether, ethyl acetate, and tert-butyl methyl ether.

[0730] <Small Process V-3>

[0731] Sub-step V-3 is a step of producing a compound represented by the following formula G-5 by esterifying the carboxylic acid of the compound represented by the following formula G-4 and then injecting water.

[0732]

[0733] The compound represented by Formula G-4, which serves as the starting material for this step, can be produced by known methods or a commercially available product. Commercially available products of the compound represented by Formula G-4 include, for example, N-acetylneuraminic acid manufactured by Tokyo Chemical Industry Co., Ltd. This step can be carried out using or applying known methods, preferably, the method described in Example 20.

[0734] <Small Process V-4>

[0735] Sub-step V-4 is a step of producing a compound represented by the following formula G-6 by selectively protecting hydroxyl groups other than the hydroxyl group bonded to the carbon 1 position in the compound represented by formula G-5 with acetyl groups.

[0736]

[0737] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method described in Example 21.

[0738] <Small Process V-5>

[0739] Sub-step V-5 is a step of reacting the compound represented by formula G-6 with 2,2,2-trifluoro-N-phenylacetimidochloride (TFPC) to produce a compound represented by the following formula G-7.

[0740]

[0741] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method shown in Example 22.

[0742] In one embodiment of the present invention, this step is to produce the compound represented by Formula G-7 by reacting the compound represented by Formula G-6 with TFPC in the presence of N-methylimidazole. Using N-methylimidazole as the base in this step reduces the amount of TFPC equivalents compared to using K₂CO₃, allowing the target product to be obtained in high yield. TFPC is an expensive reagent, so improving the yield in this step is highly beneficial for commercial production.

[0743] The solvent in this step is not limited as long as it allows the reaction to proceed, and examples thereof include dichloromethane, toluene, ethyl acetate, acetonitrile, and tetrahydrofuran, with dichloromethane being preferred.

[0744] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 0°C to 40°C, more preferably 0°C to 35°C, and particularly preferably 0°C to 30°C.

[0745] This step is preferably carried out in the presence of a dehydrating agent. The dehydrating agent in this step is not limited as long as it allows the reaction to proceed, and examples thereof include molecular sieves, preferably molecular sieve 4A powder having a particle size of 10 μm or less.

[0746] <Small Process V-6>

[0747] Sub-step V-6 is a step of producing a compound represented by the following formula G-8 by protecting the nitrogen atom in the acetylamino group of the compound represented by the formula G-7 with a tert-butoxycarbonyl group.

[0748]

[0749] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method shown in Example 23.

[0750] In this step, the compound represented by Formula G-8 can be used directly in the next step after dissolving in a solvent, or it can be isolated and purified by recrystallization. A major advantage of the compound represented by Formula G-8 is that it can be isolated and purified by crystallization. Crystallization can yield a compound represented by Formula G-8 with a purity of 99% or greater as measured by HPLC. Since it contains no impurities, the glycosylation reaction in the next step can be stably carried out. Isolation and purification by recrystallization can be performed, for example, by adding heptane to a solution of cyclopentyl methyl ether for crystallization.

[0751] <Small Process V-7>

[0752] Sub-step V-7 is a step of producing a compound represented by the following formula G-9 by forming an α-2,6-glycosidic bond between the compound represented by formula G-8 and the compound represented by formula G-3.

[0753]

[0754] Selectively bonding galactose derivatives and N-acetylneuraminic acid derivatives through α-2,6-glycosidic bonds is difficult. For example, a method for synthesizing disaccharides by reacting the compound represented by Formula G-3 with the compound represented by Formula G-7 has been reported (J. Org. Chem., 2016, 81, 10600-10616). However, reproducibility of the reaction is difficult, and the desired yield and selectivity cannot be achieved. Furthermore, this reaction suffers from problems such as decreasing selectivity with increasing scale, a narrowing of the reaction temperature range, and a significant effect of reaction heat. The compound represented by Formula G-7, one of the starting compounds in this reaction, is very expensive, making the low reproducibility, yield, and selectivity of this reaction a major problem, particularly in commercial production requiring scale-up. On the other hand, using the compound represented by Formula G-8, which has a tert-butoxycarbonyl group attached, instead of the compound represented by Formula G-7 as the starting compound, reproducibly achieves high selectivity for α-2,6-glycosidic bonds (α:β = 93:7), and also improves yield. Furthermore, the permissible temperature range is expanded, and high reproducibility, yield, and selectivity can be achieved even when scaled up, which is extremely beneficial for commercial production.

[0755] This step can be preferably carried out in the presence of a Lewis acid. The Lewis acid in this step is not limited as long as it allows the reaction to proceed. Examples thereof include trimethylsilyl trifluoromethanesulfonate, triisopropylsilyl trifluoromethanesulfonate, and tert-butyldimethylsilyl trifluoromethanesulfonate. Trimethylsilyl trifluoromethanesulfonate is preferred.

[0756] The solvent in this step is not limited as long as it allows the reaction to proceed. Examples thereof include cyclopentyl methyl ether, diisopropyl ether, tert-butyl methyl ether, diethyl ether, dibutyl ether, dipropyl ether, 1,4-dioxane, dichloromethane, 1,2-dichloroethane, toluene, chlorobenzene, trifluoromethylbenzene, propionitrile, and acetonitrile. Preferably, cyclopentyl methyl ether is used.

[0757] The reaction temperature in this step is not limited as long as the reaction proceeds, and is, for example, -78°C to 0°C, preferably -78°C to -20°C, more preferably -78°C to -30°C, and particularly preferably -78°C to -40°C.

[0758] In this step, preferably 1 to 3 equivalents of the compound represented by formula G-3 are added per 1 equivalent of the compound represented by formula G-8, more preferably 1.4 to 2 equivalents of the compound represented by formula G-3 are added per 1 equivalent of the compound represented by formula G-8.

[0759] This step is not limited as long as the reaction proceeds. For example, it can be carried out by dripping a mixed solution of the compound represented by Formula G-8 and the compound represented by Formula G-3 (preferably a mixed solution of cyclopentyl methyl ether) for a long period of time into a solution containing a Lewis acid (preferably a solution of cyclopentyl methyl ether), or by dripping a solution of the compound represented by Formula G-8 (preferably a solution of cyclopentyl methyl ether) for a long period of time into a solution containing a Lewis acid and the compound represented by Formula G-3 (preferably a solution of cyclopentyl methyl ether). Preferably, it can be carried out by dripping a solution of the compound represented by Formula G-8 (preferably a solution of cyclopentyl methyl ether) for a long period of time into a solution containing a Lewis acid and the compound represented by Formula G-3 (preferably a solution of cyclopentyl methyl ether). The dripping time is not limited as long as the reaction proceeds, and is, for example, 30 minutes to 5 hours, preferably 1 hour to 4 hours, more preferably 2 hours to 3.5 hours, and particularly preferably about 3 hours.

[0760] In one embodiment of the present invention, this step includes the following: solid-phase extraction of the compound represented by Formula G-9 by contacting a solvent containing the compound represented by Formula G-9 with silica gel. N-phenyltrifluoroacetamide, a byproduct of the glycosylation reaction, and other trace impurities in the toluene solvent that are not adsorbed to the silica gel are not adsorbed to the silica gel. Therefore, this step allows for efficient purification of the compound represented by Formula G-9.

[0761] Examples of the solvent for dissolving the compound represented by Formula G-9 include toluene, heptane, dichloromethane, chloroform, or a combination thereof. Preferred examples include toluene, dichloromethane, chloroform, or a combination thereof, and particularly preferred is toluene, but the invention is not limited thereto.

[0762] Examples of the silica gel used in this step include 2 to 5 times the amount of the raw material, preferably 2 to 4 times the amount of the raw material, and more preferably about 3.5 times the amount of the raw material.

[0763] In this step, the solvent used to elute the compound represented by formula G-9 adsorbed on silica gel is not particularly limited as long as it does not dissolve silica gel and can elute the target substance. Examples thereof include ethyl acetate, cyclopentyl methyl ether, and tert-butyl methyl ether, with ethyl acetate being preferred.

[0764] This step can be performed, for example, by the method shown in Example 24.

[0765] <Small Process V-8>

[0766] Sub-step V-8 is a step of producing a compound represented by the following formula G-10 by removing a tert-butoxycarbonyl group from the compound represented by the formula G-9.

[0767]

[0768] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method shown in Example 25.

[0769] <Small Process V-9>

[0770] Sub-step V-9 is a step of further protecting the hydroxyl group and the nitrogen atom in the acetylamino group of the compound represented by formula G-10 with acetyl groups to produce the compound represented by the following formula G-11.

[0771]

[0772] This step can be performed by utilizing or applying a known method, and preferably, for example, it can be performed by the method shown in Example 26.

[0773] In one embodiment of the present invention, this step includes the step of subjecting the compound of Formula G-11 to solid-phase extraction by contacting a solvent containing the compound of Formula G-11 with silica gel. Byproducts such as the diacetyl form of the compound of Formula G-3, produced by acetylation of the compound of Formula G-3 used in excess in the upstream glycosylation reaction, are not adsorbed to the silica gel. Therefore, this step allows for efficient purification of the compound of Formula G-11.

[0774] Examples of the solvent for dissolving the compound represented by Formula G-11 include toluene, heptane, dichloromethane, chloroform, or a combination thereof. Preferred examples include toluene, dichloromethane, chloroform, or a combination thereof, and particularly preferred is toluene, but the solvent is not limited thereto.

[0775] Examples of the silica gel used in this step include 2 to 5 times the amount of the raw material, preferably 2 to 4 times the amount of the raw material, and more preferably about 3.5 times the amount of the raw material.

[0776] In this step, the solvent used to elute the compound represented by formula G-11 adsorbed on silica gel is not particularly limited as long as it does not dissolve silica gel and can elute the target substance. Examples thereof include ethyl acetate, cyclopentyl methyl ether, and tert-butyl methyl ether, with ethyl acetate being preferred.

[0777] <Small Process V-10>

[0778] Sub-step V-10 is a step of producing a compound represented by the following formula G-12 by removing the allyl group bonded to the carbon 1 position of D-galactopyranoside in the compound represented by formula G-11.

[0779]

[0780] This step can be performed using or applying known methods, preferably the method described in Example 27-1. Alternatively, as illustrated in Example 27-2, the compound represented by Formula G-12 can be produced by stirring a solution containing the compound represented by Formula G-11 and tris(triphenylphosphine)ruthenium(II) carbonylchlorohydrogen, then adding water and 1,3-dibromo-5,5-dimethylhydantoin, iodine, or N-bromosuccinimide to the solution and stirring, thereby removing the allyl group bonded to the carbon 1 of the D-galactopyranoside in the compound represented by Formula G-11 through the oxygen. The solvent used in this step can also be tetrahydrofuran, tetrahydropyran, cyclopentyl methyl ether, toluene, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, or tert-butyl methyl ether. The isomerization temperature can be, for example, 40 to 65°C.

[0781] <Small Process V-11>

[0782] Sub-step V-11 is a step for reacting the compound represented by formula G-12 with 2,2,2-trifluoro-N-phenylacetimidochloride (TFPC) to produce the compound represented by formula A-16. This step can be performed using or applying known methods, preferably, the method described in Example 28.

[0783] <Purification of the compound represented by Formula A-17>

[0784] In step I-4-1, the compound represented by Formula A-17 can be obtained in a purified form by the following purification method. This purification method comprises: after terminating the reaction between the compound represented by Formula A-15 and the compound represented by Formula A-16, adding a hydrophobic carrier and water to a water-soluble organic solvent containing the generated compound represented by Formula A-17 and impurities, allowing the compound represented by Formula A-17 to adsorb onto the hydrophobic carrier. The impurities are then removed by filtering and washing the hydrophobic carrier with a mixed solution of the water-soluble organic solvent and water. Subsequently, the compound represented by Formula A-17 is eluted from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by Formula A-17. Similar to the purification method for the compound represented by Formula A-6 in step I-1-1, this purification method utilizes a small amount of hydrophobic carrier, enabling the efficient production of high-quality oligosaccharides in large quantities during liquid-phase synthesis of oligosaccharide chains.

[0785] It should be noted that the purification of the compound represented by the above-mentioned formula A-17 is not limited to the purification in this step. Therefore, in one embodiment of the present invention, the following method is also provided, which comprises: adding a hydrophobic carrier and water to a water-soluble organic solvent containing the compound represented by the above-mentioned formula A-17 and impurities, allowing the compound represented by the above-mentioned formula A-17 to be adsorbed on the hydrophobic carrier, then filtering and washing the hydrophobic carrier with a mixed solution of the above-mentioned water-soluble organic solvent and water to remove the impurities, and then eluting the compound represented by the above-mentioned formula A-17 from the hydrophobic carrier using an organic solvent, thereby purifying the compound represented by the above-mentioned formula A-17.

[0786] The "impurities" mentioned above refer to compounds and reagents other than the protected oligosaccharide (the compound represented by Formula A-17 in this step), primarily including reagents used in the synthesis reaction of the protected oligosaccharide, their residues, sugars other than the protected oligosaccharide, such as monosaccharide or disaccharide compounds used in the elongation reaction of the protected oligosaccharide, and byproducts generated by the deprotection reaction of the protected oligosaccharide. It should be noted that the "hydrophobic support" (such as a resin for reversed-phase partition chromatography packing), "water-soluble organic solvent," "organic solvent," and purification temperature used in this step are the same as those described in the purification method for the compound represented by Formula A-6 in Step I-1-1 above.

[0787] <Process I-4-2>

[0788] This step is a step for removing the amino protecting group and the acyl protecting group of the hydroxyl group from the compound represented by Formula A-17 to produce the compound represented by Formula A-18. The removal (deprotection) of the amino protecting group can be performed using or applying known methods, preferably the methods described in Examples 43 or 67. For example, the removal can be performed by sequentially adding 1,2-dimethoxyethane and aqueous solutions of potassium hydroxide, sodium hydroxide, or lithium hydroxide, but is not limited to these methods.

[0789] In the compound represented by formula A-18, "M + "Preferably, it is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation, but is not limited thereto. On the other hand, the compound represented by formula A-18 obtained in the above process can be isolated in the form of its free form (the compound represented by formula A-18-FF) by a known or publicly known method. The compound represented by formula A-18 is a form of "salt" of the compound represented by formula A-18-FF, but as mentioned above, compounds formed by forming salts at hydroxyl groups, amino groups, etc. in the compound are also included in the scope of the present invention. It should be noted that the same is true for the compounds represented by formula A-19 and formula A-20 described later (it should be noted that their free forms are compounds represented by formula A-19-FF and formula A-20-FF, respectively).

[0790] The following steps I-4-3 to I-4-4 are exemplary embodiments for producing the compound represented by formula A-20 from the compound represented by formula A-18, but the present invention is not limited to these production steps.

[0791] <Process I-4-3>

[0792] This step is a step of protecting the amino group of the compound represented by formula A-18 with an acetyl group to produce a compound represented by the following formula A-19.

[0793]

[0794] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + The protection of the amino group by the acetyl group can be carried out by utilizing or applying a known method, preferably by the method shown in Example 44 or 68.

[0795] <Process I-4-4>

[0796] This step involves removing the benzyl group from the benzyloxy group of the compound represented by Formula A-19 to produce the compound represented by Formula A-20. Removal of the benzyl group can be performed using or applying known methods, preferably the methods described in Examples 45 or 69. For example, N-methylpyrrolidone and Pd / C can be added to the compound represented by Formula A-19, followed by decompression followed by nitrogen replacement and hydrogen pressurization followed by decompression, but the present invention is not limited to these methods.

[0797] <Process I-5>

[0798] Step I-5 is a step comprising reacting the compound represented by the above formula A-20 with the compound represented by the following formula A-21 (11-azido-3,6,9-trioxaundecane-1-amine) as an azidoPEG linker to produce the oligosaccharide represented by the above formula A-22.

[0799]

[0800] The bonding of the compound represented by the above-mentioned formula A-20 and the compound represented by the formula A-21 can be carried out by utilizing or applying a known method, preferably by the method shown in Example 51 or 70. For example, it can be carried out by sequentially adding the compound represented by the formula A-21, N-ethyldiisopropylamine, and (benzotriazole-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate, tripyrrolidinophosphonium bromide hexafluorophosphate, or 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride to a solution containing the compound represented by the formula A-20 and then stirring, but is not limited to this.

[0801] <Purification of the compound represented by Formula A-21>

[0802] In one embodiment of the present invention, the compound represented by the above-mentioned formula A-21 is obtained by a purification method comprising the following steps: adding the compound represented by the following formula E-1 to a solution containing the crude compound represented by the formula A-21 to generate a crystalline compound represented by the following formula E-2; isolating the crystalline compound, and then extracting the compound represented by the formula A-21 from the isolated crystalline compound.

[0803]

[0804] (wherein R7 is a hydrogen atom, a methyl group, or a methoxy group).

[0805]

[0806] (wherein R7 is a hydrogen atom, a methyl group, or a methoxy group). By this purification method, a compound represented by formula A-21 with high purity can be obtained. As the purity when measured by HPLC (also referred to as "HPLC purity" in this specification), the compound represented by formula A-21 has a purity of preferably 95% or more, more preferably 96% or more or 97% or more, and further preferably 98% or more or 99% or more. In this way, the purpose of refining the compound represented by A-21 is because the commercially available reagents of the compound are mixed with various other impurities represented by dimers. In addition, in the prior art, their purification requires strict distillation purification or complex column purification. Moreover, in the case of containing an azide structure, there is a disadvantage that the distillation operation requiring heating cannot be applied due to its explosion hazard. The present inventors investigated methods for obtaining a highly pure compound represented by Formula A-21. They discovered that, when using three tartaric acid derivatives represented by Formula E-1 (where R7 is a hydrogen atom, a methyl group, or a methoxy group), the compound represented by Formula A-21 can form a one-to-one salt with these tartaric acid derivatives and be isolated as crystals. The resulting compound represented by Formula E-2 is a novel crystalline compound. After isolation, separation using, for example, ethyl acetate / aqueous hydrochloric acid solution, followed by freeing and extraction, can yield the compound represented by Formula A-21 with a higher HPLC purity (preferably 95% or greater) than before purification.

[0807] The above purification method is described as an exemplary method as follows. First, to a solution of a solvent such as acetonitrile and water of the compound represented by formula A-21, the compound represented by formula E-1 is added, stirred, and after confirming dissolution, a solvent such as acetonitrile is added. The obtained slurry is concentrated under reduced pressure, and the crystals precipitated by stirring the slurry are filtered. The filtered crystals are washed with acetonitrile and dried under reduced pressure to obtain crystals of the compound represented by formula E-2 (crystal formation process). Next, concentrated hydrochloric acid is added to the obtained solution of ethyl acetate and water of the crystalline compound, stirred and separated, the obtained aqueous layer is washed with ethyl acetate or the like, adjusted to alkalinity with an aqueous sodium hydroxide solution or the like, and then sodium chloride or the like is added to dissolve it. A solvent such as dichloromethane is added, stirred and separated, and the obtained organic layer is concentrated under reduced pressure. A solvent such as acetonitrile is added, and after concentrated under reduced pressure, the obtained solution is filtered, washed with a solvent such as acetonitrile, and the obtained solution is concentrated under reduced pressure (extraction process), and the compound represented by formula A-21 with high purity by HPLC can be obtained. More preferably, it can be carried out, for example, by the method shown in Examples 46 to 50.

[0808] It should be noted that the purification of the compound represented by the above formula A-21 is not limited to the purification in this step. Therefore, in one embodiment of the present invention, a purification method for the compound represented by the formula A-21 is also provided, which comprises: adding the compound represented by the above formula E-1 (wherein R7 is a hydrogen atom, a methyl group, or a methoxy group) to a solution containing the crude compound represented by the above formula A-21 to produce the crystalline compound represented by the above formula E-2 (wherein R7 is a hydrogen atom, a methyl group, or a methoxy group); isolating the crystalline compound, and then extracting the compound represented by the formula A-21 from the isolated crystalline compound.

[0809] <New Compound>

[0810] The method of the present invention, comprising steps I-1 to I-5, can produce the target oligosaccharide represented by formula A-22. Intermediates of the oligosaccharide represented by formula A-22 are useful in the production of the oligosaccharide, but are not limited to the production of the oligosaccharide itself and can be used in a variety of applications. Therefore, the present invention provides the oligosaccharide represented by formula A-22 and its intermediates.

[0811] In one embodiment of the present invention, there is provided an oligosaccharide represented by the following formula A-22.

[0812]

[0813] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0814] In one embodiment of the present invention, there is provided an oligosaccharide represented by the following formula A'-22.

[0815]

[0816] In one embodiment of the present invention, an oligosaccharide represented by the following formula A"-22 is provided.

[0817]

[0818] In one embodiment of the present invention, a compound represented by the following formula A-8 is provided.

[0819]

[0820] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0821] In one embodiment of the present invention, a compound represented by the following formula A-10 is provided.

[0822]

[0823] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0824] In one embodiment of the present invention, a compound represented by the following formula A-11 is provided.

[0825]

[0826] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0827] In one embodiment of the present invention, a compound represented by the following formula A-13 is provided.

[0828]

[0829] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0830] The following compound A-14 is provided.

[0831]

[0832] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0833] In one embodiment of the present invention, a compound represented by the following formula A-14-FMA is provided.

[0834]

[0835] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0836] In one embodiment of the present invention, a compound represented by the following formula A-15 is provided.

[0837]

[0838] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0839] In one embodiment of the present invention, a compound represented by the following formula A-17 is provided.

[0840]

[0841] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group (COOAr), an acetyl group (Ac), or a 2,2,2-trichloroethoxycarbonyl group (Troc), and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group).

[0842] In one embodiment of the present invention, there is provided a compound represented by the following formula A-18-FF or a salt thereof.

[0843]

[0844] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0845] In one embodiment of the present invention, a compound represented by the following formula A-18 is provided.

[0846]

[0847] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0848] In one embodiment of the present invention, there is provided a compound represented by the following formula A-19-FF or a salt thereof.

[0849]

[0850] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0851] In one embodiment of the present invention, a compound represented by the following formula A-19 is provided.

[0852]

[0853] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0854] In one embodiment of the present invention, there is provided a compound represented by the following formula A-20-FF or a salt thereof.

[0855]

[0856] (wherein R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups).

[0857] In one embodiment of the present invention, there is provided a compound represented by the following formula A'-20-FF or a salt thereof.

[0858]

[0859] In one embodiment of the present invention, a compound represented by the following formula A-20 is provided.

[0860]

[0861] (wherein, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1-3 C1-C6 alkoxy groups, M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation).

[0862] <Glycoproteins, etc. and methods for producing the same>

[0863] In one embodiment of the present invention, novel glycoproteins and the like are provided, which utilize biantennary glycans (i.e., oligosaccharides represented by formula A-22) as donor molecules for synthesizing glycoproteins, etc. (particularly, sugar chain-remodeled antibodies or molecules comprising their Fc regions, or antibody-drug conjugates), and novel methods for producing the same. The biantennary glycans have an α2,6-sialic acid structure at their non-reducing ends. As described in detail below, the oligosaccharide represented by formula A-22 obtained by the production method of the present invention can be used for the production of glycoproteins (especially sugar chain remodeled antibodies or molecules containing Fc regions, or antibody-drug conjugates) (for the method of coupling oligosaccharides to antibodies using the "one-pot method", refer to WO2018 / 003983, WO2022 / 050300, PLoS ONE2018,13,e0193534. In addition, for the method of coupling oligosaccharides to antibodies using the oxazolidinylation method, refer to WO2019 / 065964, WO2020 / 050406, etc.), but is not limited to this and can also be used for other purposes.

[0864] In recent years, methods have been reported for uniformly introducing functional group-containing sugar chains by remodeling heterogeneous antibody sugar chains through enzymatic reactions (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Attempts have been made to use this sugar chain remodeling technology to site-specifically introduce drugs and synthesize uniform antibody-drug conjugates (ADCs) (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0865] Sugar chain reconstruction begins by using a hydrolase to remove the heterogeneous sugar chains added to proteins (e.g., antibodies) leaving only the terminal N-acetylglucosamine (GlcNAc) residues. This creates a homogeneous protein portion with GlcNAc residues (hereinafter referred to as the "acceptor molecule"). Next, a separate, arbitrary sugar chain (hereinafter referred to as the "donor molecule") is prepared, and the acceptor and donor molecules are linked using a glycosyltransferase. This allows the synthesis of uniform glycoproteins with arbitrary sugar chain structures.

[0866] In one embodiment of the present invention, the oligosaccharide represented by formula A-22 produced by the novel production method of the present invention can be used as a donor molecule for the synthesis of the above-mentioned uniform glycoprotein (particularly, a sugar chain-remodeled antibody or a molecule comprising the Fc region thereof) by activating its terminal structure.

[0867] In the present invention, a "sugar chain" refers to a structural unit composed of two or more monosaccharides linked via glycosidic bonds. Specific monosaccharides or sugar chains are sometimes represented by abbreviations such as "GlcNAc-." When structural formulas are described using these abbreviations, the oxygen or nitrogen atoms at the reducing end that are part of the glycosidic bond to another structural unit are not included in the abbreviation representing the sugar chain, unless otherwise specified.

[0868] In the present invention, when describing the monosaccharide that serves as the basic unit of the sugar chain, for convenience, the carbon atom that is bonded to the oxygen atom that constitutes the ring and directly bonded to the hydroxyl group (or the oxygen atom belonging to the glycosidic bond) in the ring structure is designated as position 1 (position 2 only in the case of sialic acid). The names of the example compounds are named based on the overall chemical structure, and this rule does not necessarily apply.

[0869] In the present invention, when a sugar chain is described as a symbol (e.g., GlcNAc), unless otherwise defined, the carbon to the reducing terminal is considered to be included in the symbol, and N or O belonging to the N- or O-glycosidic bond is considered to be excluded.

[0870] In the present invention, "glycoprotein" refers to a substance in which a part of amino acids constituting protein is bound to a sugar chain.

[0871] In one embodiment of the present invention, a "glycoprotein" is a glycoprotein-remodeled antibody or a molecule comprising its Fc region. Therefore, in one embodiment of the present invention, a method for producing a glycoprotein-remodeled antibody or a molecule comprising its Fc region is provided, comprising: a step of obtaining a glycoprotein donor molecule, including the novel method for producing oligosaccharides of the present invention; and a step of reacting the glycoprotein donor molecule with an acceptor molecule, wherein the acceptor molecule is an antibody or a molecule comprising its Fc region having a core GlcNAc residue optionally supplemented with fucose as an N297-linked sugar chain. In the present invention, "core GlcNAc residue" refers to the GlcNAc residue at the reducing end of the N297-linked sugar chain.

[0872] In the present invention, "sugar chain donor molecule" refers to a molecule that plays the role of conferring sugar chains on acceptor molecules. In one embodiment of the present invention, the sugar chain donor molecule is an oligosaccharide represented by formula A-22. In addition, in a preferred embodiment of the present invention, the oligosaccharide represented by formula A-22 is an oligosaccharide in which R1 is 4-methoxyphenyl or isopropyl, but is not limited to this. It should be noted that in the present invention, the oligosaccharide in which R1 is substituted by isopropyl from 4-methoxyphenyl can be prepared by using a compound (compound represented by formula A"-9) in which the 4-methoxyphenyl of the compound is substituted by isopropyl instead of the compound represented by formula A'-9 (refer to Example 13, Example 60, etc.).

[0873] In the present invention, an "acceptor molecule" refers to a molecule that accepts sugar chains from a sugar chain donor molecule. The "acceptor molecule" is not particularly limited as long as it can accept sugar chains from a sugar chain donor molecule. Preferred examples include deglycosylated antibodies obtained by cleaving most of the sugar chains from an antibody, or molecules comprising the Fc region thereof. More preferred examples include antibodies having a core GlcNAc, or molecules comprising the Fc region thereof. The partially deglycosylated sugar chains in deglycosylated antibodies are either N-linked or O-linked, with N-linked sugar chains being preferred.

[0874] N-linked sugar chains are linked to the amino acid side chains of antibodies via N-glycosidic bonds, and O-linked sugar chains are linked to the amino acid side chains of antibodies via O-glycosidic bonds.

[0875] The antibody serving as the "receptor molecule" is preferably IgG, more preferably IgG1, IgG2, or IgG4.

[0876] It is known that the asparagine residue at position 297 (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain of IgG has a highly conserved N-linked sugar chain (hereinafter referred to as "Asn297-linked sugar chain or N297-linked sugar chain"), which contributes to the activity, dynamics, etc. of the antibody molecule (Eon-Duval, A. et al, Biotechnol. Prog. 2012, 28, 608-622, Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0877] The amino acid sequence in the constant region of IgG is highly conserved. In a report by Edelman et al. (Proc. Natl. Acad. Sci. USA, 63, 78-85, (1969)), each amino acid is identified by the EU numbering system (EU INDEX). For example, Asn297, which is attached to an N-linked sugar chain in the Fc region, corresponds to position 297 in the EU numbering system. Even if the actual amino acid position varies due to fragmentation or deletion of a region of the molecule, the amino acid is unambiguously identified by the EU numbering system.

[0878] In one embodiment of the present invention, a "receptor molecule" is an antibody having a core GlcNAc as an N297-linked sugar chain, or a molecule comprising the Fc region thereof. Other monosaccharides or sugar chains, such as fucose, may be added to the core GlcNAc as an N297-linked sugar chain. Therefore, in one embodiment of the present invention, a "receptor molecule" is an antibody having a core GlcNAc as an N297-linked sugar chain, optionally with fucose, or a molecule comprising the Fc region thereof.

[0879] The step of reacting a sugar chain donor molecule with an acceptor molecule, which is an antibody having a core GlcNAc optionally with fucose added as an N297-linked sugar chain or a molecule comprising the Fc region thereof, can employ any reaction method and reaction conditions as long as the reaction proceeds.

[0880] In the present invention, the sugar chain remodeled antibody or a molecule comprising the Fc region thereof can be produced by the method shown in the following formula according to the methods described in WO2018 / 003983 and WO2022 / 050300, for example.

[0881]

[0882] (D-1 process)

[0883] This step is a step of producing a sugar chain-cleaved antibody by hydrolyzing the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing terminal chitobiose structure of the target antibody, which is linked to the asparagine at position 297 of the antibody's amino acid sequence, using a known enzymatic reaction. The target antibody (1d) is subjected to a hydrolysis reaction of the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing terminal chitobiose structure in a buffer (phosphate buffer, etc.) at 0°C to 40°C using a hydrolase such as wild-type Endo-S enzyme. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 16 hours. 0.005 mg to 10 mg, preferably 0.01 mg to 3 mg, of wild-type Endo-S enzyme is used per 100 mg of antibody (1d). After the reaction is completed, the (Fucα1,6)GlcNAc antibody (2d) is purified by a purification method suitable for the reaction scale (affinity chromatography, hydroxyapatite column, cation exchange chromatography, multimodal chromatography, etc.).

[0884] (D-2 process)

[0885] This step is a step in which the (Fucα1,6)GlcNAc antibody (2d) obtained in step D-1 is reacted with the oligosaccharide represented by formula A-22 by a sugar chain transfer reaction using two Endo enzymes to prepare a reaction solution containing the sugar chain-remodeled antibody (3d), and then purified by an appropriate method to produce the target product (3d).

[0886] The two Endo enzymes used may be an appropriate combination of enzyme A (Endo-S-like enzyme) and enzyme B (Endo-M-like enzyme).

[0887] In the present invention, "enzyme-A" refers to an endo-β-N-acetylglucosaminidase that uses N297-linked sugar chains of Fc-containing molecules (e.g., antibodies) as substrates. Examples of enzyme A include Endo-S, Endo-S2 (EndoS-49), Endo-Si, Endo-Sd, Endo-Se, or Endo-Sz, as well as Endo-S mutants, Endo-S2 (Endo-S49) mutants, Endo-Si mutants, Endo-Sd mutants, Endo-Se mutants, or Endo-Sz mutants that have reduced hydrolytic activity.Preferred enzymes as enzyme A include Endo-SD233Q, Endo-SD233Q / Q303L, Endo-SD233Q / E350A, Endo-SD233Q / E350Q, Endo-SD233Q / E350D, Endo-SD233Q / E350N, Endo-SD233Q / D405A, Endo-S2 D184M, Endo-S2 T138Q, Endo-S2 D184Q, Endo-S2 D184Q / Q250L, Endo-S2 D184Q / E289Q, Endo-S2 D184M / Q250L, Endo-S2 D184M / E289Q, Endo-S2 D182Q, Endo-S2 D226Q, Endo-S2 T227Q, and Endo-S2 T228Q, Endo-Si D241Q / Q311L, Endo-Si D241Q / E360Q, Endo-Si D241M, Endo-Si D241M / Q311L, Endo-Si D241M / E360Q, Endo-Si T190Q, Endo-Si T190 / D241Q, Endo-Si T190Q / D241M, Endo-Si D241X1 (X1 represents any amino acid residue other than K, R and D, specifically A, N, C, Q, E, G, H, I, L, M, F, P, S, T, W, Y or V, preferably A, Q, E, H, I, L, M, F, P, S, T, W, Y or V), Endo-Si T190X2 (X2 represents an amino acid residue of F, H, K, M, Q, R, W or Y), Endo-Si Q311X3 (X3 represents an amino acid residue of F, N or Y), Endo-SiE360K, Endo-Sd D232Q, Endo-Sd D232M, Endo-Sd D232Q / Q302L, Endo-Sd D232M / Q302L, Endo-Se D233Q, Endo-Se D233M, Endo-Se D233Q / Q303L, Endo-Se D233M / Q303L, Endo-Sz D234Q, Endo-Sz D234M, Endo-Sz D234Q / Q304L, or Endo-Sz D234M / Q304L.

[0888] In the present invention, "enzyme-B" refers to an endo-β-N-acetylglucosaminidase that uses the sugar chains of sugar chain donor molecules as substrates, particularly an endo-β-N-acetylglucosaminidase that uses the sugar chains of sugar chain donor molecules as substrates but not N297-linked sugar chains (in other words, has low reactivity, preferably extremely low reactivity, with N297-linked sugar chains). Examples of enzyme B include Endo-M, Endo-Om, Endo-CC, or Endo-Rp, as well as Endo-M mutants, Endo-Om mutants, Endo-CC mutants, or Endo-Rp mutants with reduced hydrolytic activity. Preferred enzymes as enzyme B include Endo-M N175Q, Endo-M N175Q / Y217F, Endo-CC N180H, Endo-Om N194Q, Endo-Rp N172Q, Endo-Rp N172H, Endo-Rp N172A, Endo-Rp N172C, Endo-Rp N172D, Endo-RpN172E, Endo-Rp N172G, Endo-Rp N172I, Endo-Rp N172L, Endo-Rp N172M, Endo-RpN172P, Endo-Rp N172S, Endo-Rp N172T, Endo-Rp N172V, Endo-Rp W278F / S216V, Endo-Rp W278F / N246D, Endo-Rp W278F / D276N, Endo-Rp W278F / A310D, Endo-Rp W278F / N172D / F307Y, Endo-Rp W278F / N172D / F307H, Endo-Rp W278F / N172D / A310D, Endo-Rp W214F / F307Y / L306I, etc.

[0889] The sugar chain transfer reaction is carried out by reacting the antibody (2d) with the oligosaccharide represented by formula A-22 in a buffer (Tris buffer, etc.) in the presence of enzyme A (Endo-S enzyme-like) and enzyme B (Endo-M enzyme-like) glycosyltransferase. The reaction temperature can be appropriately selected according to the optimal temperature of the enzyme used, usually 15 to 50°C, preferably 25 to 40°C. The reaction time can be appropriately selected between 2 hours and 48 hours. After the reaction is completed, a purification method (affinity chromatography, hydroxyapatite column, cation exchange chromatography, multimodal chromatography, etc.) and an ultrafiltration method (ultrafiltration membrane) suitable for the reaction scale are selected to obtain the sugar chain remodeled antibody (3d).

[0890] In the preparation of the above-mentioned sugar chain-remodeled antibody, concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be performed according to the common procedures A to C described in WO2020 / 050406.

[0891] As described above, the method for producing a sugar chain remodeled antibody or a molecule comprising the Fc region thereof in the present invention may further include the following step: reacting each or both of the two azide groups (N3-) at the terminal of the compound represented by formula A-22 with a molecule having an alkyne structure. This reaction step can be performed before or after the step of reacting the sugar chain donor molecule with the acceptor molecule. In the present invention, the "molecule having an alkyne structure" can be any molecule as long as it has an alkyne structure, for example, chemotherapeutic agents, molecular targeted drugs, immunostimulants, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins and hormones. In the present invention, the "step of reacting an azide group (N3-) with a molecule having an alkyne structure" can use all reaction methods and reaction conditions as long as the reaction is carried out. For example, it can be performed according to known methods (WO2019 / 065964, WO2020 / 050406, etc.). For example, the "step of reacting an azide group (N3-) with a molecule having an alkyne structure" can be performed as follows: the sugar chain-remodeled antibody (3d) obtained in the above-mentioned step D-2 is reacted with a molecule having an alkyne structure through SPAAC (strain-promoted azide-alkyne cycloaddition: J. Am. Chem. Soc. 2004, 126, 15046-15047) to bind.

[0892] In one embodiment of the present invention, a method for producing an antibody-drug conjugate is also provided, comprising a method for producing the aforementioned sugar-chain remodeled antibody or a molecule comprising its Fc region. In the present invention, the antibody and drug contained in the "antibody-drug conjugate" are not limited as long as they exert the desired effect, and all substances can be used depending on the target. In the present invention, as an example of an antibody-drug conjugate production method, a production method comprising the aforementioned step D-1, step D-2, and SPAAC reaction can be cited, but is not limited thereto.

[0893] In the present invention, an "antibody-drug conjugate" refers to a conjugate in which a drug is bound to an antibody via a linker. By using the method of the present invention, a drug can be site-specifically introduced, and a uniform antibody-drug conjugate can be synthesized.

[0894] In the present invention, the "antibody drug conjugate" is represented by the following formula (XIII).

[0895]

[0896] m 1 The range is 1 to 10, indicating the number of drug-bound molecules per antibody molecule in the antibody-drug conjugate. Ab represents an antibody or a functional fragment of the antibody, L represents a linker connecting Ab to D, and D represents the drug.

[0897] As the antibody used in the present invention, a complete antibody (full-body) and a functional fragment of an antibody can be listed. "Functional fragment of an antibody" is also referred to as "antigen-binding fragment of an antibody", which refers to a partial fragment of an antibody having binding activity with an antigen, including Fab, F(ab')2, Fv, scFv, double-chain antibodies (diabody), linear antibodies, and multispecific antibodies formed by antibody fragments. In addition, the monovalent fragment Fab' of the variable region of an antibody after F(ab')2 is treated under reducing conditions is also included in the antigen-binding fragment of an antibody. Among them, as long as there is binding ability with an antigen, it is not limited to these molecules. In addition, among these antigen-binding fragments, not only molecules after the full-length molecules of the antibody protein are treated with appropriate enzymes are included, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0898] In the present invention, the antibody may be derived from any species, preferably humans, rats, mice, and rabbits. In the case of species other than humans, chimerization or humanization is preferably performed using known techniques. In addition, in the present invention, the antibody may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, humanized antibodies, functional fragments thereof, or modified forms thereof.

[0899] The antibodies used in the present invention are not limited as long as they exhibit the desired effects, and examples thereof include anti-HER2 antibodies, anti-HER3 antibodies, anti-DLL3 antibodies, anti-FAP antibodies, anti-CDH11 antibodies, anti-CDH6 antibodies, anti-A33 antibodies, anti-CanAg antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD98 antibodies, anti-TROP2 antibodies, anti-CEA antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUC1 antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-Mesothelin antibodies, anti-ENPP3 antibodies, anti-CD47 antibodies, anti-EGFR antibodies, anti-GPR20 antibodies, and anti-DR5 antibodies.

[0900] The antibodies used in the manufacture of the antibody-drug conjugates of the present invention can be obtained by immunizing animals with a polypeptide serving as an antigen, collecting and purifying the antibodies produced in the organism using methods commonly used in the art. The source of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice and rats. In this case, antibodies that bind to the obtained heterologous antigens can be tested for cross-reactivity with human antigens to select antibodies suitable for use in human diseases.

[0901] Alternatively, monoclonal antibodies can be obtained by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells to establish hybridomas according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497, Kennett, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0902] It should be noted that antigens can be obtained by using genetic manipulation to produce genes encoding antigenic proteins in host cells.

[0903] The humanized antibodies used for the production of the antibody-drug conjugates of the present invention can be obtained according to known methods (eg, Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984), Nature (1986) 321, pp. 522-525, WO90 / 07861).

[0904] For example, anti-HER2 antibodies (US5821337, WO2004 / 008099, WO2020 / 050406, etc.), anti-CD33 antibodies (WO2014 / 057687, WO2020 / 050406, etc.), anti-EphA2 antibodies (WO2009 / 028639, WO2020 / 050406, etc.), anti-CDH6 antibodies (WO2018 / 212136, WO2020 / 050406, etc.), anti-CD70 antibodies (WO2004 / 073656, WO2007 / 038637, etc.), anti-TROP2 antibodies (WO2015 / 098099, etc.), and anti-EGFR antibodies (WO1998 / 050433, WO2002 / 092771, etc.) can be obtained by known means.

[0905] The drugs used in the present invention are not limited as long as they exert the desired effect, and include pharmaceutically active compounds such as chemotherapeutic agents, molecular targeted drugs, immunostimulants, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins, hormones, etc.

[0906] Drug D is represented by, for example, the following formula.

[0907]

[0908]

[0909] (wherein the asterisk indicates binding to the linker L).

[0910] In the present invention, the linker L that links the antibody Ab and the drug D is represented by the following formula.

[0911] -Lb-La-Lp-Lc-*

[0912] (Here, the asterisk indicates binding to drug D).

[0913] Lp represents a linker consisting of an amino acid sequence that can be cleaved in target cells, or Lp is absent. Specific examples thereof include -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, or -GGPP-.

[0914] La represents a member selected from -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -(CH2)n 4 -O-C(=O)-, or -(CH2)n 9 -C(=O)-. (Here, n 2 Indicates an integer from 1 to 3, n 3 Integer from 1 to 5, n 4Integer from 0 to 2, n 9 represents an integer from 2 to 7).

[0915] Lb represents a spacer that binds La to the sugar chain or remodeled sugar chain of Ab, and specific examples thereof include:

[0916]

[0917] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain or remodeled sugar chain of Ab).

[0918] Lc represents -NH-CH2-, -NH-B-CH2-O(C=O)-, or Lc is absent. Here, B is 1,4-phenyl, 2,5-pyridyl, 3,6-pyridyl, 2,5-pyrimidinyl, or 2,5-thienyl.

[0919] The present invention will be described below by way of examples, but the present invention is not limited to these examples. It should be noted that, for example, "Compound A-1" or "A-1" in the synthesis route represents the compound corresponding to "the compound represented by formula A-1", and the same applies to the compound numbers thereafter.

[0920] In the following examples, room temperature means 15°C to 35°C. Silica gel chromatography was performed using Biotage Sharp HC D (20 μm, manufactured by Biotage), reverse phase column chromatography was performed using Universal Column ODS Premium 30 μm L size (manufactured by Yamazen Co., Ltd.) and Inject column ODS L size (manufactured by Yamazen Co., Ltd.), and preparative HPLC was performed using Agilent Preparative HPLC System (manufactured by Agilent Technology). The preparative column used was XBridge Prep OBD (5 μm, C18, 250×30 mm, made by Waters).

[0921] The following equipment was used to measure various spectral data. 1 H-NMR and 13 C-NMR spectra were measured using ECZ500R and ECX400P manufactured by JEOL, and mass spectra were measured using Shimadzu LCMS-2010 and LCMS-2020 (manufactured by Shimadzu Corporation), XEVO Q-Tof MS (Waters), and Q-Exactive (Thermo Fisher).

[0922] <Synthesis of the compound represented by formula A-4>

[0923] The compound represented by Formula A-4 was synthesized according to the following synthetic route 1.

[0924] [Synthesis Path 1]

[0925]

[0926] Example 1

[0927] 1,2:5,6-bis-O-(1-methylethylidene)-3-O-(2-naphthylmethyl)-α-D-glucofuranose (compound represented by formula C-2)

[0928]

[0929] After cooling a solution of sodium hydride (55.32 g, 1.38 mol, content: 50%-72%) in tetrahydrofuran (900 mL) to 0°C, a 1,2:5,6-bis-O-(1-methylethylidene)-α-D-furanose solution (the compound represented by Formula C-1) (300.00 g, 1.15 mol) in tetrahydrofuran (1.05 L) was added dropwise over 1 hour. The temperature was then raised to 25°C, and 1,3-dimethyl-2-imidazolidinone (150 mL) and 2-bromomethylnaphthalene (280.31 g, 1.27 mol) were added. After stirring at 25°C for 6 hours, the reaction was confirmed to be complete by HPLC, and ethylenediamine (anhydrous) (13.85 g, 230.52 mmol) was added, followed by further stirring for 1 hour. The solution was cooled to 0°C, and 10% aqueous citric acid (1.2 L) was added over 1 hour. The reaction mixture was diluted with heptane (3 L) and separated into an organic layer and an aqueous layer. The organic layer was washed with water (900 mL) and concentrated under reduced pressure to 900 mL. Acetonitrile (3 L) was further added and the mixture was concentrated again to 900 mL. The crude product, 1,2:5,6-bis-O-(1-methylethylidene)-3-O-(2-naphthylmethyl)-α-D-furanose (the compound represented by Formula C-2), was obtained as an acetonitrile solution. This product was used directly in the next step.

[0930] Example 2

[0931] 3-O-(2-naphthylmethyl)-D-glucopyranose (compound represented by formula C-3)

[0932]

[0933] Acetonitrile (1.5 L), water (600 mL) and concentrated hydrochloric acid (17.51 ​​g, 172.89 mmol) were added to a solution (900 mL) of the compound represented by the crude product formula C-2 obtained in Example 1, and the mixture was stirred at 55°C for 18.5 hours. After the reaction was confirmed to be complete by HPLC, the reaction solution was cooled to 0°C and the pH in the system was adjusted to 6.25 with 4N aqueous sodium hydroxide solution (43.22 mL). The reaction solution was diluted with heptane (900 mL) and separated into an acetonitrile layer and a heptane layer. Ethyl acetate (2.4 L) and water (600 mL) were added to the acetonitrile layer for liquid separation to obtain an organic layer A and an aqueous layer. A mixed solution of ethyl acetate (1.5 L) and tetrahydrofuran (1.5 L) was added to the aqueous layer for liquid separation to obtain an organic layer B and an aqueous layer. Organic layers A and B were mixed, washed with saturated brine (600 mL), and then concentrated under reduced pressure to a volume of 1.5 L (precipitation of crystals was confirmed during the concentration stage). Ethyl acetate (4.5 L) was further added, and the suspension was concentrated again to a volume of 3 L. Ethyl acetate (1.5 L) and cyclopentyl methyl ether (1.5 L) were added to the suspension, and the mixture was stirred at 55°C for 1 hour. Heptane (3 L) was added dropwise over 1.5 hours, stirred for 1 hour, and then cooled to 0°C. The precipitated crystals were then filtered and washed with a mixed solution of ethyl acetate (1.2 L) and heptane (600 mL) cooled to 0°C. The resulting crystals were dried at 40°C under reduced pressure to obtain 3-O-(2-naphthylmethyl)-D-pyranose glucose (compound represented by Formula C-3) (356.95 g, 96.7% yield).

[0934] 1 H-NMR (500MHz, DMSO-d6) δ7.85-7.90 (m, 4H), 7.59 (dd, J=8.0, 1.5Hz, 1H), 7. 46-7.51 (m, 2H), 6.69 (d, J=6.0Hz, 1H), 5.12 (dd, J=5.0, 3.0Hz, 2H), 4.94-5.0 0 (m, 2H), 4.53 (t, J=6.0Hz, 1H), 4.35 (dd, J=8.0, 6.5Hz, 1H), 3.70 (ddd, J=11 .5, 5.0, 2.0Hz, 1H), 3.45-3.50 (m, 1H), 3.25-3.31 (m, 2H), 3.11-3.16 (m, 2H).

[0935] 13 C-NMR (125MHz, DMSO-d6) δ137.3, 132.8, 132.3, 127.6, 127.5, 127.4, 126.1, 126.0, 125.6, 125.5, 96.9, 85.4, 76.7, 74.8, 73.7, 69.9, 61.1.

[0936] HRMS(ESI － )[M-H] － C 17 H 19 Theoretical value of O6: 319.1187; experimental value 319.1175.

[0937] Example 3

[0938] 2,4,6-tri-O-acetyl-3-O-(2-naphthylmethyl)-D-glucopyranose (compound represented by formula C-5)

[0939]

[0940] To a solution of 3-O-(2-naphthylmethyl)-D-glucopyranose (the compound represented by Formula C-3) (150.00 g, 468.25 mmol) in tetrahydrofuran (675 mL) were added triethylamine (236.92 g, 2.34 mol) and 4-dimethylaminopyridine (0.29 g, 2.34 mmol). After cooling to 0°C, acetic anhydride (195.99 g, 1.92 mol) was added dropwise over 30 minutes. The temperature was then raised to 25°C and stirred for 3 hours. Completion of the reaction was confirmed by HPLC. The reaction mixture was cooled to 10°C and 1-methylpiperazine (60.97 g, 608.73 mmol) was added. After stirring at 35°C for 18 hours, completion of the reaction was confirmed by HPLC and the mixture was cooled to 0°C. The pH was adjusted to 6.36 with 6N hydrochloric acid (480 mL), the mixture was diluted with heptane (375 mL), and the organic and aqueous layers were separated. The organic layer was washed with saturated sodium bicarbonate aqueous solution (450 mL) and water (450 mL), then concentrated under reduced pressure to 450 mL. Ethyl acetate (2.25 L) was added, and the solution was concentrated again to 450 mL. The same procedure was repeated. Dichloromethane (2.25 L) was added to the solution, and the solution was concentrated to 450 mL. The same procedure was repeated again, yielding the crude product, 2,4,6-tri-O-acetyl-3-O-(2-naphthylmethyl)-D-glucopyranose (the compound represented by Formula C-5), as a dichloromethane solution. This product was used directly in the next step.

[0941] Note that the step of converting the compound represented by Formula C-3 to the compound represented by Formula C-5 was carried out by a one-pot method.

[0942] Example 4

[0943] 2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-1-O-(2,2,2-trichloroethaneimido)-D-glycerol-hexopyranose (compound represented by formula C-6)

[0944]

[0945] To a solution (450 mL) of the crude product 2,4,6-tri-O-acetyl-3-O-(2-naphthylmethyl)-D-glucopyranose (the compound represented by Formula C-5) obtained in Example 3, dichloromethane (450 mL) and trichloroacetonitrile (338.03 g, 2.34 mol) were added. After cooling to 0°C, 1,8-diazabicyclo[5.4.0]-7-undecene (5.70 g, 37.46 mmol) was added dropwise. After stirring at 0°C for 14.5 hours, completion of the reaction was confirmed by HPLC, and acetic acid (2.25 g, 37.46 mmol) was added. Silica gel 60N (manufactured by Kanto Chemical, particle size: 40-50 μm, 150 g) was added to the solution, stirred for 1.5 hours, and then filtered. The silica gel was washed with dichloromethane (1.5 L), and the filtrate was concentrated under reduced pressure to a volume of 450 mL. Dichloromethane (1.5 L) was then added and the mixture was concentrated to 450 mL. This yielded 2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-1-O-(2,2,2-trichloroethaneimido)-D-glycero-hexopyranose (compound represented by Formula C-6) as a dichloromethane solution. This product was used directly in the next step.

[0946] Example 5

[0947] 4-Methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (compound represented by formula C-8)

[0948]

[0949] To a solution (450 mL) of 2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-1-O-(2,2,2-trichloroethaneimido)-D-glycero-hexopyranose (compound represented by Formula C-6) obtained in Example 4 were added 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula C-7) (276.66 g, 468.25 mmol), dichloromethane (4.2 L), and molecular sieves 4A powder (10 μm or less, 83.00 g), and the mixture was cooled to -5°C. Trimethylsilyl trifluoromethanesulfonate (10.41 g, 46.83 mmol) was added dropwise to the suspension over 20 minutes, followed by stirring for 3 hours. After confirming the completion of the reaction by HPLC, triethylamine (23.69 g, 234.13 mmol) was added. The suspension was filtered and washed with ethyl acetate (2.8 L). The filtrate was concentrated under reduced pressure to a volume of 1.4 L. Ethyl acetate (4.2 L) was further added and concentrated to a volume of 1.4 L. The same procedure was repeated once more. Ethyl acetate (2.8 L) was added to the solution, washed with saturated sodium bicarbonate aqueous solution (830 mL) and water (830 mL), and the organic layer was concentrated under reduced pressure to a volume of 830 mL. 2-Propanol (4.2 L) was further added and concentrated to a volume of 1.4 L. The suspension was then temperature-adjusted to 65°C. Ethyl acetate (830 mL) was added and stirred at 65°C for 2 hours. 2-Propanol (5.53 L) was then added dropwise over 2 hours. The suspension was cooled to 0°C, the crystals were filtered, and the crystals were washed with 2-propanol (1.4 L) cooled to 0°C. The obtained crystals were dried at 40° C. under reduced pressure to obtain a crude product of 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-β-D-pyranoglucopyranosyl}-β-D-pyranoglucopyranoside (the compound represented by Formula C-8) (355.34 g, a yield of 90.5%, based on the compound represented by Formula C-3).

[0950] Methyl isobutyl ketone (2.1 L) was added to the resulting crude product, the compound represented by Formula C-8 (350.00 g), and the mixture was dissolved at 50°C. Ethylcyclohexane (1.4 L) was then added dropwise over 1 hour. 4-Methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (the compound represented by Formula C-8) (70.00 mg) was added and stirred for 1 hour. After confirming the precipitation of crystals, ethylcyclohexane (4.9 L) was added dropwise over 2 hours. The suspension was cooled to room temperature and stirred for 14.5 hours. The precipitated crystals were filtered and washed with a mixed solution of methyl isobutyl ketone (350 mL) and ethylcyclohexane (1.4 L). The resulting crystals were dried at 40°C under reduced pressure to obtain 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (compound represented by Formula C-8) (331.74 g, yield 94.8%).

[0951] 11H-NMR (500 MHz, CDCl3) δ 7.81 - 7.84 (m, 4H), 7.69 (br, 1H), 7.65 (br, 3H), 7.46 - 7.51 (m, 2H), 7.28 - 7.36 (m, 6H), 7.00 (dd, J = 7.0, 1.5 Hz, 2H), 6.77 - 6.84 (m, 5H), 6.66 - 6.69 (m, 2H), 5.59, (d, J = 9.0 Hz, 1H), 5.09 - 5.15 (m, 2H), 4.82 (d, J = 12.5 Hz, 1H), 4.77 (d, J = 12.0 Hz, 1H), 4.71 - 4.77 (m, 2H), 4.60 (d, J = 8.0 Hz, 1H), 4.50 (d, J = 12.5 Hz, 1H), 4.45 (d, J = 13.0 Hz, 1H), 4.36 (dd, J = 11.0, 8.5 Hz, 1H), 4.28 (dd, J = 11.0, 8.5 Hz, 1H), 4.20 (dd, J = 12.5, 5.0 Hz, 1H), 4.10 (dd, J = 10.0, 8.5 Hz, 1H), 3.99 (dd, J = 12.0, 2.0 Hz, 1H), 3.80 (br, 2H), 3.69 (s, 3H), 3.58 - 3.62 (m, 2H), 3.44 (ddd, J = 10.0, 4.5, 2.5 Hz, 1H), 1.98 (s, 3H), 1.938 (s, 3H), 1.937 (s, 3H).

[0952] 13 13C-NMR (125 MHz, CDCl3) δ 171.0, 169.5, 169.1, 155.6, 151.0, 138.7, 138.2, 135.5, 133.9, 133.4, 133.2, 128.7, 128.4, 128.23, 128.20, 128.06, 128.05, 127.9, 127.2, 126.5, 126.2, 125.7, 123.5, 118.9, 114.5, 100.7, 97.8, 80.6, 78.5, 76.8, 75.2, 74.8, 74.1, 73.8, 73.1, 72.1, 69.9, 67.8, 62.2, 55.78, 55.75, 21.1, 20.94, 20.85.

[0953] HRMS (ESI + ) [M + H] + C 58 H 58 NO 16 Calculated: 1024.3750; Found: 1024.3706.

[0954] The spectrum was confirmed to be consistent with the following literature.

[0955] Reference 1) Org. Biomol. Chem., 2018, 16, 4720-4727.

[0956] Example 6

[0957] 4-Methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (compound represented by formula C-9)

[0958]

[0959] To a solution of 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{2,4,6-tri-O-acetyl-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (the compound represented by Formula C-8) (30.00 g, 29.29 mmol) in tetrahydrofuran (150 mL) were added methanol (90 mL) and methyl trifluoroacetate (3.75 g, 29.29 mmol). After stirring at 25°C for 10 minutes, potassium tert-butoxide (1 mol / L tetrahydrofuran solution) (14.7 mL, 14.65 mmol) was added. The temperature was then raised to 55°C and stirred for 2 hours. Completion of the reaction was confirmed by HPLC. The reaction mixture was cooled to 25°C, and acetic acid (1.76 g, 29.29 mmol) and ethyl acetate (300 mL) were added sequentially. The solution was washed twice with 1% aqueous sodium chloride solution (300 mL) and then concentrated under reduced pressure to 90 mL. Ethyl acetate (450 mL) was added, and the solution was concentrated again to 90 mL. Acetonitrile (450 mL) was further added and concentrated to 90 mL. This yielded 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (compound represented by Formula C-9) as an acetonitrile solution. This product was used directly in the next step.

[0960] Example 7

[0961] 4-Methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula C-10)

[0962]

[0963] Acetonitrile (210 mL), benzaldehyde dimethyl acetal (5.13 g, 33.69 mmol), and p-toluenesulfonic acid monohydrate (0.17 g, 0.88 mmol) were added to a solution (90 mL) of 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-4-O-{3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-β-D-glucopyranoside (the compound represented by Formula C-9), and the mixture was stirred at 25°C for 30 minutes. Toluene (600 mL) was added to the solution, and the solution was concentrated to 300 mL. At this point, completion of the reaction was confirmed by HPLC. 1-Methylimidazole (12.03 g, 146.47 mmol) was further added, and the solution was concentrated to 90 mL. This afforded 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula C-10) as a toluene solution containing 1-methylimidazole. This product was used directly in the next step.

[0964] Example 8

[0965] 4-Methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-2-O-(trifluoromethanesulfonyl)-β-D-glucopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula C-11 (wherein X1 is a Tf group))

[0966]

[0967] To a solution (90 mL, containing 1-methylimidazole) of 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-glucopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula C-10) obtained in Example 7 was added ethyl acetate (210 mL), and the mixture was cooled to 0°C. Trifluoromethanesulfonic anhydride (16.53 g, 58.59 mmol) was added dropwise to the solution over 1 hour, followed by stirring for 30 minutes. After confirming the completion of the reaction by HPLC, water (300 mL) was added, and the mixture was separated into an organic layer and an aqueous layer. The organic layer was washed twice with water (300 mL) and once with a saturated aqueous sodium chloride solution (150 mL), and then concentrated under reduced pressure to a volume of 90 mL. Ethyl acetate (300 mL) was added, and the solution was concentrated again to 90 mL. This yielded 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-2-O-(trifluoromethanesulfonyl)-β-D-glucopyranosyl}-2-deoxy-2-(1,3-diazolyl-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula C-11 (where X1 is a Tf group)) as an ethyl acetate solution. This product was used directly in the next step.

[0968] Example 9

[0969] 4-Methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-(2-carboxybenzamide)-2-deoxy-β-D-glucopyranoside (compound represented by formula C-13)

[0970]

[0971] To a solution (90 mL) of 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-2-O-(trifluoromethanesulfonyl)-β-D-glucopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula C-11 (wherein X1 is a Tf group)) obtained in Example 8 was added dimethyl sulfoxide (150 mL) and tetrabutylammonium acetate (17.67 g, 58.59 mmol). The mixture was heated to 30°C and stirred for 17 hours. Completion of the reaction was confirmed by HPLC. Toluene (150 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure to a volume of 165 mL. Methanol (45 mL) and 50% aqueous sodium hydroxide solution (3.52 g, 87.88 mmol) were added and stirred at 25 ° C for 1.5 hours. After the reaction was confirmed to be complete by HPLC, ethyl acetate (450 mL) and water (300 mL) were added for liquid separation. Water (300 mL) was added to the organic layer, cooled to 0 ° C, and the pH was adjusted to 2.73 with 6N hydrochloric acid under strong stirring. Tetrahydrofuran (300 mL) was added to the organic layer after liquid separation and concentrated under reduced pressure to a liquid volume of 150 mL. Tetrahydrofuran (300 mL) was added and concentrated again to a liquid volume of 90 mL, and the temperature was adjusted to an internal temperature of 45 ° C. After adding tetrahydrofuran (60 mL), the mixture was cooled to 25°C, and 2-propanol (150 mL) and water (15 mL) were added. 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-(2-carboxybenzamide)-2-deoxy-β-D-glucopyranoside (the compound represented by Formula C-13) (30 mg) was added. Stirring was continued at 25°C for 14 hours. After confirming the precipitation of crystals, 2-propanol (210 mL) was added dropwise over 1 hour, and the mixture was cooled to 0°C. After stirring for 2 hours, the precipitated crystals were filtered and washed with 2-propanol (150 mL) cooled to 0°C. The obtained crystals were dried at 40°C under reduced pressure to give 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-pyranosylmannoside}-2-(2-carboxybenzamide)-2-deoxy-β-D-pyranoglucoside (the compound represented by formula C-13) (27.74 g, yield 94.3%, based on the compound represented by formula C-8).

[0972] ※The step of converting the compound represented by Formula C-12 to the compound represented by Formula C-13 is carried out by a one-pot method.

[0973] ※ As seed crystals, a small portion of the reaction solution was concentrated to precipitate a solid.

[0974] *This reaction proceeds well when using tetrabutylammonium acetate manufactured by Tokyo Chemical Industry Co., Ltd. (product number: T2694, purity: >90.0%) and Sigma-Aldrich Co. (product number: 86849, purity: >90%). Using tetrabutylammonium acetate from other manufacturers may cause a delay in the reaction. In such cases, an alternative method using cesium acetate is preferred (details are described below).

[0975] The conversion of the compound represented by Formula C-11 to the compound represented by Formula C-12 can also be carried out using cesium acetate (3 equivalents) and dimethyl sulfoxide at 50°C for 24 hours. Subsequently, the compound represented by Formula C-13 can be obtained by carrying out the same reaction (compound represented by Formula C-12 to compound represented by Formula C-13) and post-treatment.

[0976] 1 H-NMR (500MHz, CDCl3) δ8.02 (dd, J=6.0, 2.0Hz, 1H), 7.69-7.83 (m, 4H), 7.38-7.49 (m, 12H), 7.32-7.34 (m, 2H), 7.16-7.29 (m, 8H), 6.97 (ddd, J=9.0, 4.0, 2.5Hz, 2H), 6.76 (ddd, J=9.5, 3.5, 2.5Hz, 2H), 5.51 (s, 1H), 5.40 (d, J =6.0Hz, 1H), 4.83-4.91 (m, 3H), 4.76 (d, J = 11.5Hz, 1H), 4.55 (d, J = 0.5Hz, 1 H), 4.49 (d, J = 12.0Hz, 1H), 4.36 (d, J = 12.0Hz, 1H), 4.26-4.30 (m, 1H), 4.1 6(t, J=6.5Hz, 1H), 4.05-4.09(m, 2H), 3.99(dd, J=3.0, 0.5Hz, 1H), 3.93(t , J=9.5Hz, 1H), 3.80-3.86 (m, 2H), 3.71 (s, 3H), 3.65-3.69 (m, 1H), 3.56 (t , J=10.0Hz, 1H), 3.51 (dd, J=10.0, 3.5Hz, 1H), 3.13 (td, J=9.5, 5.0Hz, 1H).

[0977] 13C-NMR (125MHz, CDCl3) δ170.9, 168.4, 155.3, 151.4, 138.7, 138.0, 137.6, 136.2, 135.4, 1 33.4, 133.3., 132.2, 132.1, 130.7, 130.3, 129.2, 128.6, 128.49, 128.45, 128.11, 128.07, 128.0, 127.89, 127.87, 127.8, 126.8, 126.4, 126.3, 126.2, 125.8, 118.6, 114.7, 101.7, 100.4, 99.1, 78.3, 76.7, 76.4, 75.1, 73.7, 73.3, 72.5, 69.9, 69.4, 68.5, 67.0, 55.8, 54.4.

[0978] HRMS(ESI + )[M+H] + C 59 H 58 NO 14 Theoretical value: 1004.3852; experimental value 1004.3873.

[0979] Example 10

[0980] 4-Methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula C-14)

[0981]

[0982] To a solution of 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-(2-carboxybenzamide)-2-deoxy-β-D-glucopyranoside (the compound represented by Formula C-13) (6.00 g, 5.98 mmol) in dichloromethane (30 mL) were added 1-hydroxybenzotriazole monohydrate (0.18 g, 1.20 mmol), N,N-diisopropylethylamine (0.85 g, 6.57 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.26 g, 6.57 mmol), the mixture was heated to 40° C. and stirred for 31 hours. After confirming the completion of the reaction by HPLC, the reaction solution was cooled to 0°C, ethyl acetate (90 mL) and water (60 mL) were added, and 6N hydrochloric acid was added with vigorous stirring until the pH reached 7. The organic and aqueous layers were separated, and the organic layer was washed with water (60 mL) and saturated brine (30 mL). The solution was concentrated under reduced pressure to a volume of 12 mL. Tetrahydrofuran (60 mL) was added, and the solution was concentrated again to a volume of 9 mL, yielding a tetrahydrofuran solution of the crude product, 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula C-14). This product was used directly in the next step.

[0983] Example 11

[0984] 4-Methoxyphenyl 3,6-di-O-benzyl-4-O-{2-O-benzyl-4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula C-15)

[0985]

[0986] To a solution of the crude product 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by formula C-14) were added N,N-dimethylacetamide (60 mL), benzyl bromide (1.53 g, 8.96 mmol), Methyl trifluoroacetate (0.15 g, 1.20 mmol) and molecular sieves 4A (1.8 g) were cooled to 0°C, and a solution of lithium tert-butoxide in tetrahydrofuran (Note: This solution was prepared by cooling a mixed solution of 2-methyl-2-propanol (0.66 g, 8.96 mmol) and tetrahydrofuran (2.4 mL) to 0°C, adding a solution of n-butyllithium in hexane (1.55 mol / L) (5.78 mL, 8.96 mmol), and stirring for 30 minutes). The mixture was stirred for 3 hours. After confirming the completion of the reaction by HPLC, ethylenediamine (anhydrous) (0.18 g, 2.99 mmol) was added, and the mixture was stirred for a further hour. Acetic acid (0.72 g, 11.95 mmol) was added to the solution, and the mixture was filtered. Molecular sieves 4A were washed with ethyl acetate (90 mL), and water (60 mL) was added for separation. The organic layer was washed twice with water (60 mL) and then concentrated under reduced pressure to a volume of 12 mL. Toluene (30 mL) was added and the mixture was concentrated again to a volume of 12 mL. Toluene (18 mL) and silica gel 60N (spherical, manufactured by Kanto Chemical, particle size: 40-50 μm) (9 g) were then added, and the mixture was stirred at 25°C for 30 minutes. The suspension was filtered, and the silica gel was washed with a mixed solution of toluene (191 mL) and ethyl acetate (19 mL). The filtrate was concentrated under reduced pressure to a volume of 9 mL, yielding a toluene solution of the crude product, 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{2-O-benzyl-4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula C-15).

[0987] Example 12

[0988] 4-Methoxyphenyl 3,6-di-O-benzyl-2-deoxy-4-O-{2,4-di-O-benzyl-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula A-4)

[0989]

[0990] To a solution of the obtained crude product 4-methoxyphenyl 3,6-di-O-benzyl-4-O-{2-O-benzyl-4,6-O-benzylidene-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-deoxy-2-(1,3-diazolyl-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula C-15) (5.50 g (5.48 mmol) based on the compound represented by Formula C-14) were added dichloromethane (16.5 mL) and molecular sieves 4A (550 mg), and the mixture was cooled to 0°C. Borane-tetrahydrofuran complex (0.91 mol / L tetrahydrofuran solution) (18.06 mL, 16.43 mmol) and copper (II) trifluoroacetate (0.59 g, 1.64 mmol) were added, and the mixture was stirred for 3 hours. After the reaction was confirmed to be complete by HPLC, methanol (5.5 mL) was added and stirred for a further 30 minutes. The solution was filtered, molecular sieve 4A was washed with ethyl acetate (110 mL), 0.5 N hydrochloric acid (55 mL) was added, and the mixture was stirred for 30 minutes. After separation of the organic layer and the aqueous layer, the organic layer was washed with 0.5 N hydrochloric acid (55 mL) and saturated brine (27.5 mL), and the solution was concentrated to dryness under reduced pressure. The product was purified by silica gel column chromatography (300 g of silica gel, hexane:ethyl acetate = 55:45 → 30:70) to obtain 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-4-O-{2,4-di-O-benzyl-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-(1,3-diazolyl-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula A-4) (4.94 g, yield 83.6%, HPLC area: 98.54%).

[0991] 1H-NMR(500MHz,CDCl3)δ7.67-7.84(m,8H),7.44-7.49(m,4H),7.40(dd,J=8.0,1.5Hz,1H),7.21-7.33(m,13H),6.87-6.93(m,5H),6.82(ddd,J=9.5,4.0,2.5Hz,2H),6.70(ddd,J=9.0,4.0,2.0Hz,2H),5.64(d,J=8.5Hz,1H),4.94(d,J=12.5Hz,1H),4.91(d,J=10.0Hz,1H),4.90(s,2H),4.66(s,2H),4.60(d,J=11.0Hz,1H),4.59(d,J=12.0Hz,1H),4.55(s,1H),4.40-4.46(m,3H),4.33(dd,J=11.0,9.0Hz,1H),4.06(dd,J=9.5,8.5Hz,1H),3.80-3.85(m,2H),3.70-3.76(m,2H),3.71(s,3H),3.61-3.68(m,2H),3.45-3.48(m,1H),3.44(dd,J=9.5,3.0Hz,1H),3.23(ddd,J=9.5,5.5,2.5Hz,1H),1.97(br-t,1H)。

[0992] 13 C-NMR(125MHz,CDCl3)δ155.6,151.1,138.9,138.64,138.56,138.0,135.9,134.0,133.5,133.2,131.8,128.7,128.6,128.4,128.3,128.20,128.19,128.15,128.1,120.04,128.01,127.9,127.7,127.6,127.3,126.4,126.3,126.1,125.8,123.6,119.0,114.6,101.2,98.0,82.6,79.0,77.2,75.9,75.4,75.3,75.2,75.1,74.8,74.7,73.8,72.1,68.7,62.6,55.82,55.78,34.4,30.5。

[0993] HRMS(ESI － )[M+HCO2] － C 67 H 64 NO 15Theoretical value: 1122.4281; experimental value 1122.4285.

[0994] <Synthesis of the compound represented by formula A-12>

[0995] The compound represented by Formula A-12 was synthesized according to the following synthetic route 2.

[0996] [Synthesis Path 2]

[0997]

[0998] Example 13

[0999] 4-Methoxyphenyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula F'-1)

[1000]

[1001] (Small process Z-1)

[1002] To a solution of 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-(1,3-diazolyl-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula A'-9) (50.0 g, 83.94 mmol) in ethyl acetate (200 mL) were added triethylamine (11.04 g, 109.12 mmol), dimethylaminopyridine (0.31 g, 2.52 mmol), and acetic anhydride (11.10 g, 109.12 mmol), and the mixture was stirred at 20°C for 4 hours. After completion of the reaction was confirmed by HPLC, ethanol (500 mL) and water (150 mL) were added dropwise. The slurry was stirred for 1 hour, and the precipitated crystals were filtered. The filtered crystals were washed with a mixture of ethanol and water (150 mL / 50 mL), and dried under reduced pressure at 40° C. to obtain 4-methoxyphenyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-diazolyl-1,3-dihydro-2H-isoindol-2-yl)-β-D-pyranoside (compound represented by formula F'-1) (49.0 g, yield 91%) as white crystals.

[1003] 1H-NMR (500MHz, CDCl3) δ7.85-7.60 (m, 4H), 7.24-7.34 (m, 5H), 7.04-7.00 (m, 2H), 6 .96-6.87(m, 3H), 6.84(dt, J=9.0, 3.0Hz, 2H), 6.67(dt, J=8.5, 2.5Hz, 2H), 5.66(t , J=4.0Hz, 1H), 5.22-5.18 (m, 1H), 4.64 (d, J=12.0Hz, 1H), 4.55-4.48 (m, 4H), 4.36 (d, J=12.0Hz, 1H), 3.90-3.84 (m, 1H), 3.68 (s, 3H), 3.68-3.64 (m, 2H), 1.98 (s, 3H).

[1004] 13 C-NMR (125MHz, CDCl3) δ169.6, 155.3, 150.6, 137.8, 137.5, 133.9, 128.2, 128.0, 127.7, 127.7, 127.5, 127.4, 123.3, 118.4, 114.3, 97.4, 76.8, 73.9, 73.7, 73.5, 72.2, 69.4, 55.4, 55.3, 20.8.

[1005] HRMS(ESI + )[M+H] + C 37 H 36 NO9 theoretical value: 638.2385; experimental value 638.2401.

[1006] Example 14

[1007] 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-D-glucopyranoside (compound represented by formula F-2)

[1008]

[1009] (Small process Z-2)

[1010] To a solution of 4-methoxyphenyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula F'-1) (49.0 g, 76.84 mmol) in dichloromethane (392 mL), hexafluoro-2-propanol (245 mL), and water (25 mL) was added [bis(trifluoroacetoxy)iodo]benzene (46.3 g, 107.58 mmol) at below 25°C, and the mixture was stirred at the same temperature for 4 hours. After confirming the completion of the reaction by HPLC, ethyl acetate (1225 mL) was added, the mixture was cooled on ice, and then poured into water (490 mL) containing sodium bicarbonate (24.5 g) and sodium sulfite (24.5 g) for separation to obtain an organic layer. The resulting organic layer was washed again with water (490 mL) containing sodium bicarbonate (24.5 g) and sodium sulfite (24.5 g), and then further washed with 20% saline (245 g). The resulting organic layer was concentrated under reduced pressure to 490 mL (crystal precipitation was observed during concentration), and heptane (735 mL) was added dropwise. The resulting slurry was cooled to 0°C to 5°C and stirred at the same temperature for 1 hour, and the precipitated crystals were filtered. The filtered crystals were washed with a mixture of ethyl acetate and heptane (39 mL / 118 mL) at 0°C to 5°C and dried under reduced pressure at 40°C to obtain 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-D-glucopyranoside (compound represented by Formula F-2) (37.5 g, 92% yield) as white crystals.

[1011] 1 H-NMR (400MHz, CDCl3) δ7.71-7.65 (m, 4H), 7.34-7.26 (m, 5H), 7.01-6.87 (m, 5H), 5.36 ( dd, J=8.0, 8.0Hz, 1H), 5.13 (dd, J=8.4, 10.0Hz, 1H), 4.59 (d, J=12.4Hz, 1H), 4.54 (s, 2H ), 4.50 (dd, J=8.4, 10.4Hz, 1H), 4.33 (d, J=12.4Hz, 1H), 4.17 (dd, J=8.4, 10.4Hz, 1H), 3 .79 (ddd, J=8.4, 5.2, 4.8Hz, 1H), 3.61-3.53 (m, 2H), 3.41 (d, J=8.0Hz, 1H), 1.93 (s, 3H).

[1012] 13C-NMR (100MHz, CDCl3) δ169.8, 168.1, 137.7, 137.7, 134.0, 131.6, 128.4, 128.2, 128. 0, 127.8.127.7, 127.5, 123.4, 116.2, 92.9, 73.9, 73.7, 73.5, 72.2, 69.3, 57.1, 20.9.

[1013] HRMS(ESI － )[M-H] － C 30 H 28 NO8 theoretical value: 530.1820; experimental value 530.1841.

[1014] Example 15

[1015] 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-O-[2,2,2-trifluoro-N-phenylethaneimido]-β-D-glucopyranoside (compound represented by formula A-12)

[1016]

[1017] (Small process Z-3)

[1018] 4-O-Acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-D-glucopyranoside (the compound represented by Formula F-2) (20.0 g, 37.63 mmol) was placed in a 500 mL eggplant flask. Dichloromethane (200 mL) and molecular sieves 4A powder (10 μm or less, 10.0 g) were also added. N-Methylimidazole (3.40 g, 41.39 mmol) and 2,2,2-trifluoro-N-phenylacetimidyl chloride (8.20 g, 39.51 mmol) were then added sequentially at 0°C under nitrogen, and the mixture was stirred at the same temperature for 18 hours. After completion of the reaction was confirmed by HPLC, the reaction mixture was filtered and rinsed with dichloromethane (100 mL). The filtrate was filtered through a neutral silica gel pad (Silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 60 g) filled with dichloromethane, and 100 mL fractions were collected. The silica gel pad was washed with dichloromethane (400 mL, 100 mL fractions) and ethyl acetate / dichloromethane (1:4, 400 mL, 100 mL fractions), and the selected fraction was concentrated to a volume of 40 mL. Toluene (200 mL) was added, and the mixture was concentrated again until the liquid volume reached 40 mL. Further toluene (200 mL) was added, and the mixture was concentrated until the liquid volume reached 40 mL. This afforded a crude product, 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-O-[2,2,2-trifluoro-N-phenylethaneimido]-β-D-glucopyranoside (compound represented by Formula A-12), as a toluene solution. This product was used directly in the next step.

[1019] <Synthesis of the compound represented by formula A-16>

[1020] The compound represented by Formula A-16 was synthesized according to the following synthetic route 3.

[1021] [Synthesis Path 3]

[1022]

[1023] First, the compound represented by formula G-1 was synthesized according to the following synthesis route.

[1024]

[1025] Example 16

[1026] Allyl α-D-galactopyranoside (compound represented by G-0)

[1027]

[1028] D-galactopyranose (20.00 g, 111.01 mmol) was added to a 500 mL four-diameter flask, followed by allyl alcohol (200.0 mL). Under a nitrogen atmosphere, p-TsOH·H2O (2.11 g, 11.10 mmol) was added at room temperature, the temperature was raised to 70°C, and the mixture was stirred for 24 hours. The reaction mixture was cooled to 40°C, triethylamine (1.69 g, 16.65 mmol) was added, and the mixture was stirred for 5 minutes. The reaction mixture was then concentrated under reduced pressure to 100 mL. nBuOH (200 mL) was added dropwise to the concentrated solution over 30 minutes, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated under reduced pressure to 80 mL and stirred overnight at room temperature. The suspension was filtered, and the crystals were washed with 0°C nBuOH (40 mL) and dried under reduced pressure at 40°C to obtain allyl α-D-galactopyranoside (compound represented by G-0) (8.41 g, yield 34.4%) as white crystals.

[1029] 1 H-NMR (500MHz, METHANOL-D4) δ5.97 (qd, J=11.2, 5.7Hz, 1H), 5.33 (dd, J=17.2, 1.7Hz, 1H), 5.17 (dd, J=10.6, 1. 4Hz, 1H), 4.22 (dd, J=13.2, 5.2Hz, 1H), 4.04 (dd, J=13.0, 6.0Hz, 1H), 3.88 (d, J=1.7Hz, 1H), 3.82-3.66 (m, 5H).

[1030] 13 C-NMR (125MHz, METHANOL-D4) δ62.74, 69.36, 70.21, 71.08, 71.51, 72.49, 99.46, 117.47, 135.69.

[1031] MS (ESI) m / z: 221 (M+H) + , 219 (M-H) － .

[1032] Example 17

[1033] Prop-2-en-1-yl 4,6-O-benzylidene-α-D-galactopyranoside (compound represented by formula G-1)

[1034]

[1035] Under a nitrogen atmosphere, acetonitrile (5.0 mL), benzaldehyde dimethyl acetal (5.18 g, 34.1 mmol), and p-TsOH·H₂O (431.9 mg, 2.27 mmol) were added to a 100 mL eggplant-shaped flask. Allyl α-D-galactopyranoside (5.00 g, 22.7 mmol) was then added gradually. The solution was heated to 40°C and stirred for 30 minutes. Triethylamine (344.6 mg, 3.41 mmol) was then added and stirred for 5 minutes. Isopropyl alcohol (75 mL) was then added dropwise at 40°C. The reaction mixture was concentrated under reduced pressure to 25 mL and stirred overnight at 0°C. Next, the suspension was filtered, and the resulting crystals were washed with isopropyl alcohol (5 mL) cooled to 0°C and dried under reduced pressure at 40°C to obtain prop-2-en-1-yl 4,6-O-benzylidene-α-D-galactopyranoside (compound represented by formula G-1) (5.35 g, yield 76.3%) as white crystals.

[1036] 1 H-NMR (500MHz, METHANOL-D4) δ7.53-7.52 (dd, J=7.5, 2.0Hz, 2H), 7.34 (m, 3H), 5.9 8 (qd, J=11.1, 5.4Hz, 1H), 5.59 (s, 1H), 5.35 (d, J=18.9Hz, 1H), 5.19 (d, J=10.3Hz, 1H), 4.95 (d, J=4.0Hz, 1H), 4.26 (d, J=3.4Hz, 1H), 4.22 (dd, J=13.2, 5.2Hz, 1H), 4. 13 (s, 2H), 4.08 (q, J=6.5Hz, 1H), 3.92 (ddd, J=24.1, 10.3, 3.4Hz, 2H), 3.74 (s, 1H).

[1037] 13 C-NMR (125MHz, METHANOL-D4) δ64.60, 69.70, 70.03, 70.08, 70.34, 78.07, 100.22, 102.28, 117.58, 127.54, 129.02, 129.86, 135.59, 139.77.

[1038] MS (ESI) m / z: 309 (M+H) + , 307(M-H) － .

[1039] Example 18

[1040] Prop-2-en-1-yl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-galactopyranoside (compound represented by formula G-2)

[1041]

[1042] (Small process V-1)

[1043] Benzoyl chloride (47.87 g, 340.54 mmol) was added dropwise to a pyridine (150 mL) solution of prop-2-en-1-yl 4,6-O-benzylidene-α-D-galactopyranoside (the compound represented by Formula G-1) (30.0 g, 97.30 mmol) at below 40°C, and the mixture was stirred at the same temperature for 2 hours. After confirming the completion of the reaction by HPLC, the mixture was cooled to 20°C to 30°C, ethanol (450 mL) was added, and water (300 mL) was added dropwise over 30 minutes. After stirring the slurry at 20°C to 30°C for 1 hour, the precipitated crystals were filtered, washed with a mixture of ethanol and water (75 / 75 mL), and dried under reduced pressure at 40°C to obtain prop-2-en-1-yl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-pyranoside (compound represented by formula G-2) (47.9 g, yield 95%) as white crystals.

[1044] 1 H-NMR (500MHz, CDCl3) δ8.03-7.98 (m, 4H), 7.55-7.46 (m, 4H), 7.40-7.30 (m, 7H), 5.90- 5.78 (m, 2H), 5.82 (s, 1H), 5.57 (s, 1H), 5.42 (d, J = 1.7Hz, 1H), 5.31 (dd, J = 17.2, 1.7Hz, 1H), 5.15 (dd, J=1.7, 10.5Hz, 1H), 4.66 (s, 2H), 4.33 (d, J=12.5Hz, 1H), 4.26 (dd, J=12. 5, 4.5Hz, 1H), 4.12 (dd, J=12.5, 1.0Hz, 1H), 4.08 (dd, J=6.5, 13.5Hz, 1H), 3.95 (s, 1H).

[1045] 13 C-NMR (125MHz, CDCl3) δ166.1, 165.8, 137.5, 133.4, 133.2, 133.1, 129.8, 129.7, 129.5, 1 29.4, 128.8, 128.3, 128.1, 126.1, 117.5, 100.6, 96.2, 74.2, 69.3, 69.1, 68.7, 68.6, 62.4.

[1046] HRMS(ESI + )[M+H] + C 30 H29 O8 theoretical value: 517.1857; experimental value 517.1880.

[1047] Example 19

[1048] Prop-2-en-1-yl 2,3-di-O-benzoyl-α-D-galactopyranoside (compound represented by formula G-3)

[1049]

[1050] (Small process V-2)

[1051] A solution of prop-2-en-1-yl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-galactopyranoside (the compound represented by Formula G-2) (47.1 g, 91.18 mmol) in acetonitrile (377 mL) was heated to 45°C, and water (24 mL) and concentrated hydrochloric acid (9.2 g, 91.18 mmol) were added. The mixture was stirred at the same temperature for 30 minutes. Water (353 mL) was added dropwise at 45°C to 50°C over 3 hours, and the mixture was stirred for a further 30 minutes. After confirming the completion of the reaction by HPLC, sodium acetate (11.22 g, 136.78 mmol) was added, and ethyl acetate (942 mL) and water (471 mL) were poured into the mixture. The mixture was cooled to below 25°C and separated to obtain an organic layer. The resulting organic layer was washed twice with water (471 mL) and then further washed with 20% saline (236 mL). The organic layer was concentrated under reduced pressure to 141 mL, toluene (707 mL) was added, and the mixture was concentrated under reduced pressure to 141 mL again. Toluene (236 mL) was added to the obtained concentrated solution, and the mixture was concentrated under reduced pressure to 141 mL. After the concentrated solution was cooled to 0°C to 5°C, a slurry of toluene (330 mL) containing neutral silica gel (silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 141 g) cooled to 0°C to 5°C was injected, and the mixture was stirred at the same temperature for 15 minutes to allow the product to be adsorbed on the silica gel, and then filtered. After washing the silica gel solid phase containing the product with toluene (942 mL) at 0°C to 5°C (the filtrate from the toluene washing was discarded), the target product was desorbed from the silica gel with cyclopentyl methyl ether (707 mL). This yielded a cyclopentyl methyl ether solution of prop-2-en-1-yl 2,3-di-O-benzoyl-α-D-galactopyranoside (compound represented by Formula G-3) (quantitative value 36.2 g, quantitative yield 93%). This solution was used in the next step.

[1052] Example 20

[1053] 5-Acetylamino-3,5-dideoxy-D-glycero-D-galacto-nona-2-pyranose methyl ester monohydrate (compound represented by formula G-5)

[1054]

[1055] (Small process V-3)

[1056] To a solution of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonan-2-pyranose acid (the compound represented by Formula G-4) (40.1 g, 129.66 mmol) and methyl orthoformate (15.60 mL, 142.59 mmol) in methanol (321 mL) was added sulfuric acid (1.0 g, 10.20 mmol), heated to 40°C, and stirred for 3 hours. After confirming the completion of the reaction by HPLC, the mixture was cooled to 25°C, dimethylacetamide (40 mL) was added, and the mixture was concentrated under reduced pressure to 160 mL. The resulting concentrate was cooled to 15°C, and water (20 mL) and ethyl acetate (722 mL) were added. After stirring at 25°C for 1 hour, the slurry was cooled to 0°C to 5°C and stirred at the same temperature for 2 hours. The precipitated crystals were filtered, washed with ethyl acetate (80 mL) at 0°C to 5°C, and dried under reduced pressure at 40°C to obtain 5-acetylamino-3,5-dideoxy-D-glycero-D-galacto-nona-2-pyranose methyl ester monohydrate (compound represented by formula G-5) (41.1 g, yield 93%) as white crystals.

[1057] 1 H-NMR (500MHz, CD3OD) δ4.07-3.98 (m, 2H), 3.85-3.77 (m, 2H), 3.78 (s, 3H), 3.72-3.68 (m, 1H), 3.62 (dd, J=10.9, 5 .7Hz, 1H), 3.48 (dd, J=9.2, 1.1Hz, 1H), 2.22 (dd, J=12.9, 4.9Hz, 1H), 2.02 (s, 3H), 1.89 (dd, J=12.6, 11.5Hz, 1H).

[1058] 13 C-NMR (125MHz, CD3OD) δ175.2, 175.1, 171.8, 96.6, 72.1, 72.0, 71.6, 70.1, 67.9, 64.8, 54.4, 54.3, 53.2, 40.7, 22.7, 22.7.

[1059] HRMS(ESI + )[M+H] + C 12 H 22 NO9 theoretical value: 324.1289; experimental value 324.1288.

[1060] Example 21

[1061] 5-Acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-nona-2-pyranone methyl ester (compound represented by formula G-6)

[1062]

[1063] (Small process V-4)

[1064] A slurry of methyl 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nona-2-pyranose monohydrate (the compound represented by Formula G-5) (40.3 g, 118.07 mmol) in acetonitrile (403 mL) was adjusted to 25°C, and acetic anhydride (60.27 g, 590.36 mmol) and p-toluenesulfonic acid monohydrate (1.12 g, 5.89 mmol) were added. The mixture was stirred at 25°C for 24 hours. The reaction mixture was then cooled to 15°C, and acetic anhydride (12.05 g, 118.03 mmol) was added. The mixture was stirred at the same temperature for 47 hours. After confirming the completion of the reaction by HPLC, methanol (40 mL) was added, the temperature was adjusted to 25°C, and the mixture was stirred at the same temperature for 2 hours. Subsequently, sodium acetate (0.97 g, 11.82 mmol) was added, and the mixture was stirred at the same temperature for another 1 hour. The reaction solution was concentrated under reduced pressure to 120 mL, cooled to 0°C to 5°C, and then ethyl acetate (403 mL) and water (161 mL) were injected. Triethylamine was added while stirring at 0°C to 5°C to adjust the pH to 7.0. The organic layer obtained by separation was washed twice with 10% saline (121 mL) and concentrated under reduced pressure to 200 mL. Ethyl acetate (605 mL) was added to the concentrated solution, and the solution was concentrated under reduced pressure again to 200 mL. Ethyl acetate (40 mL) was added to the concentrated solution, seeded with crystals, and stirred at 25°C for 4 hours. Heptane (302 mL) was then added dropwise over 30 minutes. After stirring the slurry at 25°C for 2 hours, the precipitated crystals were filtered and washed with a mixture of ethyl acetate and heptane (67 / 135 mL), and dried under reduced pressure at 35°C to obtain 5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-nonane-2-pyranone sugar acid methyl ester (compound represented by formula G-6) (44.1 g, yield 76%) as white crystals.

[1065] 1H-NMR (500MHz, CDCl3) δ6.28 (d, J=10.3Hz, 1H), 5.41 (dd, J=4.6, 2.3Hz, 1H), 5.28-5 .23 (m, 1H), 5.21-5.14 (m, 1H), 5.09 (s, 1H), 4.62 (dd, J=12.3, 2.6Hz, 1H), 4.28 (dd, J=10.3, 2.3Hz, 1H), 4.21-4.11 (m, 1H), 4.04 (dd, J=12.3, 8.3Hz, 1H), 3.85 (s, 3H), 2 .24-2.20 (m, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H).

[1066] 13 C-NMR (125MHz, CDCl3) δ171.5, 171.1, 170.8, 170.3, 170.2, 168.9, 94.9, 72 .1, 71.4, 69.1, 68.3, 62.5, 53.2, 49.1, 36.1, 23.0, 21.0, 20.8, 20.7, 20.7.

[1067] HRMS(ESI + )[M+H] + C 20 H 30 NO 13 Theoretical value: 492.1712; experimental value: 492.1712.

[1068] Example 22

[1069] 5-Acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-glycero-β-D-galacto-nona-2-pyranone acid methyl ester (compound represented by formula G-7)

[1070]

[1071] (Small process V-5)

[1072] A slurry of 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-nona-2-pyranose acid methyl ester (the compound represented by Formula G-6) (44.0 g, 89.53 mmol) and molecular sieves 4A powder (powder particle size 10 μm or less) (22 g) in dichloromethane (352 mL) was adjusted to 20°C and stirred at the same temperature for 30 minutes. Then, 2,2,2-trifluoro-N-phenylacetimidochloride (26.02 g, 125.35 mmol) was added. N-methylimidazole (11.03 g, 134.33 mmol) was then added dropwise, and the mixture was stirred at 20°C for 7.5 hours. After confirming the completion of the reaction by HPLC, the reaction solution was filtered and washed with dichloromethane (88 mL) to obtain a filtrate. The obtained filtrate was cooled to 0°C, cold water (440 mL) was added, and triethylamine was added while stirring at 0°C to 5°C to adjust the pH to 7.5. After stirring at 0°C to 5°C for 30 minutes, the liquid was separated, and the obtained organic layer was washed twice with cold water (440 mL), washed with cooled 20% saline (220 mL), and concentrated under reduced pressure to 88 mL. Ethyl acetate (440 mL) was added to the concentrated solution, and the solution was concentrated under reduced pressure to 88 mL again. Tert-butyl methyl ether (308 mL) was added to the concentrated solution, seeded with crystals, and stirred at 20°C for 4 hours. Heptane (264 mL) was added dropwise to the resulting slurry over a period of 1 hour. After stirring at the same temperature for 2 hours, the precipitated crystals were filtered and washed with a mixture of tert-butyl methyl ether and heptane (132 / 88 mL). The filtered crystals were then dried under reduced pressure at 35°C to obtain 5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-glycero-β-D-galacto-nonane-2-pyranone sugar acid methyl ester (compound represented by formula G-7) (39.5 g, yield 67%) as white crystals.

[1073] 1H-NMR (500MHz, CDCl3) δ7.30-7.24 (m, 2H), 7.09 (t, J=7.4Hz, 1H), 6.72 (d, J=8.0Hz, 2H), 5.76 (d, J=9.7Hz , 1H), 5.48-5.45 (m, 1H), 5.30 (td, J=10.9, 4.8Hz, 1H), 5.15-5.10 (m, 1H), 4.60 (dd, J=12.6, 2.3Hz, 1H), 4. 30(q, J=10.3Hz, 1H), 4.23(dd, J=10.3, 2.3Hz, 1H), 4.11(dd, J=12.3Hz, 7.7Hz, 1H), 3.81(s, 3H), 2.79(dd , J=13.5, 4.9Hz, 1H), 2.21-2.15 (m, 1H), 2.16 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 1.90 (s, 3H), 1.75 (s, 3H).

[1074] 13 C-NMR (125MHz, CDCl3) δ171.0, 170.7, 170.4, 170.2, 170.1, 165.3, 142.6, 141.0 (q, 2 J C－F =36.0Hz), 128.8, 124.6, 119.0, 115.1(q, 1 J C－F =284.4Hz), 99.7, 73.6, 71.9, 68.3, 68.0, 62.4, 53.1, 48.6, 35.6, 23.0, 20.8, 20.8, 20.7, 20.3.

[1075] HRMS(ESI + )[M+NH4] + C 28 H 37 F3N3O 13 Theoretical value: 680.2273; experimental value 680.2314.

[1076] Example 23

[1077] 4,7,8,9-tetra-O-acetyl-5-[acetyl(tert-butoxycarbonyl)amino]-3,5-dideoxy-2-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-glycero-β-D-galacto-nona-2-pyranose methyl ester (compound represented by formula G-8)

[1078]

[1079] (Small process V-6)

[1080] To a solution of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-glycero-β-D-galacto-nona-2-pyranose (the compound represented by Formula G-7) (39.0 g, 58.86 mmol) in tetrahydrofuran (390 mL) were added di-tert-butyl dicarbonate (27.05 g, 123.94 mmol) and dimethylaminopyridine (1.80 g, 14.73 mmol). The mixture was heated to reflux. After stirring at reflux for 30 minutes, the reaction was confirmed to be complete by HPLC, and the reaction solution was concentrated under reduced pressure to 117 mL. Toluene (195 mL) was added to the concentrated solution, and the solution was concentrated under reduced pressure again to 117 mL. The concentrate was filtered through a silica gel-filled funnel (Silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 117 g, wet-packed with toluene) and washed with a toluene / ethyl acetate mixture (8 / 2) (975 mL) to obtain a filtrate. The resulting filtrate was concentrated under reduced pressure (to a weight of 59 g) and cyclopentyl methyl ether (23 mL) was added. The solution temperature was adjusted to 20°C, and heptane (156 mL) was added dropwise over 15 minutes. The mixture was stirred at the same temperature for 1 hour. After confirming the precipitation of crystals, heptane (312 mL) was added dropwise over 1 hour, the precipitated crystals were filtered, washed with heptane (78 mL), and dried under reduced pressure at 35° C. to obtain 4,7,8,9-tetra-O-acetyl-5-[acetyl(tert-butoxycarbonyl)amino]-3,5-dideoxy-2-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-glycero-β-D-galacto-nona-2-pyranose acid methyl ester (compound represented by formula G-8) (37.0 g, yield 82%) as white crystals.

[1081] 1 H-NMR (500MHz, CDCl3)

[1082] Note: Detected as a mixture of ca. 1 / 4 isomers.

[1083] Major isomers: δ7.27 (t, J = 8.9 Hz, 2H), 7.09 (t, J = 7.2 Hz, 1H), 6.73 (d, J = 8.0 Hz, 2H), 5.75-5.65 (m, 1H), 5.31 (d, J = 4.6 Hz, 1H), 5.18-5.14 (m, 1H), 5.15 (d, J = 6.0 Hz, 2H), 4.54 (dd, J = 12.0, 2.0 Hz, 1 H), 4.08 (dd, J=12.6, 6.9Hz, 1H), 3.84 (s, 3H), 2.90 (dd, J=13.7, 5.2Hz, 1H), 2.39 (s, 3H), 2.2 5 (dd, J=13.7, 11.2Hz, 1H), 2.09 (s, 3H), 2.07 (s, 3H), 1.99 (s, 3H), 1.77 (s, 3H), 1.62 (s, 9H).

[1084] Minor isomers: δinter alia 6.76 (d, J = 8.0 Hz, 2H), 5.85-5.80 (m, 1H), 5.29-5.25 (m, 1H), 5.22-5.19 (m, 1H), 4.44 (d, J = 11.0 Hz, 1H), 4.15-4.11 (m, 1H), 3.03 (dd, J = 14.0, 5.0 Hz, 1H), 2.41 (s, 3H), 2.12 (s, 3H), 2.00 (s, 3H), 1.88 (s, 3H), 1.74 (s, 3H), 1.54 (s, 9H).

[1085] 13 C-NMR (125 MHz, CDCl3) mixture: δ 173.7, 170.4, 170.2, 170.0, 169.9, 165.4, 151.7, 142.8, 128.7, 124.5, 119.1, 100.6, 85.2, 72.8, 71.3, 67.7, 65.9, 62.0, 53.1, 52.0, 36.7, 27.9, 27.7, 26.6, 20.8, 20.7, 20.6, 20.3.

[1086] HRMS(ESI + )[M+NH4] + C 33 H 45 F3N3O 15 Theoretical value: 780.2797; experimental value 780.2801.

[1087] Example 24

[1088] Prop-2-en-1-yl 2,3-di-O-benzoyl-6-O-{4,7,8,9-tetra-O-acetyl-5-[acetyl(tert-butoxycarbonyl)amino]-3,5-dideoxy-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl}-α-D-galactopyranoside (compound represented by formula G-9)

[1089]

[1090] (Small process V-7)

[1091] A cyclopentyl methyl ether solution of prop-2-en-1-yl 2,3-di-O-benzoyl-α-D-galactopyranoside (compound represented by Formula G-3) (quantitative value: 31.46 g, 73.43 mmol) was concentrated under reduced pressure to 105 mL and then added to a cyclopentyl methyl ether solution of methyl 4,7,8,9-tetra-O-acetyl-5-[acetyl(tert-butoxycarbonyl)amino]-3,5-dideoxy-2-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-glycero-β-D-galacto-nona-2-pyranose (compound represented by Formula G-8) (35.0 g, 45.89 mmol). Cyclopentyl methyl ether was then added to the resulting mixed solution to adjust the total volume to 350 mL (a mixed solution of the compounds represented by Formula G-3 and Formula G-8 in cyclopentyl methyl ether). In a separate container, cyclopentyl methyl ether (525 mL) and molecular sieve 4A powder (powder particle size 10 μm or less) (17.5 g) were added. After cooling to -60°C, trimethylsilyl trifluoromethanesulfonate (4.2 mL, 23.24 mmol) was added. To this solution, a mixed solution of the compound represented by Formula G-3 and the compound represented by Formula G-8 in cyclopentyl methyl ether was added dropwise over 4.5 hours at -60°C with vigorous stirring, and then stirred at the same temperature for 2 hours. After confirming the completion of the reaction by HPLC, triethylamine (4.5 mL, 32.12 mmol) was added, and the reaction solution was warmed to 0°C. Celite 545 (35.00 g) was then added, and the reaction solution was filtered and washed with cyclopentyl methyl ether (175 mL). Water (350 mL) was added to the filtrate for separation. Subsequently, 0.5N aqueous hydrochloric acid (350 mL) was added to the organic layer, and the mixture was stirred at 20°C for 2 hours. After confirming the decomposition of the by-products by HPLC, the organic layer was separated. After washing the organic layer with water (350 mL) and 20% saline (175 mL), it was concentrated under reduced pressure to 70 mL. Toluene (700 mL) was added to the concentrate and concentrated under reduced pressure to 70 mL. Toluene (700 mL) and neutral silica gel (silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 158 g) were added to the concentrate again and stirred at 20°C for 30 minutes. After the product was adsorbed on the silica gel, it was filtered, and the silica gel solid phase containing the product was washed with toluene (1575 mL) (the filtrate during the toluene washing was discarded), and the target product was desorbed from the silica gel with ethyl acetate (875 mL). The obtained ethyl acetate solution was concentrated under reduced pressure to 70 mL, toluene (175 mL) was added, and the mixture was concentrated under reduced pressure again to 70 mL to obtain a toluene solution of prop-2-en-1-yl 2,3-di-O-benzoyl-6-O-{4,7,8,9-tetra-O-acetyl-5-[acetyl(tert-butoxycarbonyl)amino]-3,5-dideoxy-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl}-α-D-galactopyranoside (compound represented by formula G-9).This solution was used in the next step.

[1092] Example 25

[1093] Prop-2-en-1-yl 6-O-{5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl}-2,3-di-O-benzoyl-α-D-galactopyranoside (compound represented by formula G-10)

[1094]

[1095] (Small process V-8)

[1096] To a toluene solution of prop-2-en-1-yl 2,3-di-O-benzoyl-6-O-{4,7,8,9-tetra-O-acetyl-5-[acetyl(tert-butoxycarbonyl)amino]-3,5-dideoxy-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl}-α-D-galactopyranoside (the compound represented by Formula G-9) obtained in Example 24, dichloromethane (525 mL) and copper (II) trifluoromethanesulfonate (8.30 g, 22.95 mmol) were added. The mixture was heated to 40°C and stirred at the same temperature for 2 hours. After confirming the completion of the reaction by HPLC, the mixture was cooled to 25°C and concentrated under reduced pressure to 70 mL. Ethyl acetate (525 mL) was added to the concentrate, and the mixture was washed three times with 5% saline (350 mL). Heptane (263 mL) was then added to the organic layer, and the mixture was washed four times with 20% methanol in water (525 mL). After confirming by HPLC that the impurities derived from the β-isoform of Compound 8, a by-product of the glycosylation reaction, had been removed from the aqueous layer, the organic layer was concentrated under reduced pressure to 70 mL. Isopropyl acetate (525 mL) was added to the concentrate, and the mixture was concentrated under reduced pressure to 350 mL. This yielded an isopropyl acetate solution of prop-2-en-1-yl 6-O-{5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl}-2,3-di-O-benzoyl-α-D-galactopyranoside (compound represented by Formula G-10). This solution was used in the next step.

[1097] Example 26

[1098] Prop-2-en-1-yl 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-α-D-galactopyranoside (compound represented by formula G-11)

[1099]

[1100] (Small process V-9)

[1101] To a solution of prop-2-en-1-yl 6-O-{5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl}-2,3-di-O-benzoyl-α-D-galactopyranoside (the compound represented by Formula G-10) obtained in Example 25 in isopropenyl acetate, p-toluenesulfonic acid monohydrate (0.88 g, 4.62 mmol) was added. The mixture was heated to reflux (internal temperature approximately 90°C) and stirred at the same temperature for 3 hours. After confirming the completion of the reaction by HPLC, the mixture was cooled to 25°C, triethylamine (0.95 mL, 6.85 mmol) was added, and the mixture was concentrated under reduced pressure to 70 mL. Toluene (350 mL) was added to the concentrated solution, and the mixture was again concentrated under reduced pressure to 70 mL. Toluene (630 mL) was added to the concentrate, and neutral silica gel (Silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 123 g) was added, and the mixture was stirred at the same temperature for 30 minutes to allow the product to adsorb on the silica gel, followed by filtration. The silica gel solid phase containing the product was washed with toluene (1925 mL) and a toluene / ethyl acetate mixture (97 / 3, 1400 mL) (the filtrate from the washing was discarded), and the target product was desorbed from the silica gel solid phase containing the product with ethyl acetate (1050 mL). The resulting ethyl acetate solution was concentrated under reduced pressure to obtain prop-2-en-1-yl 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranonosyl]-α-D-galactopyranoside (compound represented by formula G-11) (30.1 g, yield 67% (based on the compound represented by formula G-8)) as a white foamy solid (containing 0.42 equivalents of toluene (approximately 4 weight%)).

[1102] 1H-NMR (500MHz, CDCl3) δ7.99 (d, J=8.4Hz, 2H), 7.88 (dd, J=8.4, 1.4Hz, 2H), 7 .53-7.47(m, 2H), 7.37(dt, J=14.7, 6.9Hz, 4H), 5.90-5.82(m, 1H), 5.82(dd, J =10.9, 3.4Hz, 1H), 5.73 (d, J = 2.9Hz, 1H), 5.58 (dd, J = 10.9, 4.0Hz, 1H), 5.51 (td, J=10.6, 5.0Hz, 1H), 5.35-5.30 (m, 3H), 5.17-5.15 (m, 2H), 4.94 (dd, J=10 .3, 1.7Hz, 1H), 4.38-4.27(m, 3H), 4.20-4.13(m, 2H), 4.08(dd, J=13.2, 5.7H z, 1H), 3.94 (dd, J=10.3, 6.3Hz, 1H), 3.82 (s, 3H), 3.50 (dd, J=9.7, 7.4Hz, 1H) , 2.73(dd, J=13.2, 5.2Hz, 1H), 2.37(s, 3H), 2.31(s, 3H), 2.19(s, 3H), 2.15( s, 3H), 2.14 (s, 3H), 2.03 (s, 3H), 1.97 (s, 3H), 1.86 (dd, J=13.2, 10.9Hz, 1H).

[1103] 13 C-NMR (125MHz, CDCl3) δ174.5, 173.6, 170.5, 170.1, 169.9, 169.8, 169.6, 1 67.3, 166.0, 165.5, 133.5, 133.3, 133.1, 129.8, 129.5, 129.4, 128.4, 128.3 , 117.5, 98.6, 95.5, 77.2, 69.8, 68.9, 68.6, 68.6, 68.3, 68.3, 67.5, 67.0, 66.7, 62.4, 61.8, 57.0, 52.9, 38.7, 27.9, 25.9, 21.0, 20.9, 20.7, 20.7, 20.6.

[1104] HRMS(ESI + )[M+H] + C 47 H 56 NO 22 Theoretical value: 986.3288; experimental value 986.3277.

[1105] The spectrum of the obtained compound was confirmed to be consistent with the following document: Document 4) J. Org. Chem. 2016, 81, 10600-10616.

[1106] Example 27-1

[1107] 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12)

[1108]

[1109] (Small process V-10)

[1110] A methanol solution (290 mL) of prop-2-en-1-yl 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-α-D-galactopyranoside (the compound represented by formula G-11) (29.00 g, 29.41 mmol), 1,3-dimethylbarbituric acid (9.19 g, 58.86 mmol) and triphenylphosphine (2.31 g, 8.81 mmol) was repeatedly subjected to reduced pressure and nitrogen replacement five times and degassed, and then palladium (II) acetate (0.66 g, 2.94 mmol) was added, and the mixture was stirred at 40°C for 12 hours. After confirming the completion of the reaction by HPLC, toluene (580 mL) and water (1015 mL) were added and separated to obtain an organic layer. The organic layer was washed four times with 20% methanol in water (580 mL). 1,3-Dimethylbarbituric acid was removed to the aqueous layer, and the mixture was concentrated under reduced pressure to 58 mL. Toluene (435 mL) was added, and the mixture was concentrated under reduced pressure again to 58 mL. Toluene (383 mL), chloroform (197 mL), and neutral silica gel (Silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 145 g) were added to the concentrate. The mixture was stirred for 30 minutes to allow the product to adsorb onto the silica gel, and then filtered. The silica gel solid phase containing the product was washed with a mixture of toluene and chloroform (2 / 1, 4350 mL) (the filtrate from the washing was discarded), and the target product was desorbed from the silica gel solid phase containing the product using ethyl acetate (870 mL). SH silica gel (29.00 g) was added to the obtained ethyl acetate solution, and after stirring for 30 minutes, the mixture was filtered and washed with ethyl acetate (145 mL) to obtain an ethyl acetate solution containing the target substance. The obtained solution was concentrated under reduced pressure to 58 mL, toluene (145 mL) was added, and the mixture was concentrated under reduced pressure to 58 mL again. The concentrate was purified by silica gel column (silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 290 g, mobile phase hexane / ethyl acetate 50 / 50-30 / 70), and the selected fraction was concentrated under reduced pressure to 29 mL. Ethyl acetate (290 mL) and activated carbon (Shirayuki A, 14.5 g) were added to the concentrate, stirred for 30 minutes, filtered, and washed with ethyl acetate (87 mL) to obtain a purified ethyl acetate solution containing the target substance. The obtained solution was concentrated under reduced pressure to obtain 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12) (18.70 g, yield 67%) as a white foamy solid.

[1111] 1H-NMR (500 MHz, CDCl3) major isomers: δ7.98-8.03 (m, 2H), 7.89 (t, 2H, J = 8.9 Hz), 7.52-7.48 (m, 2H), 7.39-7.34 (m, 4H), 5.92 (dd, 1H, J = 10.3, 2.9 Hz), 5.82 (d, 1H, J = 3.4 Hz), 5.68 (t, 1H, J = 2.3 Hz), 5.59-5.48 (m, 2H), 5.37 (td, 1H, J = 7.5, 2.5 Hz), 5.17 (d, 1H, J = 7.5 Hz), 5.02 (d, 1H, J=10.5Hz), 4.74-4.71 (m, 1H), 4.44-4.38 (m, 1H), 4.16-4.08 (m, 2H), 3.85 (s, 3H), 3.80 (m, 1H), 3.60-3.54 (m, 1H), 2.76 (dd, 1H, J=13.0, 6.0Hz), 2.38 (s, 3H), 2.33 (s, 3H), 2.31 (s, 3H), 2.15 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H), 1.93-1.87 (m, 1H).

[1112] 13 C-NMR (125 MHz, CDCl3) α / β mixture: 174.5, 173.8, 173.6, 171.7, 171.0, 170.5, 170.3, 170.3, 169.9, 169.8, 169.8, 169.6, 167.6, 167.3, 166.3, 166.0, 165.6, 165.4, 133.3, 133.2, 133.2, 133.1, 129.8, 129.7, 129.5, 129.4, 129.4, 129.3, 129.0, 128.4, 128.3, 99.1, 98.8, 95.9, 91.0, 72.2, 71.6, 71.5, 69.9, 69.9, 69.3, 69.3, 68.8, 68.5, 68.1, 67.5, 67.5, 67.3, 67.0, 66.6, 62.9, 62.6, 62.4, 60.3, 57.2, 56.8, 53.0, 52.9, 38.6, 38.3, 27.9, 27.8, 26.1, 25.7, 21.0, 20.9, 20.8, 20.7, 20.7, 20.6, 20.5.

[1113] HRMS(ESI － )[M+HCOO] － C 45 H 52 NO 24Theoretical value: 990.2885; experimental value 990.2873.

[1114] Example 27-2

[1115] 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12)

[1116] The compound represented by the above formula G-12 was synthesized in this example instead of Example 27-1. A solution of prop-2-en-1-yl 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-α-D-galactopyranoside (the compound represented by the formula G-11) (5.0 g, 5.07 mmol) in tetrahydrofuran (25 mL) was subjected to degassing by repeated pressure reduction and nitrogen replacement three times. Tris(triphenylphosphine)ruthenium(II)carbonylchlorohydride (24.1 mg, 0.03 mmol) was then added, and the mixture was stirred at 55-60°C for 3 hours. After confirming the completion of the isomerization reaction by HPLC, the mixture was cooled to 20-25°C, and water (5 mL) and 1,3-dibromo-5,5-dimethylhydantoin (870 mg, 3.04 mmol) (washed thoroughly with 5 mL of tetrahydrofuran) were added. The mixture was stirred at 20-30°C for 1 hour. After confirming the completion of the reaction by HPLC, 1% saline (25 mL) containing sodium sulfite (255 mg) and ethyl acetate (50 mL) were added. After stirring, the mixture was separated. The resulting organic layer was washed with 5% saline (25 mL), and purified egret (2.0 g) was added to the organic layer. After stirring for 1 hour, the mixture was filtered and washed with ethyl acetate (50 mL). The filtrate was concentrated under reduced pressure to 6.5 mL, ethyl acetate (25 mL) was added, and the mixture was concentrated under reduced pressure again to 6.5 mL for dehydration. 2-Propanol (100 mL) was added, seeded, and stirred for 16 hours to confirm the precipitation of crystals. The resulting slurry was then concentrated under reduced pressure to 30 mL. The slurry was cooled to 0-5°C and filtered. The obtained solid was washed with 2-propanol (25 mL) at 0-5°C and dried under reduced pressure at 35°C to obtain 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12) (3.76 g, yield 78%) (α / β mixture) as white crystals.

[1117] 1 H-NMR (500 MHz, CDCl3) major isomers: δ7.98-8.03 (m, 2H), 7.89 (t, 2H, J = 8.9 Hz), 7.52-7.48 (m, 2H), 7.39-7.34 (m, 4H), 5.92 (dd, 1H, J = 10.3, 2.9 Hz), 5.82 (d, 1H, J = 3.4 Hz), 5.68 (t, 1H, J = 2.3 Hz), 5.59-5.48 (m, 2H), 5.37 (td, 1H, J = 7.5, 2.5 Hz), 5.17 (d, 1H, J = 7.5 Hz), 5.02 (d, 1H, J=10.5Hz), 4.74-4.71 (m, 1H), 4.44-4.38 (m, 1H), 4.16-4.08 (m, 2H), 3.85 (s, 3H), 3.80 (m, 1H), 3.60-3.54 (m, 1H), 2.76 (dd, 1H, J=13.0, 6.0Hz), 2.38 (s, 3H), 2.33 (s, 3H), 2.31 (s, 3H), 2.15 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H), 1.93-1.87 (m, 1H).

[1118] 13 C-NMR (125 MHz, CDCl3) α / β mixture: 174.5, 173.8, 173.6, 171.7, 171.0, 170.5, 170.3, 170.3, 169.9, 169.8, 169.8, 169.6, 167.6, 167.3, 166.3, 166.0, 165.6, 165.4, 133.3, 133.2, 133.2, 133.1, 129.8, 129.7, 129.5, 129.4, 129.4, 129.3, 129.0, 128.4, 128.3, 99.1, 98.8, 95.9, 91.0, 72.2, 71.6, 71.5, 69.9, 69.9, 69.3, 69.3, 68.8, 68.5, 68.1, 67.5, 67.5, 67.3, 67.0, 66.6, 62.9, 62.6, 62.4, 60.3, 57.2, 56.8, 53.0, 52.9, 38.6, 38.3, 27.9, 27.8, 26.1, 25.7, 21.0, 20.9, 20.8, 20.7, 20.7, 20.6, 20.5.

[1119] HRMS(ESI － )[M+HCOO] － C 45 H53 NO 24 Theoretical value: 991.2958; experimental value 990.2789.

[1120] Purification method of 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12) by crystallization

[1121]

[1122] 4-O-Acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12) (3.00 g, 3.17 mmol, sialic acid group α / β ratio = 95.7 / 4.3) was dissolved in ethyl acetate (4 mL). 2-Propanol (60 mL) was added, and the mixture was stirred at 25°C and concentrated under reduced pressure to 18 mL. The slurry was stirred at 0°C for 3 hours, and the precipitated crystals were filtered. The filtered crystals were washed with cold 2-propanol (9 mL) and dried under reduced pressure at 40°C to obtain 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (compound represented by formula G-12) as white crystals (2.66 g, yield 88.7%, sialic acid group α / β ratio => 99.9 / ND).

[1123] [Analysis conditions]

[1124] Chromatographic column: CAPCELL PAK ADME φ4.6×150 mm, film thickness 3 μm.

[1125] Wavelength: 220nm.

[1126] Oven: 40℃.

[1127] Eluent: (A) 0.1% trifluoroacetic acid aqueous solution, (B) acetonitrile.

[1128] Gradient: 0-150 min (B) concentration 40%.

[1129] 150.1 min (B) concentration 95%.

[1130] 155 minutes (B) concentration 95%.

[1131] 155.1 minutes (B) concentration 40%.

[1132] 160 minutes (B) concentration 40%.

[1133] Flow rate: 1 mL / min.

[1134] Injection: 5 μL.

[1135] Example 28

[1136] 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-1-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-galactopyranose (compound represented by formula A-16)

[1137]

[1138] (Small process V-11)

[1139] 4-O-Acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-D-galactopyranose (the compound represented by Formula G-12) (20.0 g, 21.1 mmol) was placed in a 500 mL eggplant flask. Dichloromethane (200 mL) and molecular sieves 4A powder (10 μm or less, 10.0 g) were added, and the mixture was cooled to 0°C. N-Methylimidazole (1.91 g, 23.3 mmol) and 2,2,2-trifluoro-N-phenylacetimidoacetyl chloride (4.39 g, 21.1 mmol) were added at the same temperature under nitrogen, and the mixture was warmed to room temperature and stirred for 24 hours. After confirming the completion of the reaction by HPLC, the reaction solution was filtered and washed with dichloromethane (100 mL). The filtrate was filtered through a neutral silica gel pad (silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 60 g) filled with dichloromethane, and 100 mL aliquots were taken at a time. The silica gel pad was washed with ethyl acetate / dichloromethane (1:9, 1000 mL, 200 mL each time), and the selected fraction was concentrated under reduced pressure to obtain 4-O-acetyl-2,3-di-O-benzoyl-6-O-[4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(diacetylamino)-1-methyl-D-glycero-α-D-galacto-nona-2-pyranosyl]-1-O-(2,2,2-trifluoro-N-phenylethaneimido)-D-galactopyranose (compound represented by Formula A-16) (20.1 g, yield 85%) as a white amorphous substance.

[1140] 1H-NMR(500MHz,CDCl3)δ7.99(dd,2H,J=8.3,1.4Hz),7.91-7.88(m,2H),7.59-7.56(m,1H),7.51(tt,1H,J=9.7,2.4Hz),7.44-7.34(m,4H),7.12(t,2H,J=7.7Hz),7.01(t,1H,J=7.4Hz),6.82(brs,1H),6.43(brs,2H),5.87-5.77(m,3H),5.52(td,1H,J=11.0,5.0Hz),5.38-5.34(m,1H),5.18(dd,1H,J=8.6,1.7Hz),4.95(dd,1H,J=10.0,2.0Hz),4.53(brs,1H),4.29(dd,1H,J=12.6,2.9Hz),4.21-4.09(m,3H),4.04(dd,1H,J=10.0,6.0Hz),3.84(s,3H),3.53(dd,1H,J=10.3,7.4Hz),2.76(dd,1H,J=12.9,5.4Hz),2.39(s,3H),2.31(s,3H),2.18(s,3H),2.17(s,3H),2.14(s,3H),2.02(s,3H),1.98(s,3H),1.86(dd,1H,J=13.0,11.0Hz)。

[1141] 13 C-NMR(125MHz,CDCl3)δ174.5,173.5,170.5,170.1,169.8,169.7,169.6,167.2,165.5,165.4,165.3,142.9,133.6,133.3,129.8,129.7,129.6,129.5,129.0,128.7,128.6,128.5,128.4,124.2,119.0,98.7,98.5,70.4,69.8,68.3,68.2,67.5,67.5,66.9,66.7,62.0,61.7,60.3,56.9,53.7,52.9,38.7,31.7,29.2,27.9,26.0,25.8,21.0,21.0,20.9,20.8,20.7,20.6,20.5。

[1142] HRMS(ESI + )[M+NH4] + C 52 H 59 F3N3O 22Theoretical value: 1134.3537; experimental value 1134.3564.

[1143] The spectrum was confirmed to be consistent with the following document: Document 4) J. Org. Chem. 2016, 81, 10600-10616.

[1144] <Synthesis of the compound represented by formula A'-22>

[1145] According to the following synthetic route 4, a compound represented by formula A-22 (ie, a compound represented by formula A′-22) in which R 1 is a methoxyphenyl group was synthesized.

[1146] [Synthesis Path 4]

[1147]

[1148]

[1149] Example 29

[1150]

[1151] 3,4,6-tri-O-benzyl-1,2-O-(1-methoxyethylidene)-β-D-mannopyranose (the compound represented by Formula A-1) (40.0 g, 78.9 mmol) was placed in a 1 L four-necked flask, followed by ethyl acetate (400 mL). Water (2 mL) and p-TsOH·H₂O (45 mg, 0.237 mmol) were added at room temperature under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 6 hours. After confirming the completion of the reaction by HPLC, triethylamine (7.99 g, 78.9 mmol) was added, and the mixture was stirred at the same temperature overnight. After confirming the completion of the acetyl group transfer by HPLC, 5% sodium bicarbonate (400 mL) was added to the reaction mixture for separation. 20% saline (200 mL) was added to the organic layer for separation. The organic layer was concentrated under reduced pressure to 80 mL, toluene (400 mL) was added, and the mixture was concentrated under reduced pressure to 80 mL. Toluene (400 mL) was added again, and the mixture was concentrated under reduced pressure to 80 mL. Dehydrated toluene (120 mL) was added to obtain a toluene solution of 2-O-acetyl-3,4,6-tri-O-benzyl-D-mannopyranose (the compound represented by Formula A-2) as a colorless solution.

[1152] Example 30

[1153]

[1154] A toluene solution (78.9 mmol) of 2-O-acetyl-3,4,6-tri-O-benzyl-D-mannopyranose (compound represented by Formula A-2) was placed in a 1L flask, followed by trichloroacetonitrile (12 mL, 118 mmol) and DBU (119 μL, 0.789 mmol). The mixture was stirred at 0°C for 2 hours under nitrogen. After confirming the completion of the reaction by HPLC, acetic acid (45 μL, 0.789 mmol) was added to the reaction mixture at 0°C to obtain a toluene solution (78.9 mmol) of 2-O-acetyl-3,4,6-tri-O-benzyl-1-O-(2,2,2-trichloroethaneimido)-D-mannopyranose (compound represented by Formula A-3) as a brown solution. This solution was used directly in the next step.

[1155] Example 31

[1156]

[1157] 4-Methoxyphenyl 3,6-di-O-benzyl-2-deoxy-4-O-{2,4-di-O-benzyl-3-O-[(naphthalen-2-yl)methyl]-β-D-mannopyranosyl}-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula A-4) (20.0 g, 18.5 mmol) was placed in a 1 L four-necked flask and dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (160 mL). Water (16 mL) was then added. 2,3-Dichloro-5,6-dicyano-p-benzoquinone (4.63 g, 20.4 mmol) was added at -20°C under nitrogen, and the mixture was stirred at the same temperature for 6 hours. After confirming the completion of the reaction by HPLC, an aqueous solution of sodium sulfite (1.17 g, 9.25 mmol) dissolved in water (10 mL) was added and stirred overnight. Dichloromethane (300 mL) was added and the solution was separated and washed with an aqueous solution of sodium sulfite (4 g) and sodium bicarbonate (4 g) dissolved in water (200 mL). The organic layer was concentrated under reduced pressure to 60 mL and ethyl acetate (300 mL) was added. The organic layer was washed with 5% sodium bicarbonate water (200 mL) and 20% saline (100 mL), concentrated under reduced pressure to 40 mL, toluene (400 mL) was added, and the solution was concentrated under reduced pressure to 40 mL. Toluene (100 mL) was added and silica gel 60N (manufactured by Kanto Chemical, particle size: 40-50 μm, 60 g) was added. Heptane (40 mL) was added dropwise over 30 minutes under stirring to allow the target product to adsorb on the silica gel and then filtered. After washing the silica gel with toluene / heptane (7 / 2, 400 mL) (the filtrate was discarded), the target product was desorbed with ethyl acetate / toluene (1 / 1, 700 mL). The filtrate was concentrated under reduced pressure to obtain 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-4-O-(2,4-di-O-benzyl-β-D-mannopyranosyl)-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula A-5) (17.8 g, yield: >99%) as a white amorphous substance.

[1158] 1H-NMR (500MHz, CDCl3) δ7.67 (br, 4H), 7.27-7.37 (m, 15H), 6.89-6.96 (m, 5H), 6.83 (ddd, J=9.5, 4.0, 2.5Hz, 2H), 6.70 (ddd, J=9.0, 4.0, 2.5Hz, 2H), 5.6 4 (d, J=9.0Hz, 1H), 5.02 (d, J=11.5Hz, 1H), 4.94 (d, J=12.0Hz, 1H), 4.82 (d, J=11.5Hz, 1H), 4.70 (d, J=12.0Hz, 1H), 4.66 (d, J=12.0Hz, 1H), 4.62 (s, 1H), 4.57(d, J=11.0Hz, 1H), 4.51(d, J=12.0Hz, 1H), 4.42-4.46(m, 2H), 4.33(dd , J=10.5, 8.5Hz, 1H), 4.09 (t, J=9.0Hz, 1H), 3.69-3.79 (m, 4H), 3.71 (s, 3H), 3.65 (d, J=3.5Hz, 1H), 3.51 (td, J=9.0, 3.5Hz, 1H), 3.45 (t, J=9.0Hz, 1H), 3 .44-3.48 (m, 1H), 3.17 (m, 1H), 2.29 (d, J = 9.0Hz, 1H), 1.92 (t, J = 6.0Hz, 1H).

[1159] 13 C-NMR (125MHz, CDCl3) δ155.6, 151.0, 138.54, 138.46, 138.3, 137.8, 134.1, 131.8, 128.8, 128.6, 128.3, 128.21, 128.19, 128.15, 128.1, 1 28.0, 127.5, 127.4, 123.6, 118.9, 114.6, 101.5, 97.9, 79.1, 78.5, 77 .3, 76.7, 75.6, 75.4, 75.3, 74.9, 74.8, 74.4, 73.9, 68.5, 62.5, 55.8.

[1160] HRMS(ESI － )[M-H] － C 55 H 54 NO 13 Theoretical value: 936.3601; experimental value 936.3592.

[1161] The spectrum was confirmed to be consistent with the following literature.

[1162] Reference 1) Org. Biomol. Chem., 2018, 16, 4720-4727.

[1163] Furthermore, the reaction conditions were changed to synthesize the compound represented by Formula A-5 from the compound represented by Formula A-4. The following table shows the reaction conditions and the yield of the compound represented by Formula A-5 (measured by HPLC).

[1164]

[1165]

[1166] Project 1: The reaction proceeded with moderate yields under conventional conditions.

[1167] Project 2: The reaction was carried out in high yield by using HFIP.

[1168] • Projects 3 and 4: Deprotection based on acid and hydrogenation resulted in low yields.

[1169] Example 32

[1170]

[1171] A toluene solution (equivalent to 66.1 mmol) of 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-4-O-(2,4-di-O-benzyl-β-D-mannopyranosyl)-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula A-5) (17.8 g, 18.9 mmol) and 2-O-acetyl-3,4,6-tri-O-benzyl-1-O-(2,2,2-trichloroethaneimido)-D-mannopyranose (the compound represented by Formula A-3) was added to a 1 L four-necked flask, and toluene (178 mL) and molecular sieves 4A powder (less than 10 μm, 2.7 g) were added. Under nitrogen, trimethylsilyl trifluoromethanesulfonate (0.683 mL, 3.78 mmol) was added dropwise at -20°C over 1 hour, and the mixture was stirred at the same temperature for 1 hour. After confirming the completion of the reaction by HPLC, triethylamine (1.06 mL, 7.56 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was filtered through celite and washed with acetonitrile (178 mL). The filtrate was concentrated under reduced pressure to obtain a concentrated residue (the concentrated residue was divided into two parts, a 12.8 g portion of the starting material and a 5.0 g portion). Acetonitrile (256 mL) was added to the residue of 12.8 g of the raw material (it should be noted that 5.0 g of the raw material was treated in the same manner), and reverse phase silica gel 120RP-18 (manufactured by Kanto Chemical, particle size: 40-50 μm, 38.4 g) was added. Water (128 mL) was added dropwise over 30 minutes to allow the target product to be adsorbed on the solid phase, and then filtered. After washing the solid phase with acetonitrile / water (2 / 1, 1.53 L) and acetonitrile / water (3 / 1, 512 mL) in this order (the filtrate was discarded), the target product was desorbed with acetonitrile (1.02 L). By concentrating the filtrate under reduced pressure, 4- Methoxyphenyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula A-6) (19.6 g (8.1 g of product synthesized by another route), isolated yield 77%).

[1172] 1H-NMR (500MHz, CDCl3) δ7.63-6.60 (m, 58H), 5.54 (d, J=8.4Hz, 1H), 5.49 (dd, J=3.2, 2.0Hz, 1H), 5.34 (brt, J=3.2Hz, 1H), 5.13 (brd, J=1.6Hz, 1H), 5.05 (d, J=12.0Hz, 1H), 4.91-4.78 (m, 6H), 4.69 (d, J=11.2Hz, 1H), 4.63-4.55(m, 6H), 4.48-4.32(m, 9H), 4.21(t, J=11.2Hz, 2H), 4.07(t, J=9.6Hz, 1H), 4.00-3.73(m, 9H), 3. 70 (s, 3H), 3.68-3.51 (m, 8H), 3.44 (brd, J = 8.0Hz, 1H), 3.21 (brd, J = 9.6Hz, 1H), 2.10 (s, 3H), 1.86 (s, 3H).

[1173] 13 C-NMR (125MHz, CDCl3) δ170.2, 170.0, 155.4, 151.0, 138.9, 138.7, 138.6, 138.5, 138.2, 138.1, 138.0, 137.9, 133 .7, 131.7, 128.7, 128.5, 128.4, 128.3, 128.2, 127.9, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.1, 123.4, 11 8.8, 114.4, 101.9, 99.8, 98.5, 97.7, 81.3, 79.5, 78.2, 78.0, 77.9, 76.9, 76.6, 75.2, 75.2, 75.1, 74.9, 74.7, 74.5, 74.4, 74.2, 74.2, 73.6, 73.5, 73.4, 72.4, 72.0, 71.9, 71.4, 69.4, 69.4, 68.8, 68.8, 68.3, 66.7, 55.7, 21.2, 20.9.

[1174] HRMS(ESI + )[M+NH4] + C 113 H 119 N2O 25 Theoretical value: 1903.8096; experimental value 1903.8075.

[1175] Example 33

[1176]

[1177] 4-Methoxyphenyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (the compound represented by Formula A-6) (27.7 g, 14.6 mmol) was added to a 1 L four-necked flask, and dichloromethane (222 mL), 1,1,1,3,3,3-hexafluoro-2-propanol (139 mL) and water (14 mL) were added. Under nitrogen, [bis(trifluoroacetyloxy)iodo]benzene (12.6 g, 29.2 mmol) and trifluoroacetic acid (3.36 mL, 43.8 mmol) were added at -2°C, and stirred at the same temperature for 21 hours. After confirming the completion of the reaction by HPLC, the mixture was washed twice with an aqueous solution prepared by dissolving sodium bicarbonate (13.9 g) and sodium sulfite (13.9 g) in water (277 mL), and then washed with 20% saline (138 mL). The obtained organic layer was concentrated under reduced pressure to 55 mL, toluene (693 mL) was added, and the mixture was concentrated under reduced pressure to 55 mL again. Toluene (97 mL) and dichloromethane (42 mL) were added, and silica gel 60N (manufactured by Kanto Chemical, particle size: 40-50 μm, 83 g) was added. Heptane (166 mL) was added dropwise over 30 minutes to allow the target substance to be adsorbed on the silica gel, and then filtered. The mixture was washed with dichloromethane / heptane (1 / 3, 277 mL) (the filtrate was discarded), and the target product was desorbed from the solid phase with ethyl acetate / dichloromethane (1 / 4, 970 mL). The filtrate was concentrated under reduced pressure to obtain 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranosyl (compound represented by Formula A-7) (28.1 g, yield >99%) as a white amorphous solid.

[1178] 1H-NMR (500MHz, CDCl3)σ7.73-7.44(m, 4H), 7.40-7.07(m, 44H), 6.84-6.60(m, 6H), 5.4 8(dd, J=3.2, 2.0Hz, 1H), 5.36 (brt, J=2.8Hz, 1H), 5.21 (brs, 1H), 5.13 (d, J=1.6Hz, 1H) , 5.00 (d, J=11.6Hz, 1H), 4.88-4.76 (m, 5H), 4.70 (d, J=11.6Hz, 1H), 4.63-4.36 (m, 14H ), 4.26-4.19(m, 2H), 4.10-3.43(m, 20H), 3.26-3.19(m, 2H), 2.09(s, 3H), 1.89(s, 3H).

[1179] 13 C-NMR (125MHz, CDCl3)σ170.1, 167.9, 149.5, 138.6, 138.6, 138.5, 138.4, 138.3, 137.9, 137.9, 137.7, 1 33.6, 131.5, 128.6, 128.3, 128.2, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4, 127.4, 127.3, 126. 9, 123.1, 116.1, 101.0, 99.5, 97.9, 92.8, 80.9, 78.3, 78.0, 77.8, 76.0, 74.9, 74.9, 74.8, 74.5, 74.2, 74.1, 74.0, 73.9, 73.5, 73.4, 73.2, 72.2, 71.8, 71.6, 71.2, 69.1, 68.6, 68.3, 68.2, 66.5, 57.5, 21.0, 20.8.

[1180] HRMS(ESI + )[M+NH4] + C 106 H 113 N2O 24 + Theoretical value: 1798.7711; experimental value: 1798.7690.

[1181] Example 34

[1182]

[1183] 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranose (the compound represented by Formula A-7) (28.1 g, 15.7 mmol) was added to a 1 L four-necked flask, and dichloromethane (422 mL) and molecular sieve 4A powder (less than 10 μm, 14.1 g) were added, and the mixture was cooled to 0°C. Under nitrogen, N-methylimidazole (1.51 mL, 18.8 mmol) and 2,2,2-trifluoro-N-phenylacetimidazole chloride (2.74 mL, 17.2 mmol) were added at the same temperature and stirred for 24 hours. Since the completion of the reaction could not be confirmed by HPLC, N-methylimidazole (125 μL, 1.57 mmol) and 2,2,2-trifluoro-N-phenylacetimidazole chloride (250 μL, 1.57 mmol) were added and stirred at 0°C for 2 hours. The reaction solution was filtered through a neutral silica gel pad (Silica gel 60N, manufactured by Kanto Chemical, particle size: 40-50 μm, 84 g) filled with dichloromethane. The silica gel pad was washed with 10% ethyl acetate / dichloromethane (840 mL, 140 mL fractions each), and the main fraction was concentrated under reduced pressure to obtain 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-diazolyl-1,3-dihydro-2H-isoindol-2-yl)-1-O-(2,2,2-trifluoro-N-phenylethaneimido)-β-D-glucopyranosyl (compound represented by Formula A-8) (28.2 g, yield 91%) as a white amorphous substance.

[1184] 1H-NMR(500MHz,CDCl3)σ7.64(m,4H),7.41-7.09(m,47H),7.01(t,J=8.0Hz,1H),6.83(d,J=6.8Hz,2H),6.69-6.59(m,5H),5.49(dd,J=3.2,2.0Hz,1H),5.35(brt,J=2.0Hz,1H),5.13(brd,J=1.6Hz,1H),5.02(d,J=8.0Hz,1H),4.91-4.78(m,5H),4.69(d,J=11.6Hz,1H),4.63-4.53(m,6H),4.48-4.36(m,8H),4.36-4.06(m,5H),3.98(dd,J=9.2,3.2Hz,1H),3.95-3.61(m,13H),3.57-3.44(m,4H),3.16(brd,J=9.6Hz,1H),2.10(s,3H),1.84(s,3H)。

[1185] 13 C-NMR(125MHz,CDCl3)σ171.1,170.0,169.8,138.6,138.5,138.4,138.3,137.9,137.9,137.8,137.8,137.7,137.6,133.6,131.4,129.0,128.6,128.5,128.4,128.3,128.3,128.2,128.1,128.0,127.9,127.8,128.8,127.7,127.7,127.7,127.6,127.5,127.5,127.4,127.3,127.3,126.9,125.2,123.3,101.6,99.6,98.2,81.1,78.6,78.0,77.7,77.2,76.0,75.5,75.0,74.9,74.9,74.8,74.7,74.5,74.3,74.2,74.0,73.5,73.3,73.3,72.2,71.8,71.8,71.2,69.3,68.6,68.6,68.1,66.4,60.3,54.6,21.4,21.0,21.0,20.7,14.1。

[1186] HRMS(ESI + )[M+NH4] + C 114 H 117 F3N3O 24 +Theoretical value: 1969.8007; experimental value 1969.7983.

[1187] Example 35

[1188]

[1189] 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-pyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-pyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-pyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-1-O-(2,2,2-trifluoro-N-phenylethanediyl) A 1L four-necked flask was charged with 4-methoxyphenyl-3,6-di-O-benzyl-2-deoxy-2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula A'-9) (10.3 g, 17.3 mmol). Dichloromethane (424 mL) and molecular sieve 4A powder (8.50 g) were then added. Under nitrogen, trimethylsilyl trifluoromethanesulfonate (392 μL, 2.16 mmol) was added dropwise over 5 minutes at -7°C, and the mixture was stirred at the same temperature for 1 hour. After confirming the completion of the reaction by HPLC, triethylamine (802 μL, 5.76 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was filtered through Celite and washed with acetonitrile (283 mL). The filtrate was concentrated under reduced pressure to 56 mL, and acetonitrile (283 mL) and reverse-phase silica gel 120RP-18 (manufactured by Kanto Chemical, particle size: 40-50 μm, 85 g) were added. Water (254 mL) was added dropwise over 30 minutes to allow the target product to adsorb onto the solid phase, which was then filtered. The product was washed with acetonitrile / water (4 / 3, 283 mL) and then acetonitrile / water (3 / 1, 848 mL) in that order (the filtrate was discarded), and the target product was desorbed with acetonitrile (1.13 L). The filtrate was concentrated under reduced pressure and azeotroped twice with toluene (283 mL) to obtain 4-methoxyphenyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→6) as a white amorphous product. 4) -3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by formula A'-10) (31.0 g, 91%).

[1190] 1H-NMR(500MHz,CDCl3)σ7.78-7.48(m,8H),7.42(d,J=7.6Hz,2H),7.35-7.01(m,49H),6.98-6.84(m,4H),6.77-6.53(m,9H),5.50(dd,J=3.2,1.6Hz,1H),5.42(d,J=8.4Hz,1H),5.32(dd,J=2.8,2.0Hz,1H),5.21(dt,J=8.4,4.4Hz,1H),5.14(d,J=1.2Hz,1H),5.04(d,J=12.0Hz,1H),4.88-4.76(m,6H),4.68(d,J=6.8Hz,1H),4.63-4.60(m,3H),4.57-4.51(m,4H),4.48-4.32(m,11H),4.25-4.13(m,4H),4.06(dt,J=10.0,4.0Hz,1H),4.00-3.57(m,19H),3.55-3.34(m,5H),3.18(brt,J=9.2Hz,2H),2.09(s,3H),1.80(s,3H)。

[1191] 13 C-NMR(CDCl3)σ170.2,169.9,168.2,167.5,155.3,150.9,138.9,138.8,138.7,138.6,138.4,138.1,138.1,138.0,138.0,137.9,133.9,133.7,133.6,131.9,131.7,131.6,128.7,128.5,128.5,128.5,128.4,128.3,128.2,127.9,127.9,127.8,127.8,127.7,127.6,127.5,127.4,127.0,123.6,123.3,123.2,118.7,114.3,102.1,99.7,98.4,97.5,97.2,81.2,79.8,78.2,78.0,77.8,77.4,76.7,76.1,75.1,75.1,74.9,74.7,74.7,74.6,74.3,74.1,73.6,73.4,73.2,72.7,72.4,72.0,71.9,71.3,69.1,68.8,68.2,68.1,67.7,66.6,56.6,55.7,55.6,31.1,21.1,20.8。

[1192] HRMS(ESI +)[M+HNEt3] + C 147 H 156 N3O 31 + Theoretical value: 2459.0717; experimental value 2459.0720.

[1193] Example 36

[1194]

[1195] 4-methoxyphenyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-pyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-pyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-pyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)- β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-β-D-glucopyranoside (compound represented by Formula A'-10) (31.0 g, 13.1 mmol) was placed in a 1L four-necked flask, followed by tetrahydrofuran (124 mL), methanol (62 mL), and methyl trifluoroacetate (1.31 mL, 13.1 mmol). After stirring at room temperature under nitrogen for 5 minutes, a 1M solution of potassium tert-butoxide in tetrahydrofuran (6.6 mL, 6.6 mmol) was added, and the mixture was stirred at 50°C for 2 hours. After confirming the completion of the reaction by HPLC, the mixture was cooled to room temperature. Acetic acid (754 μL, 13.1 mmol) was added, followed by ethyl acetate (311 mL), and the organic layer was washed twice with 5% brine (311 mL). The organic layer was concentrated under reduced pressure to 62 mL, toluene (466 mL) was added, and the mixture was concentrated under reduced pressure to 62 mL. Toluene (466 mL) was added, and the mixture was concentrated under reduced pressure to 62 mL again. Toluene (155 mL) was added, and silica gel 60N (93.3 g, manufactured by Kanto Chemical, particle size: 40-50 μm) was added. Heptane (93 mL) was added dropwise over 30 minutes to allow the target substance to be adsorbed on the solid phase, and then filtered. After washing with toluene / heptane (7 / 3, 622 mL) (the filtrate was discarded), the target substance was desorbed from the solid phase using ethyl acetate / toluene (1 / 2, 1.1 L). The obtained desorbed liquid was concentrated under reduced pressure to obtain 4-methoxyphenyl 3,4,6-tr...

Claims

1. A method for producing an oligosaccharide represented by the following formula A-22, In formula A-22, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. The method comprises steps I-1, I-2, I-3, I-4, and I-5. The step I-1 is a step of generating a compound represented by the following formula A-8, and includes the following step: generating a compound represented by the following formula A-6 by forming an α-1,3-glycosidic bond and an α-1,6-glycosidic bond between the compound represented by the following formula A-3 and the compound represented by the following formula A-5, The step I-2 is a step of generating a compound represented by the following formula A-11, and includes the step of generating a compound represented by the following formula A-10 by forming a β-1,4-glycosidic bond between the compound represented by the following formula A-8 and the compound represented by the following formula A-9, In formula A-9, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. In formula A-10, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. In formula A-11, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. The step I-3 is a step of producing a compound represented by the following formula A-15, wherein: The compound represented by the formula A-11 is formed into a β-1,2-glycosidic bond with the compound represented by the formula A-12 to generate the compound represented by the formula A-13 below. Then, the phthaloyl group as the amino protecting group and the acetyl group on the hydroxyl group on the compound represented by the formula A-13 are removed to generate the compound represented by the formula A-14 below. Then, an aryloxycarbonyl group COOAr, an acetyl group Ac, a 2,2,2-trichloroethoxycarbonyl group Troc, and a phthaloyl group are added to the compound. The protecting group in the formyl group Phth protects the amino group in the compound represented by the formula A-14 to generate the compound represented by the following formula A-15, wherein R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc, and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group; or, The compound represented by the following formula A-15 is generated by removing the acetyl group Ac from the compound represented by the following formula A-13, wherein R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group, In formula A-13, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. In formula A-14, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. The step I-4 is a step of generating a compound represented by the following formula A-20, and comprises: generating a compound represented by the following formula A-17 by forming a β-1,4-glycosidic bond between the compound represented by the following formula A-15 and the compound represented by the following formula A-16, and then generating a compound represented by the following formula A-18 by removing the amino protecting group and the acyl protecting group of the hydroxyl group in the compound represented by the following formula A-17. In formula A-17, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc, and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group, In formula A-18, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, and M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation, In formula A-20, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, and M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation, The step I-5 is a step of producing the oligosaccharide represented by the formula A-22 by reacting the compound represented by the formula A-20 with the compound represented by the following formula A-21.

2. The method according to claim 1, wherein: include: The compound represented by the following formula A-4 is reacted with 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group in the compound represented by the formula A-4, thereby generating the compound represented by the formula A-5.

3. The method according to claim 2, wherein: The fluorine-containing alcohol is selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butyl alcohol, and combinations thereof.

4. The method according to claim 2 or 3, wherein: The reaction is carried out at -35°C to 70°C.

5. The method according to claim 2 or 3, wherein: The reaction is carried out at -30°C to -10°C.

6. The method according to any one of claims 1 to 5, wherein: The step I-2 includes: reacting the compound represented by the formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid to generate the compound represented by the formula A-11.

7. The method according to claim 6, wherein: The alkyl ester of the perfluorocarboxylic acid is methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, or butyl undecanoate.

8. The method according to claim 6 or 7, wherein: The strong base is selected from the sodium salt, lithium salt, and potassium salt of metal amide; C1-C 20 Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyl lithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene DBU, diazabicyclononene DBN, and 1,1,3,3-tetramethylguanidine TMG; and the group consisting of combinations thereof.

9. The method according to claim 6 or 7, wherein: The strong base is potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, or lithium hexamethyldisilazide LHMDS.

10. The method according to any one of claims 6 to 9, wherein: The step of reacting the compound represented by the formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid to produce the compound represented by the formula A-11 is carried out in a single step of C1 to C 10 In alcohol solvent, or in C1~C 10 The reaction is carried out in a mixed solvent of an alcohol solvent and an amide solvent, an ether solvent, an ester solvent, an aromatic solvent, a halogen solvent, a hydrocarbon solvent, or a nitrile solvent.

11. The method according to any one of claims 1 to 10, wherein: In the step I-3, the amino group in the compound represented by the formula A-14 is protected with an aryloxycarbonyl group COOAr to produce the compound represented by the formula A-15.

12. The method according to any one of claims 1 to 11, wherein: The step of generating the compound represented by the formula A-15 from the compound represented by the formula A-14 in the step I-3 is performed in an aqueous solution of sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate.

13. The method according to any one of claims 1 to 11, wherein: The R 1 It is isopropyl or 4-methoxyphenyl.

14. The method according to any one of claims 1 to 13, wherein: comprising a step for producing the compound represented by the formula A-16, The process is as follows: stirring a solution containing a compound represented by the following formula G-11 and carbonyl chlorohydrotris(triphenylphosphine)ruthenium (II), then adding water and 1,3-dibromo-5,5-dimethylhydantoin, iodine, or N-bromosuccinimide to the solution and stirring, thereby causing the allyl group bonded to the carbon 1 position of D-galactopyranoside in the compound represented by the formula G-11 to be detached via oxygen to generate a compound represented by the following formula G-12, and further, converting the hydroxyl group on the carbon 1 position of D-galactopyranoside in the compound represented by the formula G-12 to a 2,2,2-trifluoro-N-phenylacetimidyl group, thereby generating a compound represented by the formula A-16, 15. A method for producing a compound represented by the following formula A-5, wherein: include: The compound represented by the following formula A-4 is reacted with 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ in a mixed solvent of a fluorinated alcohol and water to remove the 2-naphthylmethyl group in the compound represented by the formula A-4, thereby generating the compound represented by the formula A-5.

16. The method according to claim 15, wherein: The fluorine-containing alcohol is selected from the group consisting of hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, nonafluoro-tert-butyl alcohol, and combinations thereof.

17. The method according to claim 15 or 16, wherein: The reaction is carried out at -35°C to 70°C.

18. The method according to claim 15 or 16, wherein: The reaction is carried out at -30°C to -10°C.

19. A method for producing a compound represented by the following formula A-11, In formula A-11, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups. The method comprises the following steps: reacting a compound represented by the following formula A-10 with a strong base in the presence of an alkyl ester of a perfluorocarboxylic acid, In formula A-10, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

20. The method according to claim 19, wherein: The alkyl ester of the perfluorocarboxylic acid is methyl trifluoroacetate, ethyl trifluoroacetate, propyl trifluoroacetate, isopropyl trifluoroacetate, butyl trifluoroacetate, methyl pentafluoropropionate, ethyl pentafluoropropionate, propyl pentafluoropropionate, isopropyl pentafluoropropionate, butyl pentafluoropropionate, methyl heptafluorobutyrate, ethyl heptafluorobutyrate, propyl heptafluorobutyrate, isopropyl heptafluorobutyrate, butyl heptafluorobutyrate, methyl nonafluorovalerate, ethyl nonafluorovalerate, propyl nonafluorovalerate, isopropyl nonafluorovalerate, butyl nonafluorovalerate, methyl undecanoate, ethyl undecanoate, propyl undecanoate, isopropyl undecanoate, or butyl undecanoate.

21. The method according to claim 19 or 20, wherein: The strong base is selected from the sodium salt, lithium salt, and potassium salt of metal amide; C1-C 20 Sodium, lithium, potassium, cesium, and barium salts of alkoxides; sodium hydride, potassium hydride, lithium hydride, butyl lithium, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, cesium phosphate, lithium phosphate, diazabicycloundecene DBU, diazabicyclononene DBN, and 1,1,3,3-tetramethylguanidine TMG; and the group consisting of combinations thereof.

22. The method according to claim 19 or 20, wherein: The strong base is potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, or lithium hexamethyldisilazide LHMDS.

23. The method according to any one of claims 19 to 22, wherein: The reaction is carried out in a single C1-C 10 In alcohol solvent, or in C1~C 10 The reaction is carried out in a mixed solvent of an alcohol solvent and an amide solvent, an ether solvent, an ester solvent, an aromatic solvent, a halogen solvent, a hydrocarbon solvent, or a nitrile solvent.

24. A method for producing a compound represented by the following formula A-17, In formula A-17, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc, and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group, The method comprises the following steps: generating a compound represented by the following formula A-15, and then forming a β-1,4-glycosidic bond with a compound represented by the following formula A-16 to generate a compound represented by the formula A-17. In formula A-15, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc, and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group, 25. The method according to claim 24, wherein: R5 is aryloxycarbonyl COOAr.

26. A method for producing a compound represented by the following formula A-18, In formula A-18, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, and M + is a sodium ion, a lithium ion, a potassium ion, or a protonated triethylamine cation, The method comprises: The amino protecting group and the acyl protecting group of the hydroxyl group in the compound represented by the following formula A-17 are removed. In formula A-17, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group.

27. An oligosaccharide represented by the following formula A-22, In formula A-22, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

28. An oligosaccharide represented by the following formula A'-22, 29. A compound represented by the following formula A-8, 30. A compound represented by the following formula A-10, In formula A-10, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

31. A compound represented by the following formula A-11, In formula A-11, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

32. A compound represented by the following formula A-13, In formula A-13, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

33. A compound represented by the following A-14, In formula A-14, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

34. A compound represented by the following formula A-14-FMA, In the formula A-14-FMA, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

35. A compound represented by the following formula A-15, In formula A-15, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group.

36. A compound represented by the following formula A-17, In formula A-17, R1 is a C1-C6 alkyl group or a phenyl group, the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups, R5 is an aryloxycarbonyl group COOAr, an acetyl group Ac, or a 2,2,2-trichloroethoxycarbonyl group Troc and R6 is a hydrogen atom, or R5 and R6 together with the nitrogen atom to which they are bonded form a phthalimide group.

37. A compound represented by the following formula A-18-FF or a salt thereof, In formula A-18-FF, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

38. A compound represented by the following formula A-19-FF or a salt thereof, In formula A-19-FF, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

39. A compound represented by the following formula A-20-FF or a salt thereof, In formula A-20-FF, R1 is a C1-C6 alkyl group or a phenyl group, and the phenyl group is optionally substituted by 1 to 3 C1-C6 alkoxy groups.

40. A compound represented by the following formula A'-20-FF or a salt thereof, 41. A method for producing a sugar chain remodeled antibody or a molecule comprising the Fc region thereof, wherein: include: A method comprising the step of obtaining an oligosaccharide represented by formula A-22 according to any one of claims 1 to 14; as well as, A step of reacting the obtained oligosaccharide represented by formula A-22 with an acceptor molecule to obtain the sugar chain remodeled antibody or a molecule comprising the Fc region thereof, wherein the acceptor molecule is an antibody or a molecule comprising the Fc region thereof having a core GlcNAc optionally with fucose added as an N297-linked sugar chain.

42. The manufacturing method according to claim 41, wherein: The method further includes a step of reacting the azido group N3- with a molecule having an alkyne structure.

43. The manufacturing method according to claim 42, wherein: The molecule having an alkyne structure is selected from chemotherapeutic agents, molecular targeted drugs, immune activators, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins and hormones.

44. A method for producing an antibody drug conjugate, wherein: The method comprises the manufacturing method as claimed in any one of claims 41 to 43.

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