Method for producing glycophospholipid

Abstract

To provide a method for synthesizing glycophospholipid in aqueous solution simply, at high throughput and high stereoselectivity in a single-stage reaction.SOLUTION: A method for producing glycophospholipid includes a condensation step of condensing saccharide and phospholipid adsorbed to a solid-phase carrier in the presence of a condensation agent represented by general formula (2). (In general formula (2), R21, R22, R23 and R24 may be the same or different from one another, each independently, are selected from the group consisting of a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent and an aryl group which may have a substituent, X denotes a halogen atom, Y- denotes anion.)SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing a glycophospholipid. [Background technology]

[0002] Glycophospholipids, which contain sugars, phosphate esters, and fatty acids in their molecules, are found in trace amounts in living organisms and have recently attracted attention for their physiological activities. For example, phosphatidylglucoside (PtdGlc) is a glycophospholipid expressed in the central nervous system during embryonic development, and because it is a pain-regulating molecule, it could be used as a therapeutic agent for painful diseases.

[0003] Non-Patent Document 1 reports a method for synthesizing a glycophospholipid, which includes protection and deprotection of a hydroxy group. Non-Patent Document 2 reports a method for synthesizing a glycophospholipid using an enzyme. Non-Patent Document 3 reports a method for synthesizing a glycophospholipid in a one-step reaction by reacting glucose activated with 2-chloro-1,3-dimethylimidazolinium chloride (DMC) with a phospholipid in an aqueous solvent. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] P. Greimel, Y. Ito, Tetrahedron Lett. 2008, 49, 3562-3566. [Non-patent document 2] A. Inoue, M. Adachi, J. Damnjanovic, H. Nakano, Y. Iwasaki, ChemistrySelect 2016, 1, 4121-4125. [Non-patent document 3] K. Kano, N. Ishii, A. Miyagawa, H. Takeda, Y. Hirabayashi, H. Kamiguchi, P. Greimel, I. Matsuo, Org. Biomol. Chem., 2023, 21, 2138-2142 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the glycophospholipid synthesis method reported in Non-Patent Document 1 requires complicated synthesis steps, including protection and deprotection of hydroxyl groups, and purification with organic solvents at each stage, resulting in problems with efficiency and environmental adaptability. The enzymatic synthesis method reported in Non-Patent Document 2 not only fails to obtain the target product in a single product, but also requires purification by chemical treatment, so the current situation is that the advantages of the enzymatic method are not fully utilized.

[0006] The method for synthesizing glycophospholipids reported in Non-Patent Document 3 is limited to a batch-type reaction in a liquid phase, and therefore there is room for improvement in productivity, stereoselectivity of glycophospholipids, etc.

[0007] The present invention is intended to solve the above-mentioned problems, and an object of the present invention is to provide a method for synthesizing a glycophospholipid in an aqueous solution by a single-step reaction in a simple manner with high throughput and high stereoselectivity. [Means for solving the problem]

[0008] As a result of investigations aimed at solving the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by a method for producing a glycophospholipid, which includes a condensation step of condensing a sugar with a phospholipid adsorbed on a solid phase carrier in the presence of a condensing agent represented by a specific formula, and have thus succeeded in completing the present invention.

[0009] That is, the present invention relates to the following [1] to [6]. [1] A sugar represented by the following general formula (1), [ka] (In general formula (1), R 11 is a hydroxy group or an acetamido group, R 12 , R 13 and R 14 may be the same or different and independently represent a hydrogen atom, a fluorine atom, a hydroxy group, a hydroxymethyl group, a carboxymethyl group, a carboalkoxymethyl group, an amino group, an acetamide group, an acylamino group, a carboxy group, an amide group, a PO4 2- , SO4 - , alkyl groups, alkenyl groups, alkynyl groups, acyl groups, alkoxy groups, acyloxy groups, azide groups, sugar residues of monosaccharides, sugar residues of oligosaccharides, and sugar residues of polysaccharides) and a phospholipid adsorbed onto a solid phase carrier. A method for producing a glycophospholipid, comprising a condensation step of condensing in the presence of a condensing agent represented by the following general formula (2): [ka] (In general formula (2), R 21 , R 22 , R 23 and R 24 may be the same or different and are each independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, and an optionally substituted aryl group; X is a halogen atom; Y - is an anion) [2] The method according to [1], wherein the solvent used in the condensation step is water. [3] The method according to [1] or [2], wherein the phospholipid is a glycerophospholipid represented by the following general formula (3): [ka] (In general formula (3), R 31 and R 32may be the same or different and are each independently selected from an optionally substituted alkyl group or an optionally substituted alkenyl group; A + is a hydrogen ion, an alkali metal ion, or a triethylammonium ion) [4] The method according to any one of [1] to [3], wherein the phospholipid is adsorbed to the solid phase carrier by hydrophobic interaction. [5] The method according to any one of [1] to [4], further comprising an adsorption step of adsorbing the phospholipid onto the solid phase carrier. [6] The method according to any one of [1] to [5], further comprising a recovery step of recovering the glycophospholipid obtained in the condensation step from the solid phase carrier. [Effects of the Invention]

[0010] According to the present disclosure, a method for synthesizing a glycophospholipid in an aqueous solution can be provided that is simple, high-throughput, and highly stereoselective in a single-step reaction. Furthermore, the method for synthesizing a glycophospholipid according to the present disclosure can use only water as the reaction solvent, thereby realizing a reaction with low environmental impact. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows the results of LCMS analysis of the glycophospholipid obtained in Example 1. [Figure 2] The upper panel shows the results of 1H-NMR analysis of the glycophospholipid obtained in Example 1, and the lower panel shows the results of 1H-NMR analysis of the glycophospholipid obtained by the liquid phase method without adsorbing the phospholipid to a solid phase support. [Figure 3] 1 shows the results of LCMS analysis of the glycophospholipid obtained in Example 2. [Figure 4] 1 shows the results of LCMS analysis of the glycophospholipid obtained in Example 3. [Figure 5] 1 shows the results of LCMS analysis of the glycophospholipid obtained in Example 4. [Figure 6]The upper panel shows the results of 1H-NMR analysis of the glycophospholipid obtained in Example 5, and the lower panel shows the results of 1H-NMR analysis of the glycophospholipid obtained by the liquid phase method without adsorbing the phospholipid to a solid phase support. DETAILED DESCRIPTION OF THE INVENTION

[0012] Unless otherwise specified, the expressions "XX to YY" or "XX to YY" representing a numerical range mean a numerical range including the lower and upper limits, which are the endpoints. When a numerical range is described in stages, the upper and lower limits of each numerical range can be combined in any way.

[0013] <Method of producing glycophospholipid> A method for producing a glycophospholipid according to one embodiment of the present invention (hereinafter simply referred to as "method for producing a glycophospholipid") ") includes a condensation step in which a sugar and a phospholipid adsorbed on a solid support are condensed in the presence of a condensing agent represented by a specific formula.

[0014] [sugar] The sugar used in the method for producing glycophospholipid (hereinafter simply referred to as "sugar") is represented by the following general formula (1).

[0015] [ka] (In general formula (1), R 11 is a hydroxy group or an acetamide group, and R 12 , R 13 and R 14 may be the same or different and independently represent a hydrogen atom, a fluorine atom, a hydroxy group, a hydroxymethyl group, a carboxymethyl group, a carboalkoxymethyl group, an amino group, an acetamide group, an acylamino group, a carboxy group, an amide group, a PO4 2- , SO4 - , alkyl groups, alkenyl groups, alkynyl groups, acyl groups, alkoxy groups, acyloxy groups, azide groups, sugar residues of monosaccharides, sugar residues of oligosaccharides, and sugar residues of polysaccharides)

[0016] In the general formula (1), the "acyl group (including the acyl group of an acylamino group and the acyl group moiety of an acyloxy group)" may include a group derived from an oxo acid such as a carboxylic acid, for example, a carboxylic acid acyl group such as an optionally substituted hydrocarbon group -CO- group, more specifically, an optionally substituted C 1-10 It may refer to an acyl group.

[0017] In the general formula (1), the "alkyl group" may be either a straight chain or a branched chain, or may be a cyclic group. The alkyl group may be, for example, C 1-22 Alkyl (methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl yl, isopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decanyl , hexadecanyl, eicosanyl, etc.), and cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. The alkyl group is preferably C 1-6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, etc.), and more preferably C 1-4 Alkyl (for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc.).

[0018] In the general formula (1), the "alkenyl group" may be either a straight chain or a branched chain, or may be cyclic. The alkenyl group may be, for example, C 2-22 Examples of the alkenyl group include cyclic alkenyl groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, and cyclohexenyl. The alkenyl group is preferably C 2-6 alkenyl (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, etc.), more preferably C 2-4 alkenyl (eg, ethenyl, propenyl, butenyl, etc.);

[0019] In the general formula (1), the "alkynyl group" may be either a straight chain or a branched chain, or may be cyclic. The alkynyl group may be, for example, C 2-22 Examples of the alkynyl group include cyclic alkynyl groups such as cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl. The alkynyl group is preferably C 2-6 Alkynyl (e.g., ethynyl, propynyl such as propargyl, butynyl, pentynyl, hexynyl, etc.), more preferably C 2-4 Alkynyl (for example, ethynyl, propynyl such as propargyl, butynyl, etc.).

[0020] In general formula (1), the "sugar residue" may include modified sugar residues. Modified sugars may be those modified during the process of isolation and purification from naturally occurring sugars, enzymatically modified, chemically modified, or modified by biological methods including microbial modification, and may include modifications known in the field of glycoscience, such as hydrolysis, oxidation-reduction, esterification, acylation, amination, etherification, nitration, dehydration, glycosylation, etc.

[0021] In general formula (1), R 12 is preferably a hydroxy group, a fluorine atom or an azide group, and R 13 is preferably a hydroxy group, and R 14 is preferably a hydroxymethyl group or an azide group.

[0022] The sugar represented by general formula (1) is not particularly limited by its origin or derivation, and may include those obtained from nature, those synthesized by genetic engineering using animal cells, plant cells, microorganisms, etc., those produced enzymatically, those produced by fermentation, or those artificially chemically synthesized, etc. Furthermore, the sugar represented by general formula (1) may include monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and may also be monosaccharides, disaccharides, oligosaccharides, and polysaccharides having a fluorine atom or an azide group.

[0023] Examples of monosaccharides include glucose, galactose, mannose, glucosamine, N-acetylglucosamine, galactosamine, N-acetylgalactosamine, mannosamine, N-acetylmannosamine, fructose, glucuronic acid, and iduronic acid.

[0024] Examples of disaccharides include maltose, isomaltose, lactose, lactosamine, N-acetyllactosamine, cellobiose, melibiose, N,N'-diacetylchitobiose, hyaluronic acid disaccharides, and glycosaminoglycan disaccharides.

[0025] Oligosaccharides are sugars usually composed of 2 to 30 monosaccharide units, typically 2 to 20 monosaccharide units, and include, for example, homo-oligomers composed of one component selected from the group consisting of glucose, galactose, mannose, glucosamine, N-acetylglucosamine, and fructose, and hetero-oligomers composed of two or more components. Specific examples include maltooligosaccharides, isomaltooligosaccharides, lacto-oligosaccharides, lactosamine oligosaccharides, N-acetyllactosamine oligosaccharides, cello-oligosaccharides, melibio-oligosaccharides, N-acetylchitotriose, N-acetylchitotetraose, and N-acetylchitopentaose.

[0026] Polysaccharides are found in a wide range of organisms, including animals, plants (including seaweed), insects, and microorganisms, and examples include sialocomplex glycans, N-linked glycans, O-linked glycans, glycosaminoglycans, starch, amylose, amylopectin, cellulose, chitin, glycogen, agarose, alginic acid, hyaluronic acid, inulin, and glucomannan.

[0027] [Phospholipids] The phospholipid used in the method for producing glycophospholipid (hereinafter simply referred to as "phospholipid") is a phospholipid adsorbed to a solid phase carrier as described below, and may be a sphingophospholipid or a glycerophospholipid represented by general formula (3).

[0028] [ka] (In general formula (3), R 31 and R 32 may be the same or different and are each independently selected from an optionally substituted alkyl group or an optionally substituted alkenyl group; A + is a hydrogen ion or an alkali metal ion)

[0029] In general formula (3), R 31 and R 32 are each independently preferably an alkyl or alkenyl group having 2 to 32 carbon atoms, more preferably an alkyl or alkenyl group having 6 to 28 carbon atoms, and even more preferably an alkyl or alkenyl group having 10 to 24 carbon atoms. + is preferably a hydrogen ion, a sodium ion or a triethylammonium ion.

[0030] In general formula (3), the alkyl group or alkenyl group may have a substituent. The substituent is not particularly limited, but examples thereof include a fluorescent group.

[0031] The fluorescent group is a group that is irradiated with light, absorbs the energy, and thereby excites electrons, and releases excess energy as electromagnetic waves when returning to the ground state. The fluorescent group is not particularly limited, but examples thereof include rhodamine-based, coumarin-based, oxazine-based, carbopyronine-based, cyanine-based, pyrromesene-based, naphthalene-based, biphenyl-based, anthracene-based, phenanthrene-based, pyrene-based, carbazole-based, Cy-based, EvoBlue-based, fluorescein-based, and derivatives thereof.

[0032] [Solid phase support] In the method for producing a glycophospholipid, the solid phase carrier to which the phospholipid is adsorbed (hereinafter also simply referred to as "solid phase carrier") can have various forms and compositions, and can be obtained from a naturally occurring material (natural material), a synthetically modified natural material, or a synthetic material.

[0033] In the method for producing glycophospholipids, by adsorbing phospholipids onto a solid support, the production amount can be increased by a continuous reaction, unlike a reaction in a liquid phase. Furthermore, since only water can be used as the reaction solvent, the reaction has a low environmental impact. In addition, the stereoselectivity of glycophospholipids can be improved.

[0034] In glycophospholipids, the bond between the sugar and phospholipid may be either an α-glycosidic bond or a β-glycosidic bond, but natural glycophospholipids mainly have a β-glycosidic bond. Therefore, β-glycosidic bonds are preferred. A glycophospholipid having an α-glycosidic bond is called an “α-type,” and a glycophospholipid having a β-glycosidic bond is called a “β-type.” In the method for producing a glycophospholipid of the present invention, it is preferable that a β-type glycophospholipid is produced with high selectivity.

[0035] Solid supports can have a variety of forms and compositions and can be derived, for example, from naturally occurring materials (natural materials), synthetically modified natural materials, or synthetic materials. Specific examples of solid phase supports include silicon; glass (e.g., microporous glass, porous glass (e.g., controlled pore glass (CPG)); metals (e.g., gold, platinum); magnetic beads with a ferrite core coated with glycine methacrylate; plastics (e.g., polyethylene glycol resin, silica gel resin, polytetrafluoroethylene resin, polyester resin, polyethylene resin, polypropylene resin, polystyrene resin, acrylonitrile butadiene styrene resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, vinyl chloride resin); polysaccharides (e.g., agarose, dextran, nitrocellulose); polyacrylamide; polyvinyl alcohol; and copolymers of hydroxyethyl methacrylate and methyl methacrylate.

[0036] The solid phase carrier is preferably a carrier capable of adsorbing phospholipids through hydrophobic interaction, and is preferably a carrier having a hydrophobic group. For example, a plastic solid phase carrier is preferred, a plastic (polymer-based) solid phase carrier other than silica gel resin is more preferred, and a polystyrene resin or polypropylene resin is even more preferred. Commercially available solid phase supports may be used, such as InertSep RP-C18 (a styrene vinyl benzene polymer with an alkyl chain introduced therein, manufactured by GL Sciences), Oasis HLB Cartridge (polypropylene, manufactured by Waters (registered trademark)), Waters Sep-Pak Vac (silica gel, manufactured by Waters (registered trademark)), and ISOLUTE C18 (silica gel, manufactured by Biotage).

[0037] The solid phase carrier may be in any shape, such as a plate (substrate), bead, thread, sphere, polygon, powder, etc. Although the solid phase carrier can be used in any shape, it is preferably used in a form packed in a container such as a column.

[0038] [Condensing agent] The condensing agent used in the method for producing glycophospholipid (hereinafter simply referred to as "condensing agent") is represented by the following general formula (2).

[0039] [ka] (In general formula (2), R 21 , R 22 , R 23 and R 24 may be the same or different and are each independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, and an optionally substituted aryl group; X is a halogen atom; Y - is an anion)

[0040] In general formula (2), the "alkyl group" can have the same structure as the "alkyl group" in general formula (1).

[0041] In the general formula (2), the "alkenyl group" may be either a straight chain or a branched chain, or may be a cyclic group. The alkyl group may be, for example, C 2-24 Examples include alkenyl (vinyl, allyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1-methyl-2-propenyl, 2-methyl-1-propenyl, etc.).

[0042] In the general formula (2), the "aryl group" is, for example, C 6-14 Examples thereof include aryl (phenyl, 1-naphthyl, 2-naphthyl, 2-biphenylyl, 3-biphenylyl, 4-biphenylyl, 2-anthryl, 3-indenyl, 5-fluorenyl, etc.).

[0043] In the general formula (2), examples of the "halogen atom" include fluorine, chlorine, bromine, and iodine.

[0044] In the general formula (2), the "anion" is, for example, a halide ion such as a chloride ion, a bromide ion, or an iodide ion, or an OH - , BF4 - , PF6 - etc.

[0045] The condensing agent is a haloformamidinium derivative, such as 2-chloro-1,3-dimethylimidazolinium salt, 1-(chloro-1-pyrrolidinylmethylene)pyrrolidinium hexafluorophosphate, 1-(chloro-1-pyrrolidinylmethylene)pyrrolidinium tetrafluoroborate, chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate, N,N,N',N'- Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, (4R,5R)-2-chloro-1,3-dimethyl-4,5-diphenyl-1-imidazolinium chloride, (4S,5S)-2-chloro-1,3-dimethyl-4,5-diphenyl-1-imidazolinium chloride, N,N,N',N'-tetramethylchloroformamidinium chloride, chloro-N,N,N',N'-bis(tetramethylene)formamidiniumhexafluorophosphate, 2-chloro-1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinium chloride, and the like.

[0046] Among these, the condensing agent is preferably a 2-chloro-1,3-dimethylimidazolinium salt, and examples thereof include 2-chloro-1,3-dimethylimidazolinium chloride (DMC), 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP), and 2-chloro-1,3-dimethylimidazolinium tetrafluoroborate (CIB). The condensing agent is more preferably 2-chloro-1,3-dimethylimidazolinium chloride (DMC).

[0047] [Adsorption process] The method for producing a glycophospholipid preferably further comprises an adsorption step of adsorbing the phospholipid to a solid phase carrier. In the adsorption step, a solution in which the phospholipid is dissolved is preferably brought into contact with the solid phase carrier to adsorb the phospholipid to the solid phase carrier. The adsorption of the phospholipid to the solid phase carrier can be confirmed by LCMS analysis.

[0048] The solvent used in the adsorption step is not particularly limited as long as the adsorption proceeds, but an aqueous solvent can be suitably used. Examples of the aqueous solvent include water; alcohols such as methanol, ethanol, n-propanol, isopropanol, cyclohexanol, furfuryl alcohol, ethylene glycol, and benzyl alcohol; ethers such as tetrahydrofuran (THF), dioxane, tetrahydrofurfuryl alcohol, diethylene glycol, cyclohexyl methyl ether, methyl cellosolve, cellosolve, butyl cellosolve, and methyl tert-butanol; ketones such as methyl ethyl ketone, furfural, methyl isobutyl ketone, mesityl oxide, diacetone alcohol, and cyclohexanone; acetone; Examples of suitable amines include nitrile compounds such as nitrile, propionitrile, and benzonitrile; sulfoxides such as dimethyl sulfoxide (DMSO) and sulfolane; amides such as formamide, N,N-dimethylformamide (DMF), and N,N-dimethylacetamide; esters such as methyl formate, ethyl formate, ethyl acetate, butyl acetate, methoxybutyl acetate, cellosolve acetate, diethyl carbonate, and glycol carbonate; organic acids such as formic acid, acetic acid, and propionic acid; heterocyclic compounds such as hexamethylphosphorotriamide, pyridine, and quinoline; aromatic amines such as aniline and N-methylaniline; and nitro compounds. These solvents can be used alone, or two or more kinds can be mixed in an appropriate ratio as required.

[0049] From the viewpoint of efficiently adsorbing phospholipids onto the solid phase carrier, the solvent used in the adsorption step preferably contains water and an alcohol, more preferably contains water and an alcohol having 1 to 5 carbon atoms, even more preferably contains water and an alcohol having 1 to 3 carbon atoms, and particularly preferably contains water and methanol. Furthermore, the ratio of water to alcohols [water / alcohols (v / v)] in the solvent used in the adsorption step is preferably 99 / 1 to 50 / 50, more preferably 97 / 3 to 75 / 25, and even more preferably 95 / 5 to 85 / 15, from the viewpoint of efficiently adsorbing phospholipids to the solid phase carrier.

[0050] The amount of phospholipid to be dissolved in 1 mL of the solvent used in the adsorption step is preferably 0.001 mg to 2.5 mg, more preferably 0.005 to 0.5 mg, and even more preferably 0.01 to 0.25 mg.

[0051] In the adsorption step, after adsorbing the phospholipid to the solid phase carrier, it is preferable to bring the solvent used in the condensation step described later into contact with the solid phase carrier to perform solvent substitution. By performing solvent substitution, the condensation reaction in the condensation step described later can be more easily progressed. The solid phase carrier with adsorbed phospholipid obtained in the adsorption step can be used in the condensation step described later immediately after adsorption, or can be used after storage such as refrigerated storage.

[0052] [Condensation process] In the condensation step (hereinafter simply referred to as "condensation step") included in the method for producing glycophospholipid, sugar and phospholipid adsorbed to a solid phase carrier are condensed in the presence of a condensing agent. The phospholipid adsorbed to the solid phase carrier is preferably the one obtained in the above-mentioned adsorption step.

[0053] The solvent used in the condensation step is not particularly limited as long as the reaction proceeds, but the aqueous solvents described above are preferably used. These solvents can be used alone, or two or more kinds can be mixed in an appropriate ratio as required.

[0054] The solvent used in the condensation step is preferably water. When the solvent is water alone, a reaction with low environmental impact can be realized. In addition, the stereoselectivity of the glycophospholipid can be more easily improved.

[0055] The solvent used in the condensation step is preferably water alone, but may contain a nitrile compound such as acetonitrile, propionitrile, benzonitrile, butyronitrile, or valeronitrile, or may contain a nitrile compound represented by the following general formula (4):

[0056] [ka] (In general formula (4), R 41 is selected from the group consisting of alkyl groups, aralkyl groups, and aryl groups each having 2 or more carbon atoms.

[0057] In the general formula (4), examples of the "alkyl group" include an ethyl group, a propyl group, an isopropyl group, a butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, and a hexyl group.

[0058] In the general formula (4), examples of the "aralkyl group" include a benzyl group.

[0059] In general formula (4), examples of the "aryl group" include mono-substituted phenyl groups such as a phenyl group, a 2-methylphenyl group, a 3-methylphenyl group, a 4-methylphenyl group, a 2-methoxyphenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl group, a 2-ethoxyphenyl group, a 3-ethoxyphenyl group, and a 4-ethoxyphenyl group, and di-substituted phenyl groups such as a 2,5-dimethylphenyl group, a 2,4-dimethoxyphenyl group, and a 2,6-dimethoxyphenyl group.

[0060] Examples of the nitrile compound represented by general formula (4) include propionitrile, butyronitrile, isobutyronitrile, valeronitrile, isovaleronitrile, trimethylacetonitrile, hexanitrile, heptanitrile, phenylacetonitrile, benzonitrile, 2-tolunitrile, 3-tolunitrile, 4-tolunitrile, 2-methoxybenzonitrile, 3-methoxybenzonitrile, 4-methoxybenzonitrile, 2-ethoxybenzonitrile, 3-ethoxybenzonitrile, 4-ethoxybenzonitrile, 2,5-dimethylbenzonitrile, 2,4-dimethoxybenzonitrile, and 2,6-dimethoxybenzonitrile.

[0061] Among them, the nitrile compound represented by the general formula (4) is preferably propionitrile, butyronitrile, valeronitrile, or the like.

[0062] It is preferable to add an amine to the solvent used in the condensation step. The amine may be any of primary amine, secondary amine, tertiary amine, and quaternary amine, and examples thereof include those having an aliphatic hydrocarbon residue, an aromatic hydrocarbon residue, and a heterocyclic residue. The aliphatic hydrocarbon residue may be linear or branched, saturated or unsaturated, and includes, for example, an alkyl group, an alkenyl group, a cycloalkyl group, an aralkyl group, and a cycloalkyl group. The aromatic hydrocarbon residue may be monocyclic or a condensed ring of two or more rings, and includes, for example, a phenyl group, a naphthyl group, and the like. The heterocyclic residue may have one or more atoms selected from the group consisting of a sulfur atom, an oxygen atom, and a nitrogen atom, and may include a pyridyl group, an imidazolyl group, a thiazolyl group, a quinolinyl group, and the like. The amine may also include piperidine, morpholine, thiomorpholine, piperazine, pyrrolidine, and the like. Representative amines include tertiary amines or diamines having an aliphatic hydrocarbon residue, such as trimethylamine, triethylamine, diethylmethylamine, dimethylethylamine, n-butyldimethylamine, diisopropylethylamine, and tetramethylethylenediamine.

[0063] In the condensation step, the amount of condensing agent added per 1 mol of sugar is preferably 0.25 mol or more and 10 mol or less, more preferably 0.5 mol or more and 7 mol or less, and even more preferably 1 mol or more and 5 mol or less. When the amount of the condensing agent added per 1 mol of sugar is within the above range, the yield of the obtained glycophospholipid can be further improved.

[0064] In the condensation step, the amount of amine added per 1 mol of sugar is preferably 0.5 mol or more and 50 mol or less, more preferably 1 mol or more and 35 mol or less, and even more preferably 3 mol or more and 25 mol or less. When the amount of amine added per 1 mol of sugar is within the above range, the yield of the obtained glycophospholipid can be further improved.

[0065] In the condensation step, the amount of sugar added per 1 mg of phospholipid adsorbed to the solid phase carrier is preferably 10 μmol to 1000 μmol, more preferably 50 μmol to 500 μmol, and even more preferably 100 μmol to 250 μmol. When the amount of sugar added per 1 mg of phospholipid adsorbed to the solid phase carrier is within the above range, the yield of the obtained glycophospholipid can be further improved.

[0066] In the condensation step, it is preferable to prepare a mixed solution of a sugar and a condensing agent, preferably a sugar, a condensing agent, and an amine, before contacting the sugar with the phospholipid adsorbed on the solid phase carrier. By preparing the mixed solution in advance, the sugar can be activated. When preparing the mixed solution, it is preferable to first mix the sugar and the amine, and then add the condensing agent. The solution temperature when preparing the mixed solution is not particularly limited as long as the sugar activation proceeds. The solution temperature can be, for example, -30°C to 30°C, -20°C to 20°C, or -10°C to 10°C.

[0067] In the condensation step, the phospholipid adsorbed on the solid support is contacted with a sugar in the presence of a condensing agent, preferably a condensing agent and an amine, to obtain a glycophospholipid. The reaction temperature in the condensation step is not particularly limited as long as the reaction proceeds. The reaction temperature can be, for example, -30°C to 80°C, -20°C to 40°C, or -10°C to 10°C. The reaction time is not particularly limited as long as the reaction proceeds. The reaction time can be, for example, 3 seconds to 5 minutes, 7 seconds to 2 minutes, or 10 seconds to 1 minute. The reaction time can also be expressed as the contact time between the solution containing at least the sugar and the condensing agent and the phospholipid adsorbed on the solid phase carrier.

[0068] In the condensation step, the solution containing at least a sugar and a condensing agent may be contacted with the phospholipid adsorbed on the solid phase carrier only once, or may be contacted two or more times. Contacting two or more times can further improve the yield of glycophospholipid.

[0069] [Recovery process] The method for producing a glycophospholipid preferably further comprises a recovery step of recovering the glycophospholipid obtained in the condensation step from the solid phase carrier. In the recovery step, the glycophospholipid is preferably recovered by contacting a recovery solvent with the solid phase carrier and dissolving the glycophospholipid in the recovery solvent. Glycophospholipid recovery was performed using LCMS analysis and 1 This can be confirmed by H-NMR analysis.

[0070] In the recovery step, it is preferable to bring the solvent used in the condensation step into contact with the solid phase carrier before bringing the recovered solvent into contact with the solid phase carrier to remove sugar, condensing agent, and amine residues.

[0071] The recovery solvent used in the recovery step is not particularly limited as long as the recovery proceeds, but preferred examples include alcohols such as methanol, ethanol, n-propanol, isopropanol, cyclohexanol, furfuryl alcohol, ethylene glycol, and benzyl alcohol; ethers such as tetrahydrofuran (THF), dioxane, tetrahydrofurfuryl alcohol, diethylene glycol, cyclohexyl methyl ether, methyl cellosolve, cellosolve, butyl cellosolve, and methyl tert-butanol; and nitrile compounds such as acetonitrile, propionitrile, and benzonitrile. These solvents can be used alone or, if necessary, two or more of them can be mixed in an appropriate ratio. The recovery solvent is preferably selected depending on the length of the lipids constituting the glycophospholipid. [Example]

[0072] The present invention will be explained in more detail below by way of examples, but modifications can be made as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0073] Example 1 [Adsorption process] 0.1 mg of the phospholipid represented by the following formula was dissolved in 2000 μL of a solvent with a ratio of water to methanol [water / methanol (v / v)] = 90 / 10. [ka]

[0074] This phospholipid solution was passed through a solid support, InertSep RP-C18 (styrene vinyl benzene polymer with alkyl chains, column volume 100 μL, manufactured by GL Sciences), to adsorb the phospholipids onto the solid support. After the phospholipids were adsorbed onto the solid support, 300 μL of water was passed through the solid support to perform solvent exchange. LCMS analysis confirmed that the phospholipids had been adsorbed onto the solid support.

[0075] [Condensation process] 75 μL of ultrapure water, 7.5 μL of 2.4 M D-glucose solution, 15 μL of 15 M triethylamine (TEA), and 7.5 μL of 7.3 M 2-chloro-1,3-dimethylimidazolinium chloride (DMC) solution were mixed in this order using a micromixer at a solution temperature of 0°C to obtain a mixed solution containing sugar, condensing agent, and amine.

[0076] The resulting mixed solution was passed through the solid phase carrier once at a reaction temperature of 0°C at a flow rate of 1 mL / min over 19 seconds to carry out a condensation reaction between the phospholipid adsorbed on the solid phase carrier and the sugar.

[0077] [Recovery process] Ultrapure water in an amount three times the column volume was passed through the solid phase carrier, and then a 50% aqueous methanol solution in an amount three times the column volume was passed through the solid phase carrier to remove sugar, condensing agent, and amine residues. After removing the residue, 1000 μL of methanol as a recovery solvent was passed through the solid phase carrier, and then 1000 μL of 2-propanol was passed through the solid phase carrier to recover the glycophospholipid. 1 As a result of H-NMR analysis and LCMS analysis, the obtained glycophospholipid was represented by the following formula, and the ratio of α-type / β-type was about 1 / 50. The LCMS analysis results of the obtained glycophospholipid are shown in Figure 1. 1 The H-NMR analysis results are shown in the upper panel of Figure 2. As a result of Example 1, it was found that although a small amount of unreacted phospholipid remained, a large amount of glycophospholipid was synthesized. 1 The bottom row of the H-NMR analysis results shows the analysis results for glycophospholipids obtained by the liquid phase method without adsorbing the phospholipids to a solid phase support. It was confirmed that the ratio of α-type was lower when the phospholipids were adsorbed to a solid phase support (top row of Figure 2) compared to the liquid phase method (bottom row of Figure 2). [ka]

[0078] <Example 2> The same glycophospholipid as in Example 1 was obtained in the same manner as in Example 1, except that Oasis HLB Cartridge (polypropylene, column volume 100 μL, manufactured by Waters (registered trademark)) was used as the solid phase carrier. The α-type / β-type ratio was about 1 / 50. The results of LCMS analysis of the obtained glycophospholipid are shown in FIG. 3.

[0079] Example 3 The same glycophospholipid as in Example 1 was obtained in the same manner as in Example 1, except that Waters Sep-Pak Vac (silica gel, column volume 100 μL, manufactured by Waters (registered trademark)) was used as the solid phase carrier. The α-type / β-type ratio was about 1 / 50. The results of LCMS analysis of the obtained glycophospholipid are shown in FIG. 4.

[0080] Example 4 The same glycophospholipid as in Example 1 was obtained in the same manner as in Example 1, except that ISOLUTE C18 (silica gel, column volume 100 μL, manufactured by Biotage) was used as the solid phase support. The α-type / β-type ratio was approximately 1 / 50. The results of LCMS analysis of the obtained glycophospholipid are shown in FIG.

[0081] <Example 5> [Adsorption process] 0.1 mg of the same phospholipid as in Example 1 was dissolved in 2000 μL of a solvent containing water and methanol in a ratio of water / methanol (v / v)=90 / 10.

[0082] This phospholipid solution was directly passed through 30 mg of InertSep RP-C18 (a styrene vinyl benzene polymer with alkyl chains, manufactured by GL Sciences) as a solid support, and the phospholipids were adsorbed onto the solid support. After the phospholipids were adsorbed onto the solid support, 1000 μL of water was passed directly through the solid support to perform solvent exchange. LCMS analysis confirmed that the phospholipids had been adsorbed onto the solid support.

[0083] [Condensation process] 75 μL of ultrapure water, 7.5 μL of 2.4 M D-glucose solution, 15 μL of 15 M triethylamine (TEA), and 7.5 μL of 7.3 M DMC solution were mixed in this order in a separate container at a solution temperature of 0°C to obtain a mixed solution containing a sugar, a condensing agent, and an amine.

[0084] The resulting mixed solution was passed directly through the solid phase carrier once at a reaction temperature of 0°C at a flow rate of 1 mL / min over 19 seconds, and a condensation reaction was carried out between the phospholipid adsorbed on the solid phase carrier and the sugar.

[0085] [Recovery process] Ultrapure water in an amount three times the volume of the solid phase carrier was directly passed through the solid phase carrier, and then a 50% aqueous methanol solution in an amount three times the volume of the solid phase carrier was directly passed through the solid phase carrier to remove residual sugars, condensing agents, and amines. After removing the residue, 1000 μL of methanol as a recovery solvent was passed through the solid phase carrier, and then 1000 μL of 2-propanol was passed through the solid phase carrier to recover the glycophospholipid. 1As a result of H-NMR analysis and LCMS analysis, the obtained glycophospholipid was as shown in the following formula, and the ratio of α-type to β-type was about 1 / 20. 1 The H-NMR analysis results are shown in the upper part of Figure 6. As a result of Example 5, it was found that the amount of unreacted phospholipid was less than that of Example 1, and glycophospholipid was synthesized in a high yield. 1 The bottom row of the H-NMR analysis results shows the analysis results for glycophospholipids obtained by the liquid phase method without adsorbing the phospholipids to a solid phase support. It was confirmed that the ratio of α-type was lower when the phospholipids were adsorbed to a solid phase support (top row of Figure 6) compared to the liquid phase method (bottom row of Figure 6). [ka]

Claims

1. A sugar represented by the following general formula (1), 【Chemistry 1】 (In general formula (1), R １１ is a hydroxy group or an acetamide group, R １２ , R １３ and R １４ may be the same or different, and each independently represents a hydrogen atom, a fluorine atom, a hydroxy group, a hydroxymethyl group, a carboxymethyl group, a carboalkoxymethyl group, an amino group, an acetamide group, an acylamino group, a carboxy group, an amide group, PO ４ ２－ , S.O. ４ － , alkyl groups, alkenyl groups, alkynyl groups, acyl groups, alkoxy groups, acyloxy groups, azide groups, sugar residues of monosaccharides, sugar residues of oligosaccharides, and sugar residues of polysaccharides. A phospholipid adsorbed on a solid phase carrier. A method for producing a glycophospholipid, comprising a condensation step of condensing in the presence of a condensing agent represented by the following general formula (2): 【Chemistry 2】 (In general formula (2), R ２１ , R ２２ , R ２３ and R ２４ may be the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, and an aryl group which may have a substituent; X is a halogen atom; Y － is an anion)

2. The method according to claim 1 , wherein the solvent used in the condensation step is water.

3. The method according to claim 1 or 2, wherein the phospholipid is a glycerophospholipid represented by the following general formula (3): 【Chemistry 3】 (In general formula (3), R ３１ and R ３２ may be the same or different and are each independently selected from an optionally substituted alkyl group or an optionally substituted alkenyl group; A ＋ is a hydrogen ion, an alkali metal ion, or a triethylammonium ion.

4. The method according to claim 1 or 2, wherein the phospholipid is adsorbed to the solid phase carrier by hydrophobic interaction.

5. The method according to claim 1 or 2, further comprising an adsorption step of adsorbing the phospholipid onto the solid phase carrier.

6. The method according to claim 1 or 2, further comprising a recovery step of recovering the glycophospholipid obtained in the condensation step from the solid phase carrier.

Images