Production of glycosyl fluorides

Abstract

The present invention relates to a novel efficient process for the manufacture of glycofluorides by fluorination of protected sugars using fluorinating agents such as poly (hydrofluoric acid), triethylamine trihydrofluoric acid and DMPU-HF, where the fluorination is carried out in the presence of a Lewis acid.

Description

Field of the Invention

[0001] The present invention relates to a method for preparing glycosyl fluoride.

[0002] background

[0003] Glycosyl fluorides such as 1-deoxy-1-fluoroglycosides are carbohydrate derivatives that can be described as α-haloethers. They are available in two anomeric forms, but the α-anomer is more stable.

[0004] Glycosyl fluorides are important building blocks for the synthesis of complex oligosaccharides and have remarkable stability. In fact, they are the only glycosyl halides that can be dissolved in water. Moreover, most glycosyl fluorides are crystalline compounds that can be stored for a long time without decomposition.

[0005] Due to their stability, this class of compounds has found widespread application in chemistry, biochemistry and biotechnology.

[0006] In particular, glycosyl fluorides have been used to synthesize glycosphingolipids, in which glycosyl fluoride donors are coupled to D-erythro-sphingosine in the presence of endoglycoceramidase glycoside synthase (EGCase) (MD Vaughan et al., J. Am. Chem. Soc., 2006, 128, 6300-6301). In addition, glycosyl fluorides can be internalized by engineered cells and converted into complex glycosyl fluorides, which are used in pharmaceuticals and biology (WO 2021 / 170620 A1).

[0007] Therefore, synthetic access to glycosyl fluorides can enable the production of biologically relevant compounds such as glycosphingolipids.

[0008] Fluoride is installed on the anomeric carbon of sugar and needs to be chemically synthesized. There are various methods for the anomeric fluorination of sugar, in which protected sugar is reacted with fluorinating agents, such as hydrofluoric acid, pyridinium polyhydrofluoric acid, DAST, Xtalfluor, deoxofluor, etc. (Uhrig et al., Org. Biomol. Chem., 2019, 17, 5173-5189). The disadvantages of these methods include the use of a large amount of fluorinating agents, such as hydrofluoric acid (DE 4021001 A1) or pyridinium polyhydrofluoric acid (J. Jünneman et al., Carbohydr. Res. 1993, 249, 91-94), or the use of unstable and / or expensive fluorinating agents, such as DAST, Xtalfluor or deoxofluor, resulting in the synthesis of glycosyl fluorides being difficult to scale up.

[0009] Therefore, there is a need to develop new methods with high technical feasibility and low cost that can efficiently and scalably manufacture glycosyl fluorides for the synthesis of biologically relevant compounds. Summary of the invention

[0010] The first aspect of the present invention relates to a method for producing a glycosyl fluoride by fluorination of a protected sugar, wherein each hydroxyl group of the protected sugar is derivatized with a protecting group, and wherein the anomeric hydroxyl group of the protected sugar is derivatized with an acyl protecting group,

[0011] The method comprises the following steps:

[0012] - reacting the protected sugar with a fluorinating agent, wherein the fluorinating agent is selected from pyridinium poly(hydrogen fluoride), triethylamine trihydrofluoride and DMPU-HF, thereby obtaining a protected glycosyl fluoride,

[0013] - deprotecting the protected glycosyl fluoride to generate a glycosyl fluoride,

[0014] The step of reacting the protected sugar with the fluorinating agent is performed in the presence of a Lewis acid. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present inventors have discovered that, unexpectedly, glycosyl fluorides can be prepared under mild conditions and without the need to use a large excess (eg, about 40 molar equivalents) of a fluorinating agent such as pyridinium poly(hydrofluoric acid).

[0017] In particular, the present inventors have found that when the fluorination reaction is carried out in the presence of a Lewis acid, a stoichiometric amount or a slight excess (e.g., about 1 to about 10 molar equivalents) of the fluorinating agent is sufficient. Thus, the methods described herein are particularly suitable for making glycosyl fluorides on an industrial scale from protected sugars, wherein each hydroxyl group of the protected sugar is derivatized with a protecting group, and wherein the anometric hydroxyl group of the protected sugar is derivatized with an acyl protecting group,

[0018] The method comprises the following steps:

[0019] - reacting the protected sugar with a fluorinating agent, wherein the fluorinating agent is selected from pyridinium poly(hydrofluoric acid), triethylamine trihydrofluoric acid and DMPU-HF, thereby obtaining a protected glycosyl fluoride,

[0020] - deprotecting the protected glycosyl fluoride to generate the glycosyl fluoride.

[0021] Non-limiting embodiments of various aspects of the invention are described below and illustrated by way of non-limiting examples.

[0022] The terms, definitions and embodiments described in the present specification relate to all aspects and embodiments of the present invention.

[0023] The term "a" is grammatically singular, but it can also refer to the plural, such as the target compound. For example, the skilled person will understand that in the expression "fluorinating agent", it refers not only to providing a single fluorinating agent, but also to providing a plurality of fluorinating agents of the same type.

[0024] The term "acyl" as used herein refers to a group derived from an oxoacid, preferably a carboxylic acid, by removing one or more hydroxyl groups. The acyl group of the present invention is generally a saturated or unsaturated C 2-6 The acyl group may be substituted or unsubstituted.

[0025] In the context of the present invention, the terms "about", "around" or "approximately" are used interchangeably for specific numerical values ​​(e.g., "a temperature of about 5°C", "a temperature of around 5°C" or "a temperature of approximately 5°C") or ranges (e.g., "an amount from about 1 to about 10", "an amount from around 1 to around 10" or "an amount approximately 1 to approximately 10") to indicate a deviation of 0.1% to 10% of this specific numerical value.

[0026] The term "fluorination" as used herein refers to a chemical reaction that introduces fluorine into an organic molecule. Generally, in the context of the present invention, a fluorination reaction refers to a chemical reaction that results in the replacement of a carbon-oxygen bond at the anomeric position of a protected sugar by a carbon-fluorine bond.

[0027] The term "O-acylation" as used herein refers to an esterification reaction in which a hydroxyl group of a sugar reacts with an organic acid anhydride or an acid chloride to form an acylester or an acylate.

[0028] The term "stereoselectivity" associated with the term O-acylation refers to the fact that the O-acylation reaction results in the preferential formation of one stereoisomer in the middle of a mixture of stereoisomers. The preferred stereoisomer can be the sole product of the reaction, or can be formed as a component of an unequal mixture of stereoisomers. Typically, in embodiments of the present invention, the O-acylation reaction is considered to be stereoselective when the preferred stereoisomer constitutes at least about 55% of the mixture of stereoisomers, preferably about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0029] The expression "the reaction is carried out under reflux" used herein means a reaction in which a mixture of reactants and a solvent is heated at about a temperature at which the solvent boils, and vapor generated by the reaction mixture is condensed back into the reaction vessel.

[0030] The term "aprotic solvent" as used herein refers to any solvent lacking unstable (acidic) hydrogen atoms. Aprotic solvents can be polar aprotic solvents or nonpolar aprotic solvents. Polar aprotic solvents are characterized by a net positive dipole moment and a relatively high dielectric constant. Examples of polar aprotic solvents include, but are not limited to, hydrofuran (e.g., tetrahydrofuran, etc.), hydropyran, organic esters (e.g., ethyl acetate, propyl acetate, butyl acetate, etc.), ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.), dichloromethane, dimethylformamide, acetonitrile, propionitrile, dimethyl sulfoxide, propylene carbonate, N-methyl-2-pyrrolidone, etc. Nonpolar aprotic solvents are characterized by a low dielectric constant and are not miscible with water. Examples of non-polar solvents include, but are not limited to, alkanes (e.g., hexane, heptane, cyclohexane, etc.), aromatic hydrocarbons (e.g., toluene, xylene, mesitylene, etc.), ethers (e.g., dioxane, methyl tert-butyl ether, diisopropyl ether, etc.), and the like.

[0031] As used herein, the term "1-β-glycosyl ester" refers to an O-acylated derivative of a sugar, wherein at least the anomeric hydroxyl group bears an acyl group and the anomeric configuration is β.

[0032] As used herein, the term "fluorinating agent" refers to a nucleophilic fluorinating agent capable of converting a carbon-oxygen bond into a carbon-fluorine bond.

[0033] In the context of the present invention, the term "Lewis acid" refers to a substance that is able to accept a pair of nonbonding electrons.

[0034] The term "protecting group" refers to a group introduced into a functional group in a compound and alters the chemical reactivity of the functional group. Typically, a protecting group alters the chemical reactivity of a functional group in such a way that the functional group becomes chemically inert to the reaction conditions used for subsequent chemical transformations of the compound.

[0035] Those skilled in the art will appreciate that a protecting group is introduced onto a functional group of a compound by reaction between an (unprotected) functional group and a protecting group precursor, thereby generating a "protected" derivative of the compound, such as a protected sugar.

[0036] The term "glycosyl moiety of ganglioside" as used herein is defined to include glycosyl moieties in which the anomeric carbon at the reducing end of the oligosaccharide moiety of ganglioside forms a glycosidic bond with another chemical entity, such as fluoride. The glycosidic bond may be an alpha glycosidic bond or a beta glycosidic bond, preferably an alpha glycosidic bond.

[0037] As used herein, the term "protected glycosyl moiety" refers to a protected derivative of a glycosyl moiety in which all of the hydroxyl groups of the glycosyl moiety are derivatized with protecting groups. The hydroxyl groups of the protected glycosyl moiety may all be derivatized with the same protecting group, or may each be independently derivatized with a different protecting group. Suitable protecting groups for use in the context of the present invention are those that are inert under the conditions of the fluorination reaction. Examples of suitable protecting groups include, but are not limited to, acyl, benzoyl, benzyl, alkylsiloxy, alkoxy, and the like.

[0038] As used herein, the term "sugar" refers to a monosaccharide, a disaccharide or an oligosaccharide (more than one monosaccharide unit). Sugars having more than one monosaccharide unit may present a linear or branched structure.

[0039] The monosaccharide unit can be any C 5-9 Sugars include aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxy sugars (e.g., L-rhamnose, L-fucose, etc.), deoxy amino sugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid, ketoaldonic acid (e.g., sialic acid). Monosaccharide units can form different ring structures, such as pyranose (six-membered) ring structure or furanose (five-membered) ring structure.

[0040] In some embodiments, the sugar is a sugar of formula (2):

[0041]

[0042] in,

[0043] R 4 Selected from -NHAc or -OR 8 , where R 8 is selected from a hydrogen atom or a glycosyl moiety,

[0044] R 5 , R 6 and R 7 are independently selected from a hydrogen atom or a glycosyl moiety.

[0045] In some embodiments, the sugar of formula (2) is a sugar of formula (3):

[0046]

[0047] in

[0048] R 4 , R 5 , R 6 and R 7 As defined in the sugar of formula (2).

[0049] In some embodiments, the sugar of formula (2) is a sugar of formula (4):

[0050]

[0051] in

[0052] R 4 , R 5 , R 6 and R 7 As defined in the sugar of formula (2).

[0053] In some embodiments, for a sugar of formula (2), (3) or (4), R 4 is -OH, R 5 is hydrogen, R 6 is selected from a hydrogen atom or a glycosyl moiety, and R 7 For hydrogen.

[0054] In some embodiments, the sugar of formula (2) is a sugar of formula (3), wherein R 4 is -OH, R 5 and R 6 is hydrogen, and R 7 The sugar moiety.

[0055] In some embodiments, for the sugar of formula (2) or (3), R 6 For hydrogen.

[0056] In some embodiments, for the sugar of formula (2) or (3), R 6 It is a glycosyl moiety selected from Galβ1-, Galβ1-3GlcNAcβ1-3Galβ1-, and Galβ1-4GlcNAcβ1-3Galβ1-.

[0057] In some embodiments, the sugar of formula (2) or (3) is glucose.

[0058] In some embodiments, the sugar of formula (2) or (3) is lactose.

[0059] In some embodiments, the sugar of formula (2) or (3) is lacto-N-tetraose.

[0060] In some embodiments, the carbohydrate of formula (2) or (3) is lacto-N-neotetraose.

[0061] In some embodiments, the sugar of formula (4) is galactose.

[0062] In some embodiments, the sugar of formula (2) is melibiose.

[0063] Sugars such as galactose, glucose, lactose, lacto-N-tetraose, lacto-N-neotetraose, and melibiose are commercially available and can be purchased from established manufacturers.

[0064] The term "protected sugar" as used herein refers to a protected derivative of a monosaccharide, disaccharide or oligosaccharide (more than one monosaccharide unit), wherein all hydroxyl groups of the sugar are derivatized by a protecting group. Protected sugars having more than one monosaccharide unit may be in a linear or branched structure.

[0065] The monosaccharide unit can be any C 5-9 Sugars include aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxy sugars (e.g., L-rhamnose, L-fucose, etc.), deoxy amino sugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid, ketonic acid (e.g., sialic acid). Monosaccharide units can form different ring structures, such as pyranose (six-membered) ring structure or furanose (five-membered) ring structure.

[0066] The hydroxyl groups of the sugar may all be derivatized with the same protecting group, or may be independently derivatized with different protecting groups. Protecting groups suitable for use in the context of the present invention are those that are inert under the conditions of the fluorination reaction. Examples of suitable protecting groups include, but are not limited to, acyl, benzoyl, benzyl, alkylsilyloxy, alkoxy, and the like.

[0067] Protected sugars suitable for use in the context of the present invention are sugars in which each hydroxyl group is derivatized with a protecting group and in which the anomeric hydroxyl group at the reducing end of the protected sugar is derivatized with an acyl protecting group.

[0068] In some embodiments, all hydroxyl groups of the sugar are derivatized with acyl protecting groups, wherein the acyl protecting groups are selected from C 2-6 Acyl or benzoyl protecting group.

[0069] In some embodiments, all hydroxyl groups of the sugar are derivatized with acyl protecting groups, wherein the acyl protecting groups are preferably C 2-6 Thus, in some embodiments, the protected sugar is a fully-O-acylated sugar.

[0070] In some embodiments, all hydroxyl groups of the sugar are derivatized with acetyl protecting groups. Thus, in some embodiments, the protected sugar is a fully-O-acetylated sugar.

[0071] The protected sugar may be an α sugar or a β sugar, preferably a β sugar.

[0072] In some embodiments, the protected sugar is glucose pentaacetate, galactose pentaacetate or lactose octaacetate, wherein the protected sugar may be an α sugar or a β sugar, preferably a β sugar.

[0073] In some embodiments, the protected sugar is β-D-lactose octaacetate.

[0074] In some embodiments, the protected sugar is β-D-glucose pentaacetate.

[0075] In some embodiments, the protected sugar is β-D-galactose pentaacetate.

[0076] In some embodiments, the protected sugar is β-D-melibiose octaacetate.

[0077] In some embodiments, the protected sugar is a protected derivative of the oligosaccharide portion of a ganglioside, preferably selected from GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, GM4, wherein all hydroxyl groups of the oligosaccharide portion of the ganglioside are derivatized with acyl protecting groups and wherein for those oligosaccharide portions of gangliosides carrying a sialic acid unit, such as an N-acetylneuraminic acid unit, the carboxyl function of the sialic acid unit is in the form of an alkyl ester.

[0078] In some embodiments, all of the hydroxyl groups of the oligosaccharide portion of the ganglioside are derivatized with acetyl protecting groups.

[0079] In some embodiments, the protected sugar is a human milk oligosaccharide, which is preferably selected from LNT, LNnT, LNH, LNnH, 2'FL, 3FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-I, 3'SL, 6'SL, FSL, LSTa, LSTb, LSTc and DSLNT, wherein all hydroxyl groups of the human milk oligosaccharide are derivatized with acyl protecting groups and wherein for those oligosaccharides carrying a sialic acid unit, such as an N-acetylneuraminic acid unit, the carboxyl function of the sialic acid unit is in the form of an alkyl ester.

[0080] In some embodiments, all hydroxyl groups of human milk oligosaccharides are derivatized with acetyl protecting groups.

[0081] In some embodiments, the protected sugar is β-D-lacto-N-neotetraosyl tetradecaacetate.

[0082] In some embodiments, the protected sugar is β-D-lacto-N-tetraosyl tetradecanoate.

[0083] As used herein, the term "glycosyl fluoride" refers to a 1-deoxy-1-fluoro glycoside in which the fluoride is covalently attached to the anomeric carbon at the reducing end of the glycosyl moiety.

[0084] The fluoride can be bound to the anomeric carbon of the glycosyl moiety via an α or β glycosidic bond. The α glycosidic bond is preferred.

[0085] The glycosyl moiety of the glycosyl fluoride may be derived from a monosaccharide or an oligosaccharide (more than one monosaccharide unit). The glycosyl moiety having more than one monosaccharide unit may present a linear or branched structure.

[0086] The monosaccharide unit can be any C 5-9 Sugars include aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxy sugars (e.g., L-rhamnose, L-fucose, etc.), deoxy amino sugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid, ketonic acid (e.g., sialic acid). Monosaccharide units can form different ring structures, such as pyranose (six-membered) ring structure or furanose (five-membered) ring structure.

[0087] The glycosyl moiety of the present invention can be illustrated as follows: Galβ1-4Glc1-, wherein the linker (-) represents the point of attachment of the glycosyl moiety, and wherein the glycosyl moiety can be attached via an α or β glycosidic bond, preferably an α glycosidic bond.

[0088] In some embodiments, the glycosyl moiety of the glycosyl fluoride is glucose, galactose and lactose, wherein the glycosyl moiety can be linked by an α or β glycosidic bond, preferably an α glycosidic bond. In the context of the present invention, the glycosyl fluoride whose glycosyl moiety is glucose, galactose, lactose and melibiose glycosyl moiety can be represented by the following formulas: Glc1-F, Gal1-F, Galβ1-4Glc1-F and Galα1-6Glc1-F, respectively.

[0089] In some preferred embodiments, the glycosyl fluoride is α-D-lactopyranosyl fluoride.

[0090] In some embodiments, the glycosyl fluoride is α-D-glucopyranosyl fluoride.

[0091] In some embodiments, the glycosyl fluoride is α-D-galactopyranosyl fluoride.

[0092] In some embodiments, the glycosyl fluoride is α-D-melibiosyl fluoride.

[0093] In some embodiments, the glycosyl moiety of the glycosyl fluoride is a glycosyl moiety of a ganglioside selected from GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, GM4. In the context of the present invention, the glycosyl fluoride whose glycosyl moiety is the glycosyl moiety of GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b and GM4 can be represented by the following formulae, respectively:

[0094] Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-F, Neu5Acα2-3Galβ1-3GalNAcβ1-4Galβ1-4Glc1-F, Neu5Acα2-3Ga1β1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-F, Ga1β1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc1-F, Neu5Acα2-8Neu5Acα2-3G3lβ1-4Glc1-F, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc1-F, Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-F, Neu5Acα2-8Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc1-F, Neu5Acα2-3Gal1-F,

[0095] The glycosyl moieties may be linked via α or β glycosidic bonds, preferably α glycosidic bonds.

[0096] In some embodiments, the glycosyl moiety of the glycosyl fluoride is a glycosyl moiety of a human milk oligosaccharide, wherein the human milk oligosaccharide is preferably selected from LNT, LNnT, LNH, LNnH, 2'FL, 3FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-I, 3'SL, 6'SL, FSL, LSTa, LSTb, LSTc and DSLNT.

[0097] In the context of the present invention, the glycosyl fluoride of the glycosyl moiety wherein the glycosyl moiety is LNT, LNnT, LNH, LNnH, 2'FL, 3FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-I, 3'SL, 6'SL, FSL, LSTa, LSTb, LSTc and DSLNT can be represented by the following formulae, respectively:

[0098]

[0099]

[0100] The glycosyl moieties may be linked via an α- or β-glycosidic bond, preferably an α-glycosidic bond.

[0101] The term "protected glycosyl fluoride" as used herein refers to a 1-deoxy-1-fluoro glycoside in which the fluoride is covalently attached to the anomeric carbon at the reducing end of the protected glycosyl moiety. The fluoride can be bound to the anomeric carbon of the protected glycosyl moiety via an α or β glycosidic bond. An α glycosidic bond is preferred.

[0102] The protected glycosyl moiety of the protected glycosyl fluoride may be derived from a monosaccharide or an oligosaccharide (more than one monosaccharide unit). A glycosyl moiety having more than one monosaccharide unit may present a linear or branched structure.

[0103] The monosaccharide unit can be any C 5-9 Sugars include aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxy sugars (e.g., L-rhamnose, L-fucose, etc.), deoxy amino sugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid, ketonic acid (e.g., sialic acid). Monosaccharide units can form different ring structures, such as pyranose (six-membered) ring structure or furanose (five-membered) ring structure.

[0104] In some embodiments, all hydroxyl groups of the glycosyl moiety of the protected glycosyl fluoride are derivatized with acyl protecting groups, wherein the acyl protecting groups are selected from C 2-6 Acyl or benzoyl protecting group.

[0105] In some embodiments, all hydroxyl groups of the glycosyl moiety of the protected glycosyl fluoride are derivatized with acyl protecting groups, wherein the acyl protecting groups are C 2-6 Acyl.

[0106] Thus, in some embodiments, the protected glycosyl fluoride is a per-O-acylated glycosyl fluoride.

[0107] In some embodiments, all hydroxyl groups of the glycosyl moiety of the protected glycosyl fluoride are derivatized with acetyl protecting groups. Therefore, in some preferred embodiments, the protected glycosyl fluoride is a fully-O-acetylated glycosyl fluoride.

[0108] The protected glycosyl fluoride may be an alpha or beta glycoside, preferably an alpha glycoside.

[0109] In some embodiments, the protected glycosyl fluoride is selected from glucosyl fluoride tetraacetate, galactosyl fluoride tetraacetate and lactosyl fluoride heptaacetate, wherein the protected glycosyl fluoride may be an α or β glycoside, preferably an α glycoside.

[0110] In some preferred embodiments, the protected glycosyl fluoride is α-D-lactopyranosyl fluoride heptaacetate.

[0111] In some embodiments, the protected glycosyl fluoride is α-D-glucopyranosyl fluoride tetraacetate.

[0112] In some embodiments, the protected glycosyl fluoride is α-D-galactopyranosyl fluoride tetraacetate.

[0113] In some embodiments, the glycosyl moiety of the protected glycosyl fluoride is a protected derivative of the glycosyl moiety of a ganglioside, preferably selected from GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, GM4, wherein all hydroxyl groups of the glycosyl moiety of the ganglioside are derivatized with acyl protecting groups, and wherein for those glycosyl moieties of gangliosides carrying a sialic acid unit, such as an N-acetylneuraminic acid unit, the carboxylic acid function of the sialic acid unit is in the form of an alkyl ester.

[0114] In some embodiments, all of the hydroxyl groups of the glycosyl moiety of the ganglioside are derivatized with acetyl protecting groups.

[0115] In some embodiments, the glycosyl moiety of the protected glycosyl fluoride is a glycosyl moiety of a human milk oligosaccharide, preferably selected from the group consisting of LNT, LNnT, LNH, LNnH, 2'FL, 3FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-1, 3'SL, 6'SL, FSL, LSTa, LSTb, LSTc and DSLNT, wherein all hydroxyl groups of the human milk oligosaccharide are derivatized with acyl protecting groups and wherein for those oligosaccharides carrying a sialic acid unit, such as an N-acetylneuraminic acid unit, the carboxyl function of the sialic acid unit is in the form of an alkyl ester.

[0116] In some embodiments, all hydroxyl groups of human milk oligosaccharides are derivatized with acetyl protecting groups.

[0117] The present invention describes a method for producing glycosyl fluoride, wherein the method comprises the following steps:

[0118] - reacting the protected sugar with a fluorinating agent, wherein the fluorinating agent is selected from pyridinium poly(hydrofluoric acid), triethylamine trihydrofluoric acid and DMPU-HF, thereby obtaining a protected glycosyl fluoride,

[0119] - deprotecting the protected glycosyl fluoride to generate a glycosyl fluoride,

[0120] The step of reacting the protected sugar with the fluorinating agent is performed in the presence of a Lewis acid.

[0121] Lewis acids suitable for use in the context of the present invention include, but are not limited to, iodine, boron trifluoride, boron trichloride, aluminum chloride, aluminum bromide, zinc chloride, zinc bromide, zinc fluoride, zinc trifluoromethanesulfonate, ferric chloride, ferric bromide, or a complex thereof. Further examples of Lewis acids include, but are not limited to, BF3-HF, BF3-NaCl, BCl3-NaCl, BF3-AgF, BF3-nitrobenzene, and the like.

[0122] In some embodiments, the Lewis acid is boron trifluoride or a complex thereof.

[0123] In some embodiments, the Lewis acid is boron trifluoride etherate.

[0124] Typically, about 1 to about 3 molar equivalents of Lewis acid are used based on the amount of protected sugar. Thus, in some embodiments, the protected sugar and fluorinating agent are reacted in the presence of about 1.0, 1.1, 1.2, 13, 1.4, 1.6, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 equivalents of Lewis acid based on the amount of protected sugar.

[0125] Suitable fluorinating agents include, but are not limited to, pyridinium poly(hydrofluoric acid), tetrabutylammonium hydrogen difluoride, triethylamine trihydrofluoric acid, DMPU-HF, etc. In some embodiments, the fluorinating agent is pyridinium poly(hydrofluoric acid).

[0126] Typically, about 1 to about 10 molar equivalents of fluorinating agent are used based on the amount of protected sugar. In some embodiments, about 3 to about 8 molar equivalents of fluorinating agent are used based on the amount of protected sugar. Thus, in some embodiments, about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 molar equivalents of fluorinating agent are used based on the amount of protected sugar.

[0127] The step of reacting the protected sugar with the fluorinating agent is usually carried out in a solvent such as dichloromethane, toluene, trifluorotoluene, tetrahydrofuran, 2-methyltetrahydrofuran, acetonitrile or a mixture thereof.

[0128] In some embodiments, the step of reacting the protected sugar with a fluorinating agent is performed in dichloromethane.

[0129] In some embodiments, the step of reacting the protected sugar with the fluorinating agent is carried out in toluene.

[0130] In some embodiments, the step of reacting the protected sugar with a fluorinating agent is performed in a mixture of dichloromethane and toluene.

[0131] In some embodiments, the step of reacting the protected sugar with a fluorinating agent is performed in acetonitrile.

[0132] The protected sugar, fluorinating agent and Lewis acid are generally reacted at a temperature of about -5°C to about 5°C for about 2 to about 4 hours. Preferably, the reaction is carried out for about 3 to about 4 hours.

[0133] The reaction of the protected sugar with a fluorinating agent in the presence of a Lewis acid results in the carbon-oxygen bond at the anomeric position of the protected sugar being replaced by a carbon-fluorine bond. The reaction between the protected sugar and the fluorinating agent may also be referred to as "fluorination".

[0134] The protected sugar is fluorinated with a fluorinating agent in the presence of a Lewis acid, resulting in the formation of a protected glycosyl fluoride.

[0135] In some embodiments, the protected glycosyl fluoride is a per-O-acylated glycosyl fluoride. Thus, in some embodiments, the step of deprotecting the protected glycosyl fluoride is a deacylation step.

[0136] In some embodiments, the protected glycosyl fluoride is a per-O-acetylated glycosyl fluoride. Thus, in some embodiments, the step of deprotecting the protected glycosyl fluoride is a deacetylation step.

[0137] The step of deprotecting the protected glycosyl fluoride is typically carried out in the presence of a base. Examples of suitable bases include, but are not limited to, alkali metal alkoxides, such as those derived from methanol, ethanol, propanol, isopropanol, butanol and isobutanol, wherein the alkali metal is selected from sodium, potassium or lithium.

[0138] In some embodiments, the step of deprotecting the protected glycosyl fluoride is performed in the presence of sodium methoxide or sodium ethoxide.

[0139] In some preferred embodiments, the step of deprotecting the protected glycosyl fluoride is performed in the presence of sodium methoxide.

[0140] The base can be used in a catalytic amount, an equimolar amount or an excess. Preferably, the base is used in an amount of about 0.1 to about 2 molar equivalents.

[0141] In some embodiments, the amount of base is about 0.2 to about 0.5 molar equivalents. Thus, in some embodiments, the amount of base is about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 031, 032, 0.33, 034, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5 molar equivalents.

[0142] The step of deprotecting the protected glycosyl fluoride is usually carried out in a solvent. Examples of suitable solvents include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and the like.

[0143] In some embodiments, the step of deprotecting the protected glycosyl fluoride is performed in methanol.

[0144] In some embodiments, the step of deprotecting the protected glycosyl fluoride is performed in ethanol.

[0145] The reaction components of the present invention may be combined in any order, and it will be appreciated that the order in which the reactants are combined may be adjusted as desired.

[0146] The glycosyl fluoride prepared by the above method can be used without purification. However, in some embodiments, the glycosyl fluoride can be purified.

[0147] Purification of the glycosyl fluorides can be carried out by standard methods known to those skilled in the art, such as extraction with organic solvents, chromatography, crystallization or precipitation.

[0148] A preferred purification method involves precipitation of the glycosyl fluoride. Precipitation of the glycosyl fluoride can be achieved by removing part of the reaction solvent, e.g. by evaporation (i.e., concentrating the reaction mixture), or by adding another solvent to the reaction mixture, or by changing the temperature or pressure, or by adding other solutes, or a combination of these.

[0149] The glycosyl fluoride of the present invention can be prepared in different polymorphic forms. The polymorphic forms referred to herein may include crystalline forms and amorphous forms as well as solvates and hydrate forms, which may be further characterized as follows:

[0150] -Crystalline forms have different arrangements and / or conformations of molecules in the crystal lattice.

[0151] -Amorphous forms consist of disordered arrangements of molecules without a distinguishable crystal lattice.

[0152] - Solvates are crystalline forms containing stoichiometric or non-stoichiometric amounts of solvent. If the added solvent is water, the solvate is often called a hydrate.

[0153] In some embodiments, the glycosyl fluoride is obtained in the form of a solvate. In some embodiments, the glycosyl fluoride is obtained in the form of a hydrate, such as a monohydrate, a dihydrate, or a trihydrate. In some embodiments, the glycosyl fluoride is obtained in a crystalline form. In some embodiments, the glycosyl fluoride is obtained in an amorphous form.

[0154] In some embodiments, the protected sugar is a fully-O-acylated sugar, and the method further comprises the step of generating the fully-O-acylated sugar. Thus, in some embodiments, the method comprises the following steps:

[0155] - reacting a sugar with a compound of formula (1) in the presence of an aliphatic amine in an aprotic solvent:

[0156]

[0157] in

[0158] R 1 is phenyl, preferably unsubstituted phenyl, or C 1-6 alkyl,

[0159] R 2 is -Cl, or -OC(=O)R 3 , where R 3 is phenyl, preferably unsubstituted phenyl, or C 1-6 alkyl,

[0160] Thus, fully-O-acylated sugars are obtained;

[0161] - reacting the per-O-acylated sugar with a fluorinating agent, wherein the fluorinating agent is selected from pyridinium poly(hydrofluoric acid), triethylamine trihydrofluoric acid and DMPU-HF, thereby obtaining a per-O-acylated sugar fluoride,

[0162] - deprotecting the per-O-acylated glycosyl fluoride to generate a glycosyl fluoride,

[0163] The step of reacting the per-O-acylated sugar with the fluorinating agent is performed in the presence of a Lewis acid.

[0164] In some embodiments, the compound of formula (1) is acetic anhydride. Therefore, in some embodiments, the step of reacting the sugar with the compound of formula (1) is an O-acetylation step.

[0165] In some embodiments, the compound of formula (1) is benzoyl chloride. Therefore, in some embodiments, the step of reacting the sugar with the compound of formula (1) is an O-benzoylation step.

[0166] The reaction between the sugar and the compound of formula (1) is usually carried out in the presence of an aliphatic amine.

[0167] In some embodiments, the aliphatic amine is selected from triethylamine or N,N-diisopropylethylamine. In some embodiments, the aliphatic amine is triethylamine.

[0168] The reaction between the sugar and the compound of formula (1) is usually carried out in an aprotic solvent.

[0169] In some embodiments, the solvent is selected from acetone, dichloromethane, toluene, and acetonitrile.

[0170] In some embodiments, the solvent is acetone.

[0171] In some embodiments, the solvent is dichloromethane.

[0172] In some embodiments, the solvent is acetonitrile.

[0173] The temperature range for carrying out the above process may be from room temperature to a temperature corresponding to the boiling point of the selected solvent, preferably from about 25°C to about 120°C.

[0174] In some embodiments, the O-acylation step is performed using acetone as a solvent, and the reaction is performed at a temperature of about 25° C. to about 60° C. Preferably, the temperature is about 50° C. to about 60° C. In some embodiments, the O-acylation step is performed at a temperature of about 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., or 60° C.

[0175] In some embodiments, the O-acylation step is performed using dichloromethane as a solvent, and the reaction is performed at a temperature of about 25° C. to about 45° C. Preferably, the temperature is about 35° C. to about 45° C. In some embodiments, the O-acylation is performed at a temperature of about 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C.

[0176] In some embodiments, the O-acylation step is carried out using toluene as a solvent, and the reaction is carried out at a temperature of about 100° C. to about 120° C. Preferably, the temperature is about 100° C. to about 110° C. In some embodiments, the O-acylation step is carried out at a temperature of about 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C., or 110° C.

[0177] The O-acylation step is carried out for a sufficient period of time to obtain the desired O-acylated product in the desired high yield. Typically, the reaction is carried out for about 1 to about 24 hours, preferably about 4 to about 24 hours. In some embodiments, the reaction is carried out for about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours.

[0178] The reaction components of the present invention may be combined in any order, and it will be appreciated that the order in which the reactants are combined may be adjusted as desired.

[0179] The fully-O-acylated sugars produced by the above method can be used without purification. However, in some embodiments, the fully-O-acylated sugars can be purified.

[0180] The step of purifying the per-O-acylated sugars can be performed by standard methods known to those skilled in the art, such as extraction with an organic solvent, chromatography, crystallization or precipitation.

[0181] The preferred purification method involves precipitation of the fully-O-acylated sugar. Precipitation of the fully-O-acylated sugar can be accomplished directly from the reaction mixture, by cooling the reaction mixture, or by adding an antisolvent to the reaction mixture. Alternatively, precipitation can be performed after extraction with an organic solvent, and then the solution containing the fully-O-acylated glycosyl ester is cooled or an antisolvent is added thereto, or a combination of these.

[0182] The fully-O-acylated sugars of the present invention can be prepared in different polymorphic forms. The polymorphic forms referred to herein may include crystalline and amorphous forms as well as solvate and hydrate forms, which may be further characterized as follows:

[0183] -Crystalline forms have different arrangements and / or conformations of molecules in the crystal lattice.

[0184] -Amorphous forms consist of disordered arrangements of molecules without a distinguishable crystal lattice.

[0185] - Solvates are crystalline forms containing stoichiometric or non-stoichiometric amounts of solvent. If the added solvent is water, the solvate is often called a hydrate.

[0186] In some embodiments, the fully-O-acylated sugar is obtained in the form of a solvate. In some embodiments, the fully-O-acylated sugar is obtained in the form of a hydrate, for example, in the form of a monohydrate, a dihydrate or a trihydrate. In some embodiments, the fully-O-acylated sugar is obtained in a crystalline form. In some embodiments, the fully-O-acylated sugar is obtained in an amorphous form. Example

[0187] The following working examples describe non-limiting embodiments of the present invention and are intended to illustrate the invention only.

[0188] General methods and materials:

[0189] Recorded using a JEOL (500.16 MHz) spectrometer or a Bruker Avance II (400 MHz) spectrometer 1 H NMR. 1 H chemical shifts are expressed in ppm (δ) relative to tetramethylsilane (δ = 0.00), D2O (δ = 4.79) or CDCl3 (δ = 7.26, δ = 77.00) as internal standards. MS analysis was performed using a Shimadzu LCMS-2020 system.

[0190] Thin layer chromatography (TLC) was performed using silica gel TLC plates (Merck, silica gel, F254) with detection by UV absorption (254 nm) where applicable and with ammonium molybdate (25 g / L) and cerium ammonium sulfate (10 g / L) in 10% H2SO4 (140°C).

[0191] Example 1. General method for sugar O-acylation

[0192] Sugar (1 equivalent) and triethylamine (4-16 equivalents) are suspended in acetone, dichloromethane or toluene. The suspension is heated to reflux, and acetic anhydride (6-18 equivalents) or benzoyl chloride (6-10 equivalents) is added to the reaction mixture. The mixture is heated to reflux until TLC shows that the raw material is completely consumed. The reaction mixture is cooled to room temperature until the product precipitates. If necessary, water is added to the cooled reaction mixture to promote precipitation. The resulting solid is filtered, washed with water, and dried in a vacuum. The O-acylated product can be crystallized from MeOH. The O-acylated product generally obtains a yield of about 65-85%.

[0193] Example 2. Production of β-D-lactose octaacetate

[0194] β-D-lactose octaacetate was generated from lactose according to the general procedure described in Example 1 using acetone as solvent.

[0195] Anomeric ratio 1:10α:β

[0196] 1H NMR (400MHz, CDCl3): δ (ppm) = 1.94 (s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.04(s,3H),2.07(s,3H),2.09(s,3H),2.13(s,3H),3.69-3.78(m,2H),3.78-3.90( m,2H),4.01-4.16(m,3H),4.39-4.49(m,2H),4.92(dd,J=10.1,3.4Hz,1H),5.08(t ,J=9.2Hz,1H),5.22(t,J=9.2Hz,1H),5.32(d,J=2.6Hz,1H),5.65(d,J=8.2Hz,1H).

[0197] Example 3. Production of β-D-lactose octabenzoate

[0198] β-D-lactose octabenzoate was generated from lactose according to the general procedure described in Example 1 using acetone as solvent.

[0199] Anomeric ratio 1:4α:β

[0200] 1 H NMR (400MHz, CDCl3): δ (ppm) = 3.71 (dd, J = 11.3, 7.1Hz, 1H), 3.78 (dd, J = 11.3, 6.4Hz, 1H), 3.89 (t, J = 7.0Hz,1H),4.03-4.11(m,1H),4.39(t,J=9.4Hz,1H),4.53(dd,J=12.4,3.8Hz,1H),4.59(dd,J=12.4 ,1.8Hz,1H),4.89(d,J=7.9Hz,1H),5.38(dd,J=10.4,3.3Hz,1H),5.71-5.716(m,2H),5.78(dd,J=9. 6,8.1Hz,1H),5.94(t,J=9.3Hz,1H),6.14(d,J=8.1Hz,1H),7.96-8.08(m,16H),7.29-7.65(m,24H).

[0201] Example 4. Formation of β-D-galactopyranosyl pentaacetate

[0202] β-D-galactose octabenzoate was generated from galactose according to the general procedure described in Example 1 using acetone as solvent.

[0203] Anomeric ratio 1:6.5α:β

[0204] 1H NMR (400MHz, MeOD): δ (ppm) = 1.99 (s, 3H), 2.04 (s, 6H), 2-12 (s, 3H), 2.16 (s, 3H), 4.05 (ddd, J = 7, 1, 6.1, 1.2Hz, 1H), 4.07-4 .20(m,2H),5.07(dd,J=10.4,3.4Hz,1H),5.33(dd,J=10.4,8.3Hz,1H),5-42(dd,J=3.5,1.2Hz,1H),5.69(d,J=8.3Hz,1H).

[0205] Example 5. Formation of β-D-lacto-N-neotetraosyl tetradecanoate

[0206] β-D-lacto-N-neotetraosyl tetradecanoate was generated from lacto-N-neotetraose according to the general procedure described in Example 1 using toluene as solvent.

[0207] Anomeric ratio 1:9α:β

[0208] 1 H NMR (400MHz, CDCl3): δ (ppm) = 1.88 (s, 3H), 1.95 (s, 3H), 2.01 (s, 6H), 2.02 (s, 3H), 2 .04(s,3H),2.05(s,3H),2.05(s,3H),2.08(s,3H),2.09-2.10(m,6H),2.11(s,3H), 2.13(s,3H),2.13(s,3H),3.47-3.52(m,2H),3.71(dd,J=10.0,3.6Hz,1H),3.73-3. 82(m,4H),3.86(ddd,J=7.5,6.2,1.3Hz,1H),3.95(dd,J=12.0,3.3Hz,1H),3.99-4.1 6(m,5H),4.32(d,J=8.0Hz,1H),4.40(dd,J=12.1,1.9Hz,1H),4.53(d,J=7.9Hz,1H) ,4.66(d,J=7.9Hz,1H),4.76(dd,J=12.0,2.6Hz,1H),4.94-4.99(m,2H),5.02(dd,J= 9.5,8.3Hz,1H),5.09(dd,J=10.5,7.9Hz,1H),5.14-5.22(m,2H),5.29(dd,J=3.6,1 .1Hz, 1H), 5.33 (dd, J = 3.4, 1.2Hz, 1H), 5.38 (d, J = 8.6Hz, 1H), 5.65 (d, J = 8.3Hz, 1H).

[0209] LCMS: 1254 [M+H] + ,1276[M+Na] + .

[0210] Example 6. Production of β-D-lacto-N-tetraosyl tetradecanoate

[0211] β-D-lacto-N-tetraosyl tetradecanoate was prepared from lacto-N-tetraose according to the general procedure described in Example 1 using toluene as solvent.

[0212] Anomeric ratio 1:8α:β

[0213] 1 H NMR (400MHz, CDCl3): δ (ppm) = 1.95 (s, 3H), 1.98 (s, 3H), 2.01 (s, 6H), 2.03 ( s,3H),2.04(s,3H),2.06(s,3H),2.08(s,3H),2.08(s,6H),2.09(s,3H),2. 10(s,3H),2.11(s,3H),2.13(s,3H),3.60(ddd,J=10.1,4.0,2.7Hz,1H),3. 72-3.80(m,4H),3.83(td,J=6.7,1.2Hz,1H),3.98(dd,J=11.6,3.6Hz,1H),4 .01-4.09(m,4H),4.15(dd,J=12.0,4.3Hz,1H),4.33(d,J=7.8Hz,1H),4.36 (dd,J=12.1,2.6Hz,1H),4.39-4.45(m,2H),4.57(dd,J=10.5,9.2Hz,1H),4. 87-4.97(m,3H),4.99-5.05(m,3H),5.11(d,J=8.1Hz,1H),5.22(dd,J=9.5, 8.8Hz, 1H), 5.30-5.34 (m, 2H), 5.66 (d, J = 8.3Hz, 1H), 5.88 (d, J = 6.9Hz, 1H).

[0214] LCMS: 1254 [M+H] + ,1276[M+Na] + .

[0215] Example 7. Preparation of β-D-melibiose octaacetate

[0216] β-D-Melibiose octaacetate was prepared from melibiose according to the general procedure described in Example 1 using acetone solvent.

[0217] 1H NMR (400MHz, CDCl3): δ (ppm) = 1.98 (s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 2.0 9(s,3H),2.12(s,3H),2.13(s,3H),3.62(dd,J=11.6,2.5Hz,1H),3.71(dd,J=11.6,4.4Hz,1H),3.77 (ddd,J=9.8,4.4,2.5Hz,1H),4.01-4.10(m,2H),4.17(td,J=6.4,1.3Hz,1H),5.01-5.18(m,4H),5. 24(t,J=9.4Hz,1H), 5.33(dd,J=10.8,3.4Hz,1H), 5.44(dd,J=3.4,1.3Hz,H), 5.66(d,J=8.3Hz,1H).

[0218] LCMS: 696 [M+NH4] + ,701[M+Na] + .

[0219] Example 8. General method for preparing glycosyl fluorides

[0220] The per-O-acetylated sugar (1 equivalent) was dissolved in dichloromethane (5 volumes). The solution was cooled to a temperature of about -10 to about -5°C.

[0221] Pyridinium poly(hydrofluoric acid) (70%, 8 eq.) and BF3.Et2O (2.2 eq.) were added and the reaction mixture was warmed to a temperature of about 0° C. to about 10° C. Upon completion, the reaction mixture was poured into ice-cold water, and the organic phase was separated, washed with water, saturated with NaHCO3, and filtered through Na2SO4.

[0222] The solvent is evaporated and methanol is added. The reaction mixture is then cooled to room temperature and NaOMe (25 wt %, 0.05 eq. in methanol) is added. The mixture is stirred until a white solid is formed. The resulting suspension is stirred at a temperature of about 0°C to about 25°C for 1 hour. The resulting solid is solid, washed with methanol, ethanol or acetonitrile, and dried to obtain the glycosyl fluoride. The yield is typically in the range of 60-85%.

[0223] Example 9. Production of α-D-lactopyranosyl fluoride

[0224] α-D-lactopyranosyl fluoride was prepared from β-D-lactose octaacetate according to the method described in Example 8.

[0225] 1H-NMR (500MHz, D2O): 5.59 (dd, J = 53.5Hz, 2.8Hz, 1H), 4.36 (d, J = 7.7Hz, 1H), 3.88-3.74 (m, 5H), 3.73-3.52 (m, 6H), 3.45 (dd, J = 10.0, 7.7Hz, 1H).

[0226] LCMS: 367[M+Na] + ,408[M+NH4HCO2+H] + .

[0227] Example 10. Production of α-D-galactopyranosyl fluoride

[0228] α-D-galactopyranosyl fluoride was prepared from β-D-galactopyranosyl pentaacetate according to the method described in Example 8.

[0229] 1 H NMR (400MHz, CD3OD): δ = 5.58 (dd, J = 54.4Hz, 1H), 3.99-3.94 (m, 2H), 3.87-3.71 (m, 4H).

[0230] Example 11. Production of α-D-pyranose fluoride

[0231] α-D-glucopyranosyl fluoride was prepared from β-D-glucopyranosyl pentaacetate according to the method described in Example 8.

[0232] 1 H NMR (400MHz, CD3OD): δ = 5.55 (dd, J = 53.9, 2.8Hz, 1H), 3.83 (m, 1H), 3.49-3.38 (m, 2H).

[0233] Example 12. Production of α-D-Melibiosyl Fluoride

[0234] α-D-Melibiose fluoride was prepared from β-D-melibiose octaacetate according to the method described in Example 8.

[0235] 1 H-NMR (400MHz, MeOD): δ = 3.39-3.57 (m, 3H), 3.61-3.83 (m, 8H), 3.85-4.02 (m, 5H), 5.56 (dd, J = 53.7, 2.7Hz, 1H).

[0236] LCMS: 408 [M+CH3CN+Na] + .

[0237] Example 13. Production of α-D-lacto-N-neotetraosyl fluoride

[0238] α-D-lacto-N-neotetraosyl fluoride was prepared from β-D-lacto-N-neotetraosyl tetradecanoate according to the method described in Example 8.

[0239] 1 H NMR (500MHz, D2O): δ = 2.05 (s, 3H), 3.51-3.92 (m, 21H), 3.92-4.02 (m, 4H), 4.17 (d, J = 3.3H z, 1H), 4.48 (dd, J = 13.2, 7.8 Hz, 2H), 4.73 ( d, J = 8.3 Hz, 1H), 5.71 ( dd, J = 53.5, 2.7 Hz, 1H).

[0240] LCMS: 732[M+Na] + .

[0241] Example 14. Production of α-D-lacto-N-tetraosyl fluoride

[0242] α-D-lacto-N-tetraosyl fluoride was prepared from β-D-lacto-N-tetraosyl tetradecanoate according to the method described in Example 8.

[0243] 1 H-NMR (400MHz, D2O): δ = 2.03 (s, 3H), 3.45-3.68 (m, 6H), 3.68-4.00 (m, 17H), 4.16 (d, J= 3.3Hz,1H),4.46(dd,J=7.8,5.7Hz,2H),4.74(d,J=8.3Hz,1H),5.70(dd,J=53.6,2.7Hz 1H).

[0244] LCMS: 710 [M+H] + ,732[M+Na] + .

[0245] The above embodiments can be combined. The following dependent claims set forth specific embodiments of the invention.

Claims

1. A method for producing a glycosyl fluoride by fluorination of a protected sugar, wherein each hydroxyl group of the protected sugar is derivatized with a protecting group, and wherein the anomeric hydroxyl group of the protected sugar is derivatized with an acyl protecting group, The method comprises the following steps: - reacting the protected sugar with a fluorinating agent, wherein the fluorinating agent is selected from pyridinium poly(hydrofluoric acid), triethylamine trihydrofluoric acid and DMPU-HF, thereby obtaining a protected glycosyl fluoride, - deprotecting the protected glycosyl fluoride to generate a glycosyl fluoride, The step of reacting the protected sugar with the fluorinating agent is performed in the presence of a Lewis acid.

2. The method of claim 1, wherein the fluorinating agent is pyridinium poly(hydrofluoric acid).

3. The method according to claim 2, wherein about 1 to about 10 molar equivalents, preferably about 3 to about 8 molar equivalents of pyridinium poly(hydrofluoric acid) are used based on the amount of protected sugar.

4. The method according to any one of claims 1 to 3, wherein about 1 to about 3 molar equivalents of Lewis acid are used based on the amount of protected sugar.

5. The method according to any one of claims 1 to 4, wherein the Lewis acid is boron trifluoride or a complex thereof.

6. The process according to any one of claims 1 to 5, wherein the Lewis acid is boron trifluoride etherate.

7. The method of any one of claims 1 to 6, wherein the step of reacting the protected sugar with a fluorinating agent is performed at a temperature of about -5°C to about 5°C.

8. The method according to any one of claims 1 to 7, wherein the step of reacting the protected sugar with a fluorinating agent further comprises using a solvent.

9. The method according to claim 8, wherein the solvent is selected from dichloromethane, toluene, trifluorotoluene, tetrahydrofuran, 2-methyltetrahydrofuran, acetonitrile or a mixture thereof, preferably, the solvent is dichloromethane.

10. The method according to any one of claims 1 to 9, wherein the step of deprotecting the protected glycosyl fluoride is performed in the presence of a base.

11. The process according to claim 10, wherein the base is an alkoxide, and wherein the alkoxide is selected from sodium methoxide or sodium ethoxide, preferably sodium methoxide.

12. The method according to any one of claims 1 to 11, wherein the step of deprotecting the protected glycosyl fluoride further comprises using a solvent.

13. The method according to claim 12, wherein the solvent is an alcohol, and wherein the alcohol is selected from methanol or ethanol, preferably methanol.

14. The method of any one of claims 1 to 13, wherein the protected sugar is a beta sugar, and wherein the glycosyl fluoride is an alpha glycosyl fluoride.

15. The method according to any one of claims 1 to 14, wherein the glycosyl fluoride is selected from the group consisting of glucopyranosyl fluoride, galactopyranosyl fluoride and lactopyranosyl fluoride.

16. The method according to any one of claims 1 to 15, wherein the glycosyl fluoride is α-D-lactopyranosyl fluoride.

17. The method according to any one of claims 1 to 16, wherein the protected sugar is a fully-O-acylated sugar.

18. The method of claim 17, wherein the fully-O-acylated sugar has C 2-6 Acyl, or benzoyl.

19. The method according to claim 17 or 18, wherein the fully-O-acylated sugar is a fully-O-acetylated sugar.

20. The method according to any one of claims 17 to 19, wherein the fully-O-acylated sugar is selected from glucose pentaacetate, galactose pentaacetate and lactose octaacetate.

21. The method according to any one of claims 17 to 20, wherein the method further comprises the step of generating fully-O-acylated sugars.

22. The method according to claim 21, wherein the fully-O-acylated sugar is produced by the step of reacting a sugar with a compound of formula (1): in R 1 is phenyl, preferably unsubstituted phenyl, or C 1-6 alkyl, R 2 is -Cl, or -OC(=O)R 3 , where R 3 is phenyl, preferably unsubstituted phenyl, or C 1-6 alkyl, The reaction is carried out in an aprotic solvent in the presence of an aliphatic amine.

23. The method of claim 22, wherein the sugar is a sugar of formula (2): in, R 4 Selected from -NHAc, or -OR 5 , where R 5 is selected from hydrogen or a glycosyl moiety, R 5 , R 6 and R 7 are independently selected from hydrogen or a glycosyl moiety.

24. The method of claim 22 or 23, wherein the sugar is selected from glucose, galactose or lactose.

25. A process according to any one of claims 22 to 24, wherein the compound of formula (1) is acetic anhydride.

26. A process according to any one of claims 22 to 24, wherein the compound of formula (1) is benzoyl chloride.

27. The process according to any one of claims 22 to 26, wherein the aprotic solvent is a solvent selected from acetone, dichloromethane, toluene and acetonitrile.

28. The process according to any one of claims 22 to 27, wherein the reaction is carried out under reflux.

29. The method of any one of claims 21 to 28, wherein the step of generating a fully-O-acylated sugar is stereoselective, and wherein said step results in the formation of a 1-β-glycosyl ester.

Images