Production of ceramides

Abstract

The present invention relates to a novel and efficient process for the production of sphingolipids via N-acylation of hemolytic sphingolipids, such as o-red-phytosphingosine, salts thereof or analogs thereof. The process comprises the use of an ester as an acylating agent in the presence of a base and in a hydrocarbon solvent, and is particularly useful for the production of ceramides.

Description

Technical Field

[0001] This invention relates to a novel and efficient method for producing ceramides or analogues via N-acylation of lysphingolipids such as D-erythrosphoprotein, their analogues or their salts. Background Technology

[0002] Sphingolipids are an important class of polar lipids, mainly found on the surface of eukaryotic cells. Structurally, sphingolipids are characterized by a sphingosine base skeleton and can be classified into different categories, such as ceramides and glycosphingolipids.

[0003] Ceramides are N-acylated sphingosine bases that lack an additional head group at the 1-position of the sphingosine base backbone, and in which the N-acyl group of the ceramide is typically derived from fatty acids. Glycosphingolipids (GSLs) are glycoconjugates derived from ceramides in which the polysaccharide moiety is linked to the 1-hydroxyl group of the ceramide via a glycosidic bond.

[0004] Sphingolipids participate in a variety of biological processes and play important structural and functional roles, such as cell-cell recognition, communication, and cell adhesion. In particular, GSLs (such as gangliosides) are found in the brain and play a role in neurological diseases, while ceramides are the main components of the lipid layer of the stratum corneum and play a major role in the water-retention properties of the epidermis and the barrier function of the skin.

[0005] Therefore, sphingolipids have great potential as therapeutic agents, cosmetics, and tools for studying important biological processes. However, they are not readily available for basic and clinical research. In fact, sphingolipids (such as ceramides and GSL) are characterized by their high structural complexity and challenging preparation.

[0006] Sphingolipids (such as ceramides and GSL) can be obtained via N-acylation of lysosphingolipids. Typically, lysosphingolipids are defined as sphingolipid degradation products that lack an amide-linked fatty acid at the 2-position of the sphingosine base backbone. Thus, for each parent sphingolipid, there exists a corresponding lysosphingolipid with the same head group at the 1-position but lacking an amide-linked fatty acid at the 2-position (Hannun et al., Science 1989, 243, 500-507).

[0007] N-acylation of lysosphingolipids can be carried out via chemical or enzymatic methods.

[0008] Current chemical N-acylation methods are typically based on the use of amide-coupled reagents such as N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbodiimide (DCC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Skolova et al., Biochim Biophys Acta Biomembr. [Chinese Journal of Biochemistry and Biophysics - Biomembranes] 2017, 1859, 824-834, WO 0172701 A1) or sulfonyl chloride (US 5631356 A). Alternatively, esters of acids have been used for the N-acylation of phytosphingosine (EP 2757090 A1).

[0009] Current chemical methods for the N-acylation of lysphingolipids have several drawbacks, including the use of expensive and / or toxic coupling agents (which require anhydrous solvents), the need to carry out the reaction under anaerobic conditions, or long reaction times.

[0010] Current enzymatic methods are based on the use of lipases (WO 1994026919 A1, US 2011077302A1). However, these lipases are typically not specific to the amino groups of lysphingolipids and may also act on the hydroxyl groups, resulting in a mixture of N- and O-acylated products, and thus requiring lengthy and expensive purification of the target compound. Summary of the Invention

[0011] A method for producing sphingolipids or their analogues having formula (1):

[0012]

[0013] in

[0014] W is either H or the glycosyl moiety.

[0015] R 1 It is H, aryl, or C. 1-50 Hydrocarbon group, preferably C 1-15 Hydrocarbon group, more preferably C 10-15 The hydrocarbon group may be saturated or contain one or more double and / or triple bonds, and / or may contain one or more functional groups, preferably selected from the group consisting of hydroxyl, alkoxy, acyloxy, amide, thiol, thioether, or phosphorus-containing functional groups.

[0016] R 2 and R 3 Selected from H, substituted or unsubstituted C 1-6 Hydrocarbon group, or substituted or unsubstituted C 1-6 Acyl group,

[0017] R4 Selected from substituted or unsubstituted aryl groups, heteroalkyl groups, and substituted or unsubstituted C groups that can be saturated or unsaturated. 1-31 Hydrocarbon group, preferably substituted or unsubstituted C, which can be saturated or unsaturated. 9-31 hydrocarbon group;

[0018] This method is carried out using lysozyme or its salt having formula (2):

[0019]

[0020] in

[0021] W, R 1 R 2 and R 3 As defined for the sphingolipid having formula (1),

[0022] The method involves reacting the lysozyme salt having formula (2) or a salt thereof with an ester having formula (3) in the presence of a base:

[0023]

[0024] in

[0025] R 4 As defined for the sphingolipid having formula (1), and

[0026] R 5 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl or isobutyl, more preferably selected from methyl or ethyl;

[0027] and

[0028] The reaction is carried out in a hydrocarbon solvent. Detailed Implementation

[0029] The inventors of this invention have for the first time established an efficient and economical method for the chemical N-acylation of lysphingolipids such as D-erythrosphospirin, their analogues, or salts thereof, wherein the N-acylation is characterized by a short reaction time (approximately 0.5-4 hours), and wherein the lysphingolipids used as starting materials are preferably obtained via synthetic and / or biotechnological methods. Surprisingly, the inventors of this invention have discovered that lysphingolipids such as D-erythrosphospirin, their analogues, or salts thereof can be N-acylated using esters having formula (3) in the presence of a base:

[0030]

[0031] in

[0032] R 3As defined for the sphingolipid having formula (1), and

[0033] R 4 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl or isobutyl, more preferably selected from methyl or ethyl;

[0034] and

[0035] The reaction is carried out in a hydrocarbon solvent.

[0036] Non-limiting embodiments of different aspects of the present invention are described below and illustrated by way of non-limiting examples.

[0037] The terms, definitions, and embodiments described throughout this specification relate to all aspects and embodiments of the invention.

[0038] The term “a / a type (a)” is grammatically singular, but it can also refer to, for example, the plural form of the intended compound. For example, those skilled in the art will understand that the expression “a hemolysin having formula (2)” means not only providing a single hemolysin having formula (2), but also providing multiple hemolysin of the same type.

[0039] In formulas representing parts or groups (e.g., hydrocarbon groups having formula (5) or (6)), the symbol It refers to the attachment point with another group or atom.

[0040] As used herein, the various functional groups or substituents represented will be understood as having an attachment point at the functional group or atom indicated by a dash (-). For example, in the case of -OH, it will be understood that the attachment point is an oxygen atom. If groups are listed without a dash, the attachment point is indicated by the ordinary and common meaning of the listed groups.

[0041] The letters H, Cl, Br, I, and F refer to hydrogen, chlorine, bromine, iodine, and fluorine atoms, respectively.

[0042] Those skilled in the art will understand that when referring to positions C-1, C-2, C-3, C-4, C-5, etc., this text always refers to the corresponding carbon atom of a sphingolipid having formula (1), or the corresponding carbon atom of a hemolysinic sphingolipid having formula (2). Positions C-1, C-2, C-3, C-4, and C-5 can also be referred to as position 1, position 2, position 3, position 4, and position 5, respectively.

[0043] As used herein, the term "alkyl" refers to an acyclic straight-chain or branched alkyl group having 1 to 50 carbon atoms, which may be saturated or contain one or more double and / or triple bonds (thus forming, for example, alkenyl or ynyl groups), and / or may be substituted or unsubstituted, as further described herein. Examples of "hydrocarbon group" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neopentyl, n-hexyl, vinyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, methylpentenyl, dimethylbutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, pentyynyl, and hexynyl, each of which may be substituted or unsubstituted. Typically, the term hydrocarbon group refers to a straight-chain acyclic hydrocarbon group having 1-31 carbons, which may be substituted or unsubstituted.

[0044] As used herein, the term "aryl" refers to an aromatic cyclic hydrocarbon group having 5-14 ring carbon atoms, which may be monocyclic or polycyclic, may contain fused rings, preferably 1 to 3 fused rings or non-fused rings, and may contain one or more heteroatoms, and / or may be substituted or unsubstituted, as further described herein. Examples of "aryl" include, but are not limited to, phenyl, naphthyl, anthraceneyl, phenantryl, pyrroleyl, imidazolyl, thiopheneyl, furanyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazineyl, and benzofuranyl, each of which may be substituted or unsubstituted. Typically, the term "aryl" refers to a substituted or unsubstituted phenyl group.

[0045] As used herein, the term "acyl" refers to a group derived by removing one or more hydroxyl groups from an oxyacid, preferably a carboxylic acid. Acyl groups according to the invention are typically saturated or unsaturated C4 groups. 2-32 Acyl groups, which can be substituted or unsubstituted.

[0046] As used herein, the term "alkylene" refers to a divalent saturated or unsaturated aliphatic group derived from a substituted or unsubstituted alkane by removing two hydrogen atoms from different carbon atoms, preferably from the terminal carbon atom. The alkylene group according to the invention is typically C1. 6-28 The alkylene group may be saturated or contain one or more double and / or triple bonds, and / or may be substituted or unsubstituted, as further described herein.

[0047] As used herein, the term "substituted" means that the group under discussion has been replaced by a group that typically alters the general chemical characteristics of the group under discussion. Substituents can be used to change characteristics of a molecule, such as molecular stability, molecular solubility, and the ability of the molecule to form crystals. Those skilled in the art will recognize other suitable substituents with similar size and charge characteristics that can be used as substitutes in a given situation.

[0048] Regarding the terms "alkyl group," "aryl group," "acyl group," and "alkylene group," the term "substitution" means that the group under discussion is substituted once or several times, preferably 1 to 3 times, by one or more groups selected from the following: hydroxyl group (which may exist in a tautomeric ketone form when combined with an unsaturated carbon atom), oxo group, C... 1-6 -alkoxy group (i.e., C) 1-6 -alkyl-oxygen), C 2-6 -Alkenyl group, carboxyl group, oxo group, C 1-6 -alkoxycarbonyl, C 1-6 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C 1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl)aminocarbonyl, amino-C 1-6 -alkyl-aminocarbonyl, mono- and di(C) 1-6 -alkyl)amino-C 1-6 -alkyl-aminocarbonyl, C 1-6 -alkylcarbonylamino, cyano, guanidinyl, urea, C 1-6 -alkyl-sulfonyl-amino, aryl-sulfonyl-amino, heteroaryl-sulfonyl-amino, C 1-6 -alkyl acyloxy, C 1-6 -alkyl-sulfonyl, C 1-6 -alkyl-sulfinyl group, C 1-6 -alkylsulfonyloxy, nitro, C 1-6 -Alkylthio group, halogen, wherein any alkyl, alkoxy, etc. representing a substituent can be replaced by a hydroxyl group, C 1-6 -alkoxy group, C 2-6 -Alkenyl group, carboxyl group, C 1-6 -alkyl carbonyl amino, halogen, C 1-6 -alkylthio group, C 1-6 -alkyl-sulfonyl-amino or guanidine-substituted.

[0049] The term "hemolysphingolipid" as used herein refers to a sphingolipid lacking an amide-linked fatty acid at the C-2 position of the sphingosine skeleton. Suitable hemolysphingolipids for the context of this invention are sphingosine, glycosylated sphingosine, and analogues thereof, and can be represented by hemolysphingolipids having formula (2):

[0050]

[0051] in

[0052] W is either H or the glycosyl moiety.

[0053] R 1 It is H, aryl, or C. 1-50 Hydrocarbon group, preferably C 1-15 Hydrocarbon group, more preferably C 10-15 The hydrocarbon group may be saturated or contain one or more double and / or triple bonds, and / or may contain one or more functional groups, preferably selected from the group consisting of hydroxyl, alkoxy, acyloxy, amide, thiol, thioether, or phosphorus-containing functional groups.

[0054] R 2 and R 3 Selected from H, substituted or unsubstituted C 1-6 Hydrocarbon group, or substituted or unsubstituted C 1-6 Acyl group.

[0055] The term "glycosyl moiety," as used herein, is defined as comprising a moiety derived from a monosaccharide or oligosaccharide (more than one monosaccharide unit), wherein the anodic carbon of the monosaccharide or the anodic carbon at the reducing end of the oligosaccharide is bonded to another chemical entity by a glycosidic bond, and unless otherwise specified, this bond may be an α- or β-glycosidic bond. A glycosyl moiety having more than one monosaccharide unit may represent a linear or branched structure.

[0056] Monosaccharide units can be any sugar with 5-9 carbon atoms, including 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.), deoxyglucoses (e.g., L-rhamnose, L-fucose, etc.), deoxy-aminoglycans (e.g., N-acetylglucosamine, N-acetymannosamine, N-acetylgalactosamine, etc.), uronic acids, and ketonic acids (e.g., sialic acid). Monosaccharide units can form different cyclic structures, such as pyranose (six-membered) cyclic structures or furanose (five-membered) cyclic structures.

[0057] The glycosyl portion according to the invention can be described in the following form: Galβ1-4Glc1-, wherein the dash (-) represents the attachment point of the glycosyl portion, and wherein the glycosyl portion can be linked via an α or β glycosidic bond, preferably a β glycosidic bond.

[0058] In the context of this invention, the terms “about,” “around,” or “approximate” are used interchangeably to describe a specific value (e.g., “temperature of about 25°C,” “temperature of about 25°C,” or “temperature of approximately 25°C”) or a range (e.g., “time of about 0.5 hours to about 4 hours,” “time of about 0.5 hours to about 4 hours,” or “time of approximately 0.5 hours to approximately 4 hours”) to indicate a deviation of 0.1% to 10% from that specific value.

[0059] Suitable esters used in the context of this invention are typically derived from carboxylic acids and are represented by formula (3).

[0060] In some embodiments, R of an ester having formula (3) 4 It is a substituted or unsubstituted C having formula (5) or (6). 9-31 Hydrocarbon group:

[0061]

[0062] in

[0063] Q is selected from H, -OH, Cl, Br, I, or F.

[0064] L is a straight chain C 6-28 Hydroxyl group (which may be saturated or contain one or more double and / or triple bonds), R 7 Is it H or -OR 8 , where R 8 Is it hydrogen or straight-chain C? 2-30 Acyl group (which may be saturated or contain one or more double bonds).

[0065] In some preferred embodiments, the R of the ester having formula (3) 4 It is a substituted or unsubstituted C with formula (5) 9-31 Hydrocarbon group.

[0066] Therefore, in some preferred embodiments, the ester having formula (3) is an ester having formula (7):

[0067]

[0068] in

[0069] R 5 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl or isobutyl, more preferably selected from methyl or ethyl;

[0070] Q is selected from H, -OH, Cl, Br, I or F, preferably from H or -OH.

[0071] L is a straight chain C 6-28 Hydroxyl group (which may be saturated or contain one or more double and / or triple bonds), R 7 Is it H or -OR 8 , where R 8 Is it hydrogen or straight-chain C? 2-30 Acyl group (which may be saturated or contain one or more double bonds).

[0072] In some embodiments, for hydrocarbon groups having formula (5) and for esters having formula (7), Q is H and L is a straight-chain saturated unsubstituted C. 14 -C 28 Hydroxyl group, and R 7 It's H.

[0073] In some embodiments, the ester having formula (3) or the ester having formula (7) is derived from C 18 -C 26 Esters of nonhydroxy fatty acids [N].

[0074] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from stearic acid [N(18:0)].

[0075] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from arachidic acid [N(20:0)].

[0076] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from betaine [N(22:0)].

[0077] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from lignin [N(24:0)].

[0078] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from hexacosanoic acid [N(26:0)].

[0079] In some embodiments, for hydrocarbon groups having formula (5) and for esters having formula (7), Q is -OH and L is a straight-chain saturated unsubstituted C. 14 -C 28 Hydroxyl group, and R 7 It's H.

[0080] In some embodiments, the ester having formula (3) or the ester having formula (7) is derived from C 18 -C 26 Esters of α-hydroxy fatty acids [A].

[0081] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from α-hydroxystearic acid [A(18:0)].

[0082] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from α-hydroxyarachidic acid [A(20:0)].

[0083] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from α-hydroxysorbic acid [A(22:0)].

[0084] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from α-hydroxyl lignin [A(24:0)].

[0085] In some embodiments, the ester having formula (3) or the ester having formula (7) is an ester derived from α-hydroxyhexadecanoic acid [A(26:0)].

[0086] In some embodiments, for hydrocarbon groups having formula (5) and for esters having formula (7), Q is H and L is a straight-chain saturated unsubstituted C. 14 -C 28 Hydroxyl group, and R 7 Yes - OR 8 , where R 8 Is it saturated or unsaturated C? 18 Acyl group.

[0087] The letters in square brackets refer to the abbreviated nomenclature developed by Motta et al. (Biochim Biophys Acta., 1993, 1182: 147-151) and expanded by Rabionet et al. (Biochim Biophys Acta., 2014, 1841: 422-434) and Masukawa et al. (Journal of Lipid Research, 2008, 49, 1466-1476). Non-hydroxy fatty acids (N), α-hydroxy fatty acids (A), and ω-linoleyloxy fatty acids (EO) can be represented by the letters N, A, and EO, respectively, and the number of carbon atoms and the degree of unsaturation can be expressed in parentheses following the letters N, A, E, and O.

[0088] In some embodiments, R having an ester of formula (3) or (7) 5 It is a methyl group.

[0089] In some embodiments, the ester having formula (3) or (7) is a methyl ester selected from the group consisting of methyl stearate, methyl arachidate, methyl benzyl ester, methyl creosote, and methyl hexadecanoate.

[0090] In some embodiments, the ester having formula (3) or (7) is a methyl ester selected from the group consisting of: methyl α-hydroxystearate, methyl α-hydroxyarachidate, methyl α-hydroxybeanate, methyl α-hydroxycarbamate, and methyl α-hydroxyhexadecanoate.

[0091] In some embodiments, R of an ester having formula (3) 4 It is a substituted or unsubstituted C with formula (6) 9-31 Hydrocarbon group.

[0092] Therefore, in some embodiments, the ester having formula (3) is an ester having formula (8):

[0093]

[0094]

[0095] in

[0096] R 5 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl or isobutyl, more preferably selected from methyl or ethyl;

[0097] L is a straight chain C 6-28 Hydroxyl group (which may be saturated or contain one or more double and / or triple bonds), R 7 Is it H or -OR 8 , where R 8 Is it hydrogen or straight-chain C? 2-30 Acyl group (which may be saturated or contain one or more double bonds).

[0098] In the context of this invention, esters may be purchased from well-known manufacturers (e.g., Merck) or may be obtained by methods known to those skilled in the art, such as by... The method described by et al., J. Chromatogr. [Journal of Chromatography] 1990, 152, 89-97, is used to synthesize it.

[0099] The present invention describes a method for producing a sphingolipid having formula (1), wherein an ester having formula (3), (7) or (8) is reacted with a lysosomal sphingolipid having formula (2) in the presence of a base.

[0100] In the context of this invention, hemolysinic sphingolipids are preferably obtained via synthetic and / or biotechnological methods (such as those described in WO2021170624 A2; or US 005618706).

[0101] In some preferred embodiments, the hemolysin with formula (2) is D-erythro-phytosphingosine.

[0102] In some embodiments, the hemolysinic lipid having formula (2) can be produced or used in salt form, preferably in the form of a pharmaceutically acceptable salt.

[0103] In some embodiments, the salt of the lysosomal sphingolipid having formula (2) can be formed from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, polyphosphoric acid, acetic acid, camphor sulfonic acid, p-toluene sulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, and perchloric acid.

[0104] In some preferred embodiments, the salt of the lysosphingolipid having formula (2) is a hydrochloride.

[0105] Typically, lysothelioids having formula (2) react with esters having formulas (3), (7) or (8) in the presence of a base such as an alkoxide.

[0106] In the context of this invention, alkoxides can be represented by alkoxides having formula (4):

[0107] R 6 -O - X +

[0108] (4),

[0109] in

[0110] R 6 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl or isobutyl, more preferably selected from methyl or ethyl;

[0111] X + It is selected from Na + K + Li + or NH4 + The cation, preferably Na + .

[0112] In some preferred embodiments, the lysothelioidin having formula (2) reacts with an ester having formula (7) or (8) in the presence of sodium methoxide.

[0113] The alkali can be used in catalytic amounts, equimolar amounts, or in excess.

[0114] In some embodiments, the sphingolipid having formula (2) is in the form of a free base, and the base is used in a catalytic amount of about 0.1 to about 0.5 molar equivalents based on the amount of the sphingolipid, preferably in a catalytic amount of about 0.1 to about 0.2 molar equivalents based on the amount of the sphingolipid.

[0115] In some embodiments, the sphingomyelin having formula (2) is in the form of a salt, and the base is used in an amount of about 0.5 to about 1.7 molar equivalents based on the amount of the sphingomyelin, preferably in an amount of about 1.2 to 1.4 molar equivalents based on the amount of the sphingomyelin.

[0116] Typically, lysins, esters, and bases are dissolved in hydrocarbon solvents such as C 5-10 The reaction takes place in a hydrocarbon solvent.

[0117] In some preferred embodiments, the reaction is carried out in heptane.

[0118] Typically, the lysphingolipid, ester, and base react at a temperature of about 85°C to about 100°C. Preferably, the reaction is carried out at a temperature of about 90°C to about 95°C. Thus, in some preferred embodiments, the reaction is carried out at a temperature of about 85°C, 86°C, 87°C, 88°C, 89°C, or 90°C.

[0119] The components of the reaction of the present invention can be combined in any order, and it will be understood that the order of combining reactants can be adjusted as needed.

[0120] For example, a base can be added to a solution of lysphingolipids and esters. As another example, a solvent can be added to a flask containing lysphingolipids and esters, followed by the addition of a base.

[0121] Lysospholipids, esters, and bases, as well as any other reagents used in the reaction, may be added to the reaction as solids or dissolved in a solvent, in any amount and manner effective for the desired results of the reaction.

[0122] In some embodiments, the alkali is added as a solid.

[0123] In some embodiments, the alkali is added as a solution.

[0124] The esters, bases, and lysphingolipids according to the invention typically react for a period of about 0.5 hours to about 4 hours.

[0125] Known methods for the N-acylation of lysphingolipids such as D-erythrosphospirin (e.g., as described in EP 2757090A1) are characterized by long reaction times (typically between about 24 and 36 hours). By performing N-acylation in heptane, the reaction time can be significantly reduced (approximately 90% conversion of the lysphingolipid was observed by LC-MS analysis after 0.5 hours), making the N-acylation reaction more economical and easier to scale up.

[0126] According to the invention, the reaction between the ester, the base and the lysosphingolipid causes the amino group at the C-2 carbon atom of the lysosphingolipid having formula (2) to be selectively N-acylated, thereby producing a sphingolipid having formula (1).

[0127] Sphingolipids of formula (1) carry acyl groups derived from carboxylic acids, preferably fatty acids.

[0128] An acyl group carried by a sphingolipid having formula (1) can be represented by an acyl group having formula (9):

[0129]

[0130] in

[0131] R 4 Selected from substituted or unsubstituted aryl, heteroalkyl, substituted or unsubstituted C 1-31 Hydrocarbon group (which may be saturated or unsaturated), preferably substituted or unsubstituted C. 9-31 Hydrocarbon group (which can be saturated or unsaturated).

[0132] Those skilled in the art will understand that esters having formula (3), (7) or (8) also carry an acyl group having formula (9).

[0133] In some embodiments, R having an acyl group of formula (9) 4 It is a substituted or unsubstituted C having formula (5) or (6). 9-31 Hydrocarbon group:

[0134]

[0135] in

[0136] Q is selected from H, -OH, Cl, Br, I, or F, preferably from H or -OH.

[0137] L is a straight chain C 6-28 Hydroxyl group (which may be saturated or contain one or more double and / or triple bonds), R 7 Is it H or -OR 8 , where R 8 Is it hydrogen or straight-chain C? 2-30 Acyl group (which may be saturated or contain one or more double bonds).

[0138] In some preferred embodiments, R having an acyl group of formula (9) 4 It is a substituted or unsubstituted C with formula (5) 9-31 Hydrocarbon group.

[0139] Therefore, in some preferred embodiments, the acyl group having formula (9) is an acyl group having formula (10):

[0140]

[0141] in

[0142] Q is selected from H, -OH, Cl, Br, I, or F, preferably from H or -OH.

[0143] L is a straight chain C 6-28 Hydroxyl group (which may be saturated or contain one or more double and / or triple bonds), R 7 Is it H or -OR 8 , where R 8 Is it hydrogen or straight-chain C? 2-30 Acyl group (which may be saturated or contain one or more double bonds).

[0144] In some embodiments, R having an acyl group of formula (9) 4 It is a substituted or unsubstituted C with formula (6) 9-31 Hydrocarbon group.

[0145] Therefore, in some embodiments, the acyl group having formula (9) is an acyl group having formula (11):

[0146]

[0147] in

[0148] L is a straight chain C 6-28 Hydroxyl group (which may be saturated or contain one or more double and / or triple bonds), R 7 Is it H or -OR 8 , where R 8 Is it hydrogen or straight-chain C? 2-30 Acyl group (which may be saturated or contain one or more double bonds).

[0149] In some embodiments, the acyl group having formula (9) or (10) is derived from C 18 -C 26 Acyl group of nonhydroxy fatty acid [N].

[0150] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from stearic acid [N(18:0)].

[0151] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from arachidic acid [N(20:0)].

[0152] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from benzyl acid [N(22:0)].

[0153] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from lignin [N(24:0)].

[0154] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from hexacosanoic acid [N(26:0)].

[0155] In some embodiments, for an acyl group having formula (10), Q is -OH and L is a straight-chain saturated unsubstituted C 14 -C 28 Hydroxyl group, and R 7 It's H.

[0156] In some embodiments, the acyl group having formula (9) or (10) is derived from C 18 -C 26 Acyl group of α-hydroxy fatty acid [A].

[0157] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from α-hydroxystearic acid [A(18:0)].

[0158] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from α-hydroxyarachidic acid [A(20:0)].

[0159] In some embodiments, the acyl group having formula (13) or (14) is an acyl group derived from α-hydroxysorbic acid [A(22:0)].

[0160] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from α-hydroxyl lignin [A(24:0)].

[0161] In some embodiments, the acyl group having formula (9) or (10) is an acyl group derived from α-hydroxyhexadecanoic acid [A(26:0)].

[0162] In some embodiments, for an acyl group having formula (10), Q is H and L is a straight-chain saturated unsubstituted C. 14 -C 28 Hydroxyl group, and R 7 Yes - OR 8 , where R 8 Is it saturated or unsaturated C? 18 Acyl group.

[0163] In some embodiments, the acyl group carried by the sphingolipid of the present invention is an acyl group having formula (10), wherein the acyl group having formula (10) is selected from the group consisting of acyl groups having formulas (12)-(20):

[0164]

[0165]

[0166] In some preferred embodiments, the W in the sphingolipid according to the invention is hydrogen. Therefore, in some preferred embodiments, the sphingolipid according to the invention is a ceramide.

[0167] In the context of this invention, ceramide means naturally occurring ceramides, their analogues, or derivatives thereof. Preferred ceramides are those naturally occurring in humans. Naturally occurring human ceramides (CERs) include, but are not limited to, CER[NS], CER[AS], CER[EOS], CER[NH], CER[AH] or CER[EOH], CER[NP], CER[AP] or CER[EOP], CER[NDS], CER[ADS] or CER[EODS], where the letters in square brackets refer to the abbreviated nomenclature developed by Motta et al. (Biochim Biophys Acta., 1993, 1182:147-151) and expanded by Rabionet et al. (Biochim Biophys Acta., 2014, 1841:422-434) and Masukawa et al. (Journal of Lipid Research, 2008, 49, 1466-1476). The letters N, A, and EO represent non-hydroxy fatty acids (N), α-hydroxy fatty acids (A), and ω-linolenic acid fatty acids (EO), respectively. The number of carbon atoms and the degree of unsaturation of the fatty acid are expressed in parentheses following N, A, E, and O. The letters S, H, P, and DS represent D-erythrosine (S), 6-hydroxy-... D -Erythrosphospirin (H) D -ribose-phytosphoprotein (P) D -Erythro-dihydrosphingosine (DS), where the number of carbon atoms in sphingosine can be expressed in parentheses following the letters S, H, P, and DS. Ceramides CER[NDS], CER[ADS], or CER[EODS] can also be referred to as CER[NG], CER[AG], or CER[EOG], respectively, where the letter G represents the INCI name of D-erythro-dihydrosphingosine.

[0168] In some embodiments, the sphingolipid having formula (1) or (11) is a ceramide selected from CER[NS], CER[AS], and CER[EOS].

[0169] In some preferred embodiments, the sphingolipid having formula (1) is ceramide CER[N(18:0)P(18)].

[0170] In some embodiments, the sphingolipid having formula (1) is ceramide CER[N(20:0)P(18)].

[0171] In some embodiments, the sphingolipid having formula (1) is ceramide CER[N(22:0)P(18)].

[0172] In some embodiments, the sphingolipid having formula (1) is ceramide CER[N(24:0)P(18)].

[0173] In some embodiments, the sphingolipid having formula (1) is ceramide CER[N(26:0)P(18)].

[0174] In some preferred embodiments, the sphingolipid having formula (1) is ceramide CER[A(18:0)P(18)].

[0175] In some embodiments, the sphingolipid having formula (1) is ceramide CER[A(20:0)P(18)].

[0176] In some embodiments, the sphingolipid having formula (1) is ceramide CER[A(22:0)P(18)].

[0177] In some embodiments, the sphingolipid having formula (1) is ceramide CER[A(24:0)P(18)].

[0178] In some embodiments, the sphingolipid having formula (1) is ceramide CER[A(26:0)P(18)].

[0179] In some embodiments, the W of the sphingolipid of the present invention and the W of the hemolysphingolipid of the present invention are glycosyl moieties, wherein the glycosyl moieties are selected from Glc1-, Gal1-, Galβ1-4Glc1-.

[0180] In some embodiments, the W of the sphingolipid of the present invention and the W of the hemolysphingolipid of the present invention are glycosyl moieties, wherein the glycosyl moieties are oligosaccharide moieties selected from gangliosides of GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, and GM4.

[0181] In the context of this invention, the oligosaccharide portions of GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, and GM4 can be represented by the following formulas:

[0182]

[0183] As used herein, the term "oligosaccharide moiety of ganglioside" is defined as comprising a glycosyl moiety derived from a ganglioside, wherein the anomeric carbon at the reduced end of the oligosaccharide moiety of the ganglioside is bonded to another chemical entity by a glycosidic bond, which may be an α- or β-glycosidic bond, preferably a β-glycosidic bond. In the context of this invention, the terms oligosaccharide moiety and glycosyl moiety may be used interchangeably.

[0184] In some embodiments, the W of the sphingolipid of the present invention and the W of the hemolysphingolipid of the present invention are glycosyl moieties, wherein the glycosyl moieties are glycosyl moieties of human milk oligosaccharides, and wherein the human milk oligosaccharides are 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.

[0185] In the context of this invention, the glycosyl moiety of LNT, LNnT, LNH, LNnH, 2'FL, 3FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-I, 3'SL, 6'SL, FSL, LSTTa, LSTb, LSTc, and DSLNT can be represented by the following formulas:

[0186] Galβ1-3GlcNAcβ1-3Galβ1-4Glc1-, Galβ1-4GlcNAcβ1-3Galβ1-4Glc1-, Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc1-, Galβ1-4GlcNAcβ1-3Galβ1-4GlucNAcβ1-3Galβ1-4Glc1-, Fucα1-2Galβ1-4Glc1-, Galβ1-4(Fucα1-3)Glc1-, Fucα1-2Galβ1-4(Fucα1-3)Glc1-, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc1-, Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc1-, Galβ1-3(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc1-, Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc1-, Fucα1-2Galβ1-3(Fucα1-4)GlcNacβ1-3Galβ1-4Glc1-, Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-6Galβ1-4Glc1-, Neu5Acα2-3Galβ1-4(Fucα1,3)Glc1-, Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc1-, Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc1-, Neu5Acα2-6Galβ1-3GlcNAcβ1-3Galβ1-4Glc1-, Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc1-, .

[0187] In some embodiments, the W of the sphingolipid of the present invention and the W of the hemolysphingolipid of the present invention are glycosyl moieties, and wherein the glycosyl moieties are oligosaccharide moieties selected from the gala series [SP0509], neogala series, globo series [SP0502], isoglobo series [SP0506], lacto series [SP0504], neolacto series [SP0505], arthro series [SP0508], muco series, schisto series, spirometo series, or protected analogues thereof.

[0188] The alphanumeric strings within square brackets indicate links on the website https: / / www.lipidmaps.org / , where detailed chemical formulas and structural information for the corresponding compounds can be found.

[0189] The sphingolipid produced by the method of the present invention can be separated from the reaction mixture. This separation can be carried out by standard methods known to those skilled in the art. A preferred method for separating the sphingolipid according to the invention is via precipitation from the reaction mixture. Precipitation can be achieved, for example, by partially removing the reaction solvent 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 methods.

[0190] In some embodiments, the sphingolipid according to the invention is separated from the reaction mixture by precipitation, wherein precipitation is achieved by adding another solvent to the reaction mixture. In some embodiments, the sphingolipid according to the invention is separated by adding C to the reaction mixture. 1-6 Alcohol precipitates. In some embodiments, the sphingolipid according to the invention is precipitated by adding C to the reaction mixture. 1-6 The sphingolipid precipitates as a mixture of alcohols. In some preferred embodiments, the sphingolipid according to the invention precipitates by adding methanol to the reaction mixture.

[0191] The sphingolipids according to the present invention can be separated in different polymorphic forms. The polymorphic forms mentioned herein may include crystalline and amorphous forms, as well as solvated and hydrated forms, which can be further characterized as follows:

[0192] i. Molecules that have different arrangements and / or conformations in a crystal lattice.

[0193] ii. Amorphous forms consist of molecules arranged in a disordered manner without a recognizable lattice.

[0194] iii. A solvate is a crystalline form containing stoichiometric or non-stoichiometric amounts of solvent. If the solvent incorporated is water, the solvate is usually called a hydrate.

[0195] Example

[0196] The following working examples describe non-limiting embodiments of the present invention, and these examples are given only to illustrate the invention.

[0197] General methods and materials:

[0198] LC-MS analysis was performed using a Merck Ascentis Express RP-amide column (15 cm x 4.6 mm, 2.7 μm) via a Shimadzu ECO 2020 LC system coupled to a Shimadzu MS-2020 system. The eluent consisted of 98% solvent A (2 mM ammonium formate, 0.2% v / v formic acid, 75% v / v MeOH, 25% v / v ACN) – 2% B (2 mM ammonium formate, 0.2% v / v formic acid in water).

[0199] Example 1. Synthesis of N-stearoyl D-erythro-phytosphingosine (21) (CER[N(18:0)P(18)])

[0200]

[0201] D-erythrophage hydrochloride was suspended in heptane. NaOMe (25% in MeOH or as powder, 1.2 equivalents) and methyl stearate (1 equivalent) were added to the suspension, and the mixture was heated at 95°C. After the reaction was complete (typically after 2 hours, monitored by LC-MS analysis), 0.25 volume of methanol was added, and the reaction mixture was cooled. The precipitated solid was filtered off and washed with a 9:1 mixture of MeOH / water. The solid was dried under vacuum to give CER[N(18:0)P(18)] (78%–85% yield) as a white powder.

[0202] ESI-MS: For [C] 36 H 73 The calculated value of NO4 is 583.5, and the measured value is 584.5[M+H]+.

[0203] This disclosure should not be construed in any way as limiting oneself to the described embodiments, and those skilled in the art will foresee many possibilities for modification thereto.

[0204] The embodiments described above are composable.

[0205] The following claims further illustrate specific embodiments of this disclosure.

Claims

1. A method for producing sphingolipids or similar substances having formula (1): in W is either H or the glycosyl moiety. R 1 It is H, aryl, or C. 1-50 Hydrocarbon group, preferably C 1-15 Hydrocarbon group, more preferably C 10-15 The hydrocarbon group may be saturated or contain one or more double and / or triple bonds, and / or may contain one or more functional groups, preferably selected from the group consisting of hydroxyl, alkoxy, acyloxy, amide, thiol, thioether, or phosphorus-containing functional groups. 2 and R 3 Selected from H, substituted or unsubstituted C 1-6 Hydrocarbon group, or substituted or unsubstituted C 1-6 Acyl group, R 4 Selected from substituted or unsubstituted aryl groups, heteroalkyl groups, and substituted or unsubstituted C groups that can be saturated or unsaturated. 1-31 Hydrocarbon group, preferably substituted or unsubstituted C, which can be saturated or unsaturated. 9-31 hydrocarbon group; This method is carried out using lysozyme or its salt having formula (2): in W, R 1 R 2 and R 3 As defined for the sphingolipid having formula (1), The method involves reacting the lysozyme salt having formula (2) or a salt thereof with an ester having formula (3) in the presence of a base: in R 4 As defined for the sphingolipid having formula (1), and R 5 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl or ethyl; and The reaction is carried out in a hydrocarbon solvent.

2. The method according to claim 1, wherein, The base is an alkoxide having formula (4): R 6 -O - X + (4)， in R 6 It is a C1-C4 hydrocarbon group, preferably selected from methyl, ethyl, propyl, isopropyl, butyl or isobutyl, more preferably selected from methyl or ethyl; X + It is selected from Na + K + Li + or NH4 + The cation, preferably Na + .

3. The method according to any one of claims 1 or 2, wherein, Use 0.1 to 1.7 molar equivalents of the base based on the amount of the lysosphingolipid having formula (2).

4. The method according to any one of claims 1 to 3, wherein, The reaction of the lysosomal sphingolipid of formula (2) or its salt and the ester of formula (3) takes place over a period of 0.5 to 4 hours.

5. The method according to any one of claims 1 to 4, wherein, The hydrocarbon solvent is C5-C. 10 Hydrocarbon solvent, preferably heptane.

6. The method according to any one of claims 1 to 5, wherein, R 2 and R 3 It is hydrogen.

7. The method according to any one of claims 1 to 6, wherein, W stands for hydrogen.

8. The method according to any one of claims 1 to 7, wherein, The hemolysin with formula (2) is D-erythro-dihydrosphingosine.

9. The method according to any one of claims 1 to 6, wherein, W is the glycosyl moiety, wherein the glycosyl moiety is preferably selected from Glc1-, Gal1-, Galβ1-4Glc1-.

10. The method according to any one of claims 1 to 9, wherein, R 4 It is a substituted or unsubstituted C having formula (5) or (6). 9-31 Hydrocarbon group: in Q is selected from H, -OH, Cl, Br, I, or F, preferably from H or -OH. L is a straight-chain C that can be saturated or contain one or more double and / or triple bonds. 6-28 Hydroxyl group, R 7 Is it H or -OR 8 , where R 8 It is hydrogen or a straight-chain carbon that can be saturated or contains one or more double bonds. 2-30 Acyl group.

11. The method according to any one of claims 1 to 10, wherein, The ester having formula (3) is a fatty acid methyl ester selected from the group consisting of: methyl stearate, methyl arachidate, methyl benzyl acid, methyl creosote, methyl hexadecanoate, methyl α-hydroxystearate, methyl α-hydroxyarachidate, methyl α-hydroxybenzyl acid, methyl α-hydroxycreosote, and methyl α-hydroxyhexadecanoate.

12. The method according to any one of claims 1 to 11, wherein, The sphingolipid having formula (1) is a ceramide selected from the group consisting of: CER[N(18:0)P(18)], CER[N(20:0)P(18)], CER[N(22:0)P(18)], CER[N(24:0)P(18)], CER[N(26:0)P(18)].

13. The method according to any one of claims 1 to 11, wherein, The sphingolipid having formula (1) is a ceramide selected from the group consisting of: CER[A(18:0)P(18)], CER[A(20:0)P(18)], CER[A(22:0)P(18)], CER[A(24:0)P(18)], CER[A(26:0)P(18)].

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