Method for the production of sphingolipids

Abstract

A method for production of at least one sphingolipid of formula (1), or a composition thereof. The method comprising reacting at least one lysosphingolipid of formula (2), or a salt thereof: with a compound of formula (3): wherein W, R1, R2, R3, and R4 are as defined as for the sphingolipid of formula (1), and wherein, said reacting is performed in the presence of a base of formula (4): wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4 +, Mg2+, or Ca2+; Y is selected from OH-, CO3 2-, HCO3 -, PO4 3-, methoxide, acetate, citrate, or succinate.

Description

[0001] METHOD FOR THE PRODUCTION OF SPHINGOLIPIDS

[0002] Filed of the invention

[0003] The present invention relates to a novel and efficient method for the production of sphingolipids or compositions thereof via the N-acylation of lysosphingolipids such as sphingoid bases and glycosylated sphingoid bases. The method is based on the use of an acid anhydride as acylating agents and is especially suitable for the production of ceramides and glycosphingolipids.

[0004] Background

[0005] Sphingolipids are an important class of polar lipids mainly found on the surface of eukaryotic cells.

[0006] Sphingolipids are structurally characterized by a sphingoid base backbone and can be divided in different classes such as, ceramides, and glycosphingolipids.

[0007] Ceramides are / V-acylated sphingoid bases lacking additional head groups at the 1-position of the sphingoid base backbone, and wherein the / V-acyl group of ceramides typically derives from a fatty acid.

[0008] Glycosphingolipids (GSLs) are glycoconjugates deriving from ceramides, wherein a glycan moiety is linked to the 1-hydroxyl group of a ceramide via a glycosidic linkage.

[0009] GSLs are involved in diverse biological processes and play important structural and functional roles such as cell-cell recognition, communication, and intercellular adhesion. Particularly, sialylated glycosphingolipids such as gangliosides are found in the brain, and can play roles in neurological diseases especially Alzheimer's, Parkinson's, and Huntington's diseases. Furthermore, certain gangliosides are found in the intestinal mucosa and can promote intestinal health, as well as act as anti-infective agents.

[0010] Ceramides are the main constituent of the stratum corneum lipid layer and have a major role in the water- retaining properties of the epidermis, as well as in the barrier function of the skin.

[0011] Sphingolipids hold great potential as therapeutics, cosmetics, and as food ingredients, however, they are not readily available for fundamental and clinical research. In fact, they are characterized by a high structural complexity and their preparation represents a challenge.

[0012] Sphingolipids such as ceramides and GSLs may be obtained via the N-acylation of lysosphingolipids.

[0013] Typically, lysosphingolipids are defined as sphingolipid breakdown products which lack the amide-linked fatty acyl group at the 2-position of the sphingoid base backbone. Accordingly, for each parental sphingolipid there is a corresponding lysosphingolipid that has an identical head group at the 1-position but lacks the amide-bound fatty acyl group at the 2-position (Hannun et al., Science 1989, 243, 500-507).

[0014] N-acylation of lysosphingolipids may be performed via chemical or enzymatic approaches. Chemical methods for the N-acylation of sphingolipids wherein an acid anhydride is used as acylating agent are known (WO2010111530, WO9943356, WO03048784). A drawback connected to these methods is the use of an organic base such as pyridine and triethyl amine during the N-acylation reaction. Particularly, organic bases such as pyridine and triethyl amine are flammable, toxic and render both the isolation of the final product and the reaction scale-up difficult. Current enzymatic approaches are based on the use of lipases (WO1994026919) which, however, are typically not specific for the amino group of a lysosphingolipid but may also act on hydroxyl groups, thus leading to the formation of mixtures of N- and O-acylation products, and thus requiring a lengthily and costly purification of the target compound. Accordingly, there is a demand for the development of novel and improved methodologies which enable the efficient and large-scale N-acylation of lysosphingolipids characterized by high technological feasibilities and low costs. Summary of the invention In a first aspect, the present invention relates to a method for production of at least one sphingolipid of formula (1), or a composition thereof: wherein, W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6 alkyl, preferably R5is hydrogen, R3is hydrogen, or a substituted or unsubstituted C1-6alkyl, or a substituted or unsubstituted C2-6acyl, preferably hydrogen, the bond is a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R4is a substituted or unsubstituted C4-31 alkyl, preferably a substituted or unsubstituted C15-19 alkyl; the method comprising reacting at least one lysosphingolipid of formula (2), or a salt thereof: with a compound of formula (3): R4-(C=O)-O-(C=O)-R4(3), wherein W, R1, R2, R3, and R4are as defined as for the sphingolipid of formula (1), and wherein, said reacting is performed in the presence of a base of formula (4): (X)n─(Y)m(4), wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4+, Mg2+, or Ca2+; Y is selected from OH-, CO32-, HCO3-, PO43-, methoxide, acetate, citrate, or succinate. In a second aspect, the present invention relates to a composition comprising at least one sphingolipid of formula (1): wherein, W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6 alkyl, preferably R5is hydrogen, R3is hydrogen, or a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6 acyl, preferably hydrogen, the bond is a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl; and a compound of formula (5), or a salt thereof: (5), wherein R4is as defined as for the sphingolipid of formula (1). Detailed Description of Invention The present inventors have established for the first time an efficient and economic method for the production of sphingolipids via the chemical N-acylation of lysosphingolipids, and wherein the lysosphingolipids used as the starting materials are preferably obtained via synthetic and / or biotechnological approaches. Surprisingly, the present inventors have found that lysosphingolipids can be N-acylated using as the acylating agent a compound of formula (3) in combination with a base of formula (4): (X)n─(Y)m(4), wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4+, Mg2+, or Ca2+; Y is selected from OH-, CO32-, HCO3-, PO43-, methoxide, acetate, citrate, or succinate. The process described in the present invention gives access to a diverse set of biologically relevant sphingolipids in high purity and yields. Furthermore, the process does not require the use of flammable and toxic organic bases such as pyridine and triethyl amine and the produced sphingolipids can be isolated directly from the reaction mixture via precipitation. The terms, definitions and embodiments described throughout the specification of the invention relate to all aspects and embodiments of the invention. The term “a” grammatically is a singular, but it may as well mean the plural of e.g., the intended compound. As used herein, the various functional groups or substituents represented will be understood to have a point of attachment at the functional group or atom having the dash (-). For example, in the case of – (C=O) it will be understood that the point of attachment is the carbon atom. If a group is listed without a dash, then the attachment point is indicated by the plain and ordinary meaning of the recited group. As used herein the letters N, C, O and H refer to a nitrogen atom, to a carbon atom, to an oxygen atom and to a hydrogen atom, respectively. The skilled person would understand that when speaking of position C-1, C-2, C-3, C-4, C-5 etc., reference is herein always made to the respective carbon atoms of sphingolipids of formula (1), (5), or (6), or lysosphingolipids of formula (2). As used herein, the term “alkyl” refers to an acyclic straight or branched hydrocarbyl radical having 1-50 carbon atoms which may be saturated or contain one or more double and / or triple bonds (so, forming for example an alkenyl or an alkynyl), and / or which may be substituted or unsubstituted, as herein further described. Examples of “alkyl” include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neo-pentyl, n-hexyl, ethenyl, 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, pentynyl, and hexynyl, each of which may be substituted or unsubstituted. Typically, the term alkyl refers to a straight acyclic hydrocarbyl group having 1-32 carbons, which may be substituted or unsubstituted. As used herein, the term “aryl” refers to an aromatic cyclic hydrocarbyl group having 5-14 ring carbon atoms, which may be mono- or polycyclic, which may contain fused rings, preferably 1 to 3 fused or unfused rings, and which may contain one or more heteroatoms, and / or which may be substituted or unsubstituted, as herein further described. Examples of “aryl” include, but are not limited to, phenyl, naphtyl, anthracyl, phenantryl, pyrrolyl, imidazolyl, thiophenyl, furanyl, oxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzofuranyl, each of which may be substitute or unsubstituted. Typically, the term “aryl” refers to a substituted or unsubstituted phenyl. As used herein, the term “acyl” refers to a group derived by the removal of one or more hydroxyl group from an oxoacid, preferably from a carboxylic acid. The acyl group according to the present invention is typically a saturated C1-30 acyl, which may be substitute or unsubstituted. As used herein, the term “substituted” means that the group in question is substituted with a group which typically modifies the general chemical characteristics of the group in question. The substituents can be used to modify characteristics of the molecule, such as molecule stability, molecule solubility and the ability of the molecule to form crystals. The person skilled in the art will be aware of other suitable substituents of a similar size and charge characteristics, which could be used as alternatives in a given situation. In connection with the terms “alkyl”, “aryl”, and “acyl” the term substituted means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from hydroxy (which when bound to an unsaturated carbon atom may be present in the tautomeric keto form), oxo, C1-6-alkoxy (i.e. C1-6-alkyl-oxy), C2-6-alkenyloxy, carboxy, oxo, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)aminocarbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6- alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkylcarbonylamino, cyano, guanidino, carbamido, C1-6-alkyl- sulphonyl-amino, aryl-sulphonyl-amino, heteroaryl-sulphonyl-amino, C1-6-alkanoyloxy, C1-6-alkyl- sulphonyl, C1-6-alkyl-sulphinyl, C1-6-alkylsulphonyloxy, nitro, C1-6-alkylthio, halogen, where any alkyl, alkoxy, and the like representing substituents may be substituted with hydroxy, C1-6-alkoxy, C2-6- alkenyloxy, carboxy, C1-6-alkylcarbonylamino, halogen, C1-6-alkylthio, C1-6-alkyl-sulphonyl-amino, or guanidino. In connection with the term “alkyl” the term “substituted” preferably means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from a hydroxyl group, an alkoxy group, an acyloxy group, an acylamido group, a thiol, a thioether or a phosphorus-containing functional group. The term “glycosyl moiety” when used herein is defined to encompass a moiety derived from a monosaccharide or from an oligosaccharide (more than one monosaccharide units), wherein the anomeric carbon of the monosaccharide or the anomeric carbon at the reducing end of the oligosaccharide is engaged in a glycosidic bond with another chemical entity, and the bond, if not further specified, may be an alpha or a beta glycosidic bond. A glycosyl moiety having more than one monosaccharide unit may represent a linear or a branched structure. The monosaccharide unit can be any 5-9 carbon atom sugar, comprising 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.), deoxysugars (e.g. L-rhamnose, L-fucose, etc.), deoxy-aminosugars (e.g. N- acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acids, ketoaldonic acids (e.g. sialic acid). The monosaccharide unit can form different cyclic structures such as pyranose (six- membered) cyclic structures or furanose (five-membered) cyclic structures. The glycosyl moieties according to the present invention may be illustrated in the following style: Galβ1-4Glc1-, wherein the dash (-) represents the point of attachment of the glycosyl moiety and wherein the glycosyl moiety may be linked via an alpha or a beta glycosidic bond, preferably a beta glycosidic bond. In the context of the present invention, the terms “about”, “around”, or “approximate” are applied interchangeably to a particular value (e.g. “a temperature of about 25oC”, “a temperature of around 25oC”, or “a temperature of approximate 25oC”), or to a range (e.g. “an amount from about 1% to about 99%”, “an amount from around 1% to around 99%”, or “an amount from approximate 1% to approximate 99%” ), to indicate a deviation from 0.1% to 10% of that particular value. The term “lysosphingolipid” when used herein refers to a sphingolipid breakdown product which lack the amide-linked fatty acyl group at the 2-position of the sphingoid base backbone. Suitable lysosphingolipids, for use in the context of the present invention, are sphingoid bases or glycosylated sphingoid bases and are represented by a lysosphingolipid of formula (2). Lysosphingolipids for use in the context of the present invention are preferably obtained via synthetic and / or biotechnological approaches such as those described in WO 2021170624 A2, WO2019238970 A1, WO2022158993 A1, WO2023118378 A1, by Sarmientos et al., Eur. J. Biochem. 1986, 160,527-535, by Vaughan et al., J. Am. Chem. Soc.2006, 128, 6300-6301 or as described in the examples below. In some embodiments, for the at least one lysosphingolipid of formula (2) the bond is a double bond and R2is hydrogen. In some preferred embodiments, for the at least one lysosphingolipid of formula (2) the bond is a double bond and R2and R3are hydrogen. In some preferred embodiments, the at least one lysosphingolipid of formula (2) is a lysosphingolipid of formula (9), or a salt thereof: (9), wherein W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl. In some embodiments, the stereochemical configuration of the C-2, C-3, and C-4 carbon atoms of the lysosphingolipid of formula (9) is (2S,3R,4E). In some embodiments, for the at least one lysosphingolipid of formula (2) the bond is a single bond, and R2is -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6alkyl, preferably R5is hydrogen. In some embodiments, for the at least one lysosphingolipid of formula (2) the bond is a single bond, R2is -OH, and R3is hydrogen. In some embodiments, for the at least one lysosphingolipid of formula (2) the bond is a single bond, and R2is hydrogen. In some embodiments, for the at least one lysosphingolipid of formula (2) the bond is a single bond, and R2and R3is hydrogen. In some embodiments, the at least one lysosphingolipid of formula (2) is a lysosphingolipid of formula (10), or a salt thereof: (10), wherein W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl. In some embodiments the stereochemical configuration of the C-2, C-3, and C-4 carbon atoms of the lysosphingolipid of formula (10) is (2S,3S,4R). In some embodiments, lysosphingolipids of formula (2), (9), or (10) may be produced or utilized in the form of salts, preferably in the form of pharmaceutical acceptable salts. In some embodiments, the salts of lysosphingolipid of formula (2), (9), or (10), may 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, methane sulfonic acid, trifluoromethanesulfonic acid, perchloric acid. The method of the present invention comprises reacting at least one lysosphingolipid of formula (2), (9), or (10) with a compound of formula (3): R4-(C=O)-O-(C=O)-R4(3), wherein R4is a substituted or unsubstituted C4-31 alkyl, preferably a substituted or unsubstituted C15-19 alkyl. In some embodiments, R4is a C15 alkyl. In some embodiment, the compound of formula (3) is palmitic anhydride. In some preferred embodiments, R4is a C17alkyl. In some preferred embodiments, the compound of formula (3) is stearic anhydride. In some embodiments, R4is a C19 alkyl. In some embodiments, the compound of formula (3) is arachidic anhydride. The reaction between the at least one lysosphingolipid of formula (2), (9), or (10) and the compound of formula (3) is performed in the presence of a base of formula (4): (X)n─(Y)m(4), wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4+, Mg2+, or Ca2+; Y is selected from OH-, CO32-, HCO3-, PO43-, methoxide, acetate, citrate, or succinate. In some embodiments, the base of formula (4) is selected from Na2CO3, NaHCO3, NaOH, Na3PO4, Na2HPO4, sodium acetate, sodium methoxide, sodium citrate, sodium succinate, K2CO3, KHCO3, KOH, K3PO4, K2HPO4, potassium acetate, potassium methoxide, potassium citrate, potassium succinate, CaCO3, Ca(HCO3)2, Ca(OH)2, Ca3(PO4)2, CaHPO4, calcium acetate, calcium methoxide, calcium citrate, calcium succinate, MgCO3, Mg(HCO3)2, Mg(OH)2, Mg3(PO4)2, MgHPO4, magnesium acetate, magnesium methoxide, magnesium citrate, magnesium succinate, (NH4)2CO3, NH4HCO3, NH4OH, (NH4)3PO4, (NH4)2HPO4, ammonium acetate, ammonium methoxide, ammonium citrate, or ammonium succinate. In some preferred embodiments, the base of formula (4) is Na2CO3. In some embodiments, the base of formula (4) is NaHCO3. In some embodiments, the base of formula (4) is NaOH. In some embodiments, the base of formula (4) is NaOAc. Typically, the reaction between the at least one lysosphingolipid according to the present invention and the compound of formula (3) is performed in a solvent such as methanol, ethanol, propanol, isopropanol, butanol, or isobutanol. In some preferred embodiment, the reaction is performed in methanol. In some embodiments the reaction is performed in a mixture of one or more polar solvents, such as a mixture of methanol and ethanol, methanol and propanol, methanol and isopropanol, methanol and butanol, methanol and isobutanol, or methanol and water. The reaction between the at least one lysosphingolipid according to the present invention and the compound of formula (3) is typically performed at temperature from about 25oC to about 65oC. Accordingly, in some embodiments, the reaction is performed at a temperature of about 25oC, 26oC , 27oC, 28oC, 29oC, 30oC, 31oC, 32oC, 33oC, 34oC, 35oC, 36oC, 37oC, 38oC, 39oC, 40oC, 41oC, 42oC, 43oC, 44oC, 45oC, 46oC, 47oC, 48oC, 49oC, 50oC, 51oC, 52oC, 53oC, 54oC, 55oC, 56oC, 57oC, 58oC, 59oC, 60oC, 61oC, 62oC, 63oC, 64oC, or 65oC. Preferably, the reaction between the lysosphingolipid and the triazine-based acylating agent, or a composition thereof is performed at temperature from about 40oC to about 50oC. Accordingly, in some preferred embodiments the reaction is performed at a temperature of 40oC, 41oC, 42oC, 43oC, 44oC, 45oC, 46oC, 47oC, 48oC, 49oC, or 50oC. The components of the reactions of the invention may be combined in any order, and it will be appreciated that the order of combining the reactants may be adjusted as needed. For example, the at least one lysosphingolipid may be added to a solution of the compound of formula (3), followed by the base of formula (4). As another example the compound of formula (3) may be added to a solution of the lysosphingolipid followed by the base of formula (4). As yet another example, a solvent may be added to a flask containing the lysosphingolipid, the compound of formula (3), and the base of formula (4). The at least one lysosphingolipid, the compound of formula (3), the base of formula (4) as well as any other reagent used during the reaction may be added to the reaction either as a solid or dissolved in a solvent, and in any quantities and manner effective for the intended result of the reaction. The reaction between the compound of formula (3) and the at least one lysosphingolipid according to the present invention, results in the selective N-acylation of the amino group at the C-2 carbon atom of said lysosphingolipid, thereby producing at least one sphingolipid of formula (1). In some embodiments, the present invention relates to a method for production of at least one sphingolipid of formula (1): wherein, W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6alkyl, preferably R5is hydrogen, R3is hydrogen, or a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6 acyl, preferably hydrogen, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl; the method comprising reacting at least one lysosphingolipid of formula (2), or a salt thereof: with a compound of formula (3): R4-(C=O)-O-(C=O)-R4(3), wherein W, R1, R2, R3, and R4are as defined as for the sphingolipid of formula (1), and wherein, said reacting is performed in the presence of a base of formula (4): (X)n─(Y)m (4), wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4+, Mg2+, or Ca2+; Y is selected from OH-, CO32-, HCO3-, PO43-, methoxide, acetate, citrate, or succinate. In some embodiments, the reaction between the compound of formula (3) and the at least one lysosphingolipid according to the present invention, results in the selective N-acylation of the amino group at the C-2 carbon atom of said lysosphingolipid, thereby producing a composition comprising at least one sphingolipid of formula (1). In some embodiments, the present invention relates to a method for production of a composition comprising at least one sphingolipid of formula (1): wherein, W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6alkyl, preferably R5is hydrogen, R3is hydrogen, or a substituted or unsubstituted C1-6alkyl, or a substituted or unsubstituted C2-6acyl, preferably hydrogen, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl; the method comprising reacting at least one lysosphingolipid of formula (2), or a salt thereof: with a compound of formula (3): R4-(C=O)-O-(C=O)-R4(3), wherein W, R1, R2, R3, and R4are as defined as for the sphingolipid of formula (1), and wherein, said reacting is performed in the presence of a base of formula (4): (X)n─(Y)m (4), wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4+, Mg2+, or Ca2+; Y is selected from OH-, CO32-, HCO3-, PO43-, methoxide, acetate, citrate, or succinate. In some preferred embodiments , the present invention relates to a method for production of a composition comprising at least one sphingolipid of formula (1) and a compound of formula (5), or a salt thereof: (5), wherein R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl. In some embodiments, for the at least one sphingolipid of formula (1) the bond is a double bond and R2is hydrogen. In some preferred embodiments, for the at least one sphingolipid of formula (1) the bond is a double bond and R2and R3are hydrogen. In some embodiments, the at least one sphingolipid of formula (1) is a sphingolipid of formula (11): (11), wherein, W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, R4is a substituted or unsubstituted C4-31 alkyl, preferably a substituted or unsubstituted C15-19 alkyl. In some embodiments, the stereochemical configuration of the C-2, C-3, and C-4 carbon atoms of the sphingolipid of formula (11) is (2S,3R,4E). In some embodiments, for the at least one sphingolipid of formula (1) the bond is a single bond, and R2is -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6 alkyl, preferably R5is hydrogen. In some embodiments, for the at least one sphingolipid of formula (1) the bond is a single bond, R2is -OH, and R3is hydrogen. In some embodiments, for the at least one sphingolipid of formula (1) the bond is a single bond, and R2is hydrogen. In some embodiments, for the at least one sphingolipid of formula (1) the bond is a single bond, and R2and R3is hydrogen. In some embodiments, the at least one sphingolipid of formula (1) is a sphingolipid of formula (12): W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, R4is a substituted or unsubstituted C4-31 alkyl, preferably a substituted or unsubstituted C15-19 alkyl. In some embodiments the stereochemical configuration of the C-2, C-3, and C-4 carbon atoms of the sphingolipid of formula (12) is (2S,3S,4R). In some embodiments W of the at least one lysosphingolipid of formula (2), (9), or (10) is hydrogen. Accordingly, in some embodiments the at least one lysosphingolipid of formula (2), (9), or (10) is a sphingoid base. In some preferred embodiments, the at least one lysosphingolipid of formula (2) or (9) is D-erythro- sphingosine. In some embodiments, the at least one lysosphingolipid of formula (2) or (10) is D-erythro- phytosphingosine. In some embodiments, the at least one lysosphingolipid of formula (2) is DL-erythro-dihydrosphingosine In some embodiments W of the at least one sphingolipid of formula (1), (11), or (12) is hydrogen. Accordingly, in some embodiments the at least one sphingolipid of formula (1), (11) or (12) is a ceramide. Ceramides denote, in the context of the present invention, naturally occurring ceramides, analogues thereof 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], wherein letters in brackets refer to the shorthand 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 by Masukawa et al., Journal of Lipid Research, 2008, 49, 1466-1476. Particularly, the letters N, A, and EO represent non-hydroxy fatty acids (N), alpha-hydroxy fatty acids (A), and omega-linoleoyloxy fatty acids (EO), respectively, wherein the number of fatty acid carbons and unsaturations may be expressed in parentheses following the letters of N, A, E, and O. The letters, S, H, P, and dS representD-erythro- sphingosine (S), 6-hydroxy-D-erythro-sphingosine (H),D-ribo-phytosphingosine (P),DL-erythro- dihydrosphingosine (dS), respectively, wherein the number of sphingoid carbons may be expressed in parenthesis following the letters S, H, P, and dS. Ceramides, CER[NdS], CER[AdS], or CER[EOdS], may also be referred to as CER[NG], CER[AG], or CER[EOG], respectively, wherein the letter G represent the INCI name forDL-erythro-dihydrosphingosine. In some embodiment, the sphingolipid of formula (1) or (11) is a ceramide selected from CER[N (16:0) S (18)], CER[A (16:0) S (18)], CER[N (18:0) S (18)], CER[A (18:0) S (18)], CER[N (20:0) S (18)], CER[A (20:0) S (18)], In some embodiment, the sphingolipid of formula (1) or (12) is a ceramide selected from CER[N (16:0) P (18)], CER[A (16:0) P (18)], CER[N (18:0) P (18)], CER[A (18:0) P (18)], CER[N (20:0) P (18)], CER[A (20:0) P (18)]. In some preferred embodiments, W of the lysosphingolipid of formula (2), (9), or (10), and W of the sphingolipid of formula (1), (11), and (12) is a glycosyl moiety. In some preferred embodiments, W of the lysosphingolipid of formula (2), (9), or (10), and W of the sphingolipid of formula (1), (11), or (12) is a glycosyl moiety selected from: Glc1-, Gal1-, Galβ1-4Glc1, Neu5Acα2-3Gal1-, Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-3Galβ1-3GalNAcβ1-4Galβ1-4Glc1-, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc1-, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc1-, or Neu5Acα2-8Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc1-. In some embodiments, W of the at least one lysosphingolipids of formula (2), (9), or (10) is Glc1-. In some embodiments, W of the at least one lysosphingolipids of formula (2), (9), or (10) is Gal1-. In some embodiments, W of the at least one lysosphingolipid of formula (2), (9), or (10) is Galβ1-4Glc1-. In some embodiments, W of the at least one lysosphingolipid of formula (2), (9), or (10) is Neu5Acα2- 3Galβ1-4Glc1-. In some embodiments, W of the at least one lysosphingolipid of formula (2), (9), or (10) is Neu5Acα2- 8Neu5Acα2-3Galβ1-4Glc1-. In some embodiments, W of the at least one lysosphingolipid of formula (2), (9), or (10) is Neu5Acα2- 8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-. In some embodiments the at least one lysosphingolipids of formula (2) is a lysosphingolipid of formula (13), or a salt thereof: (13), wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl. In some embodiments the at least one lysosphingolipids of formula (2) is a lysosphingolipid of (14), or a salt thereof: (14), wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, In some embodiments the at least one lysosphingolipids of formula (2) is a lysosphingolipid of formula (15), or a salt thereof: (15). wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl. In some embodiments the at least one lysosphingolipids of formula (2) is a lysosphingolipid of formula (16), or a salt thereof: (16), wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl. In some embodiments the at least one lysosphingolipid of formula (2) or (13) is lyso-GM3, or a salt thereof. In some embodiments the at least one lysosphingolipid of formula (2) or (14) is lyso-GD3, or a salt thereof. In some embodiments the at least one lysosphingolipid of formula (2) or (15) is lactosyl sphingosine In some embodiments the at least one lysosphingolipid of formula (2) or (16) is lyso-GT3. In some embodiments the at least one lysosphingolipids of formula (2) is a composition comprising a lysosphingolipid of formula (13) and a lysosphingolipid of formula (15), or salts thereof. In some embodiments the at least one lysosphingolipids of formula (2) is a composition comprising a lysosphingolipid of formula (13) and a lysosphingolipid (14), or salts thereof. In some embodiments the at least one lysosphingolipids of formula (2) is a composition comprising a lysosphingolipid of formula (13), a lysosphingolipid (14) and a lysosphingolipid of formula (15), or salts thereof. In some embodiments the at least one lysosphingolipids of formula (2) is a composition comprising a lysosphingolipid of formula (13), a lysosphingolipid (14), and a lysosphingolipid of formula (16), or salts thereof. In some embodiments the at least one lysosphingolipids of formula (2) is a composition comprising a lysosphingolipid of formula (13), a lysosphingolipid (14), a lysosphingolipid of formula (16), and a lysosphingolipid of formula (15) or salts thereof. In some embodiments, the at least one lysosphingolipids of formula (2) is a composition comprising about 75-80 wt.% of N-lyso-GM3, about 7-9 wt.% of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosyl D-erythro-sphingosine. In some embodiments, the at least one lysosphingolipids of formula (2) is a composition comprising about 40-55 wt.% of N-lyso-GD3 and about 10-15 wt.% of N-lyso-GM3, about 3-6 wt.% of N-lyso-GT3, about 4-6 wt.% of lactosylD-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosylD-erythro-sphingosine. In some embodiments, the at least one lysosphingolipids of formula (2) is a composition comprising about 15-20 wt.% of N-lyso-GD3 and about 50-60 wt.% of N-lyso-GM3, about 0.1-0.5 wt.% of N-lyso-GT3, about 4-7 wt.% of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosyl D-erythro-sphingosine. In some embodiments, the at least one lysosphingolipids of formula (2) is a composition comprising about 35-40 wt.% of N-lyso-GD3 and about 25-40 wt.% of N-lyso-GM3, and wherein the composition further comprising about 5-6 wt.% of lactosyl D-erythro-sphingosine, and about 0.5-1.0 wt.% of glucosyl D-erythro- sphingosine. In some embodiments, W of the at least one sphingolipids of formula (1), (11), or (12) is Glc1-. In some embodiments, W of the at least one sphingolipids of formula (1), (11), or (12) is Gal1-. In some embodiments, W of the at least one sphingolipid of formula (1), (11), or (12) is Galβ1-4Glc1-. In some embodiments, W of the at least on sphingolipid of formula (1), (11), or (12) is Neu5Acα2-3Galβ1- 4Glc1-. In some embodiments, W of the at least on sphingolipid of formula (1), (11), or (12) is Neu5Acα2- 8Neu5Acα2-3Galβ1-4Glc1-. In some embodiments, W of the at least on sphingolipid of formula (1), (11), or (12) is Neu5Acα2- 8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-. In some embodiments, the at least one sphingolipid of formula (1) is a sphingolipid of formula (6), or a salt thereof: (6) wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl; R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl. In some embodiments, the at least one sphingolipid of formula (1) is a sphingolipid of formula (7), or a salt thereof: (7) wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl; R4is a substituted or unsubstituted C4-31 alkyl, preferably a substituted or unsubstituted C15-19 alkyl. In some embodiments, the at least one sphingolipid of formula (1) is a sphingolipid of formula (8): wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl; R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl. In some embodiments, the at least one sphingolipid of formula (1) is a sphingolipid of formula (17): wherein, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl; R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl. In some embodiments the at least one sphingolipid of formula (1) or (6) is GM3, or a salt thereof. In some embodiments the at least one sphingolipid of formula (1) or (7) is GD3, or a salt thereof. In some embodiments the at least one sphingolipid of formula (1) or (8) is lactosyl ceramide. In some embodiments the at least one sphingolipid of formula (1) or (17) is GT3. In some embodiments, the present invention relates to a composition comprising at least one sphingolipid of formula (1), and a compound of formula (5), or salts thereof. In some embodiments, the composition comprising a sphingolipid of formula (6), and a compound of formula (5), or a salt thereof. In some preferred embodiments, the composition comprising a sphingolipid of formula (6) or a salt thereof, a sphingolipid of formula (8), and a compound of formula (5) or a salt thereof. In some embodiments the composition comprising a sphingolipid of formula (7), and a compound of formula (5), or salts thereof. In some embodiments, the composition comprising a sphingolipid of formula (6), a sphingolipid of formula (7), and a compound of formula (5), or salts thereof. In some embodiments, the composition comprising a sphingolipid of formula (6) or a salt thereof, a sphingolipid of formula (7) or a salt thereof, a sphingolipid of formula (8), and a compound of formula (5), or a salt thereof. In some embodiments, the composition comprising a sphingolipid of formula (6), a sphingolipid of formula (7), a sphingolipid of formula (17), and a compound of formula (5), or salts thereof. In some embodiments, the composition comprising a sphingolipid of formula (6) or a salt thereof, a sphingolipid of formula (7) or a salet thereof, a sphingolipid of formula (17) or a salt thereof, a sphingolipid of formula (8) or a salt thereof, and a compound of formula (5) or salts thereof. In some embodiments, the composition comprising a sphingolipid of formula (8), and a compound of formula (5), or a salt thereof. In some embodiments, the composition comprising at least about 70 wt.% of the sphingolipid of formula (6), and at least about 4 wt.% of a compound of formula (5), or salts thereof. In some embodiments, the composition comprising at least about 70 wt.% of the sphingolipid of formula (6) or a salt thereof, at least about 5 wt% of a sphingolipid of formula (8), and at least about 4 wt.% of a compound of formula (5), or a salt thereof. In some preferred embodiments, the composition comprising about 70-80 wt.% of the sphingolipid of formula (6) or the salt thereof, about 5-10% wt.% of sphingolipid of formula (8), and about 4-10 wt.% of a compound of formula (5), or a salt thereof. In some embodiments the composition comprising at least about 75 wt.% of the sphingolipid of formula (8), and at least about 1 wt.% of the compound of formula (5), or the salt thereof. In some embodiments, R4of the sphingolipid of formula (1), (6), (7), (8), (11), (12), or (16), and of the compound of formula (5) is a C15 alkyl. In some preferred embodiments, R4of the sphingolipid of formula (1), (6), (7), (8), (11), (12), or (16), and of the compound of formula (5) is a C17 alkyl. In some preferred embodiments, R4of the sphingolipid of formula (1), (6), (7), (8), (11), (12), or (16), and of the compound of formula (5) is a C19alkyl. In some embodiments the compound of formula (5) is palmitic acid, or a salt thereof. In some embodiments the compound of formula (5) is stearic acid, or a salt thereof. In some embodiments the compound of formula (5) is arachidic acid, or a salt thereof. In some preferred embodiments the composition comprising about 70-80 wt.% of GM3 or a salt thereof, about 5-10% wt.% of lactosyl ceramide, and about 4-10 wt.% of stearic acid, or a salt thereof. In some embodiments the composition comprising about 75-90% wt.% of lactosyl ceramide, and about 1- 5 wt.% of stearic acid, or a salt thereof. In some embodiments, the sphingolipid of formula (1), (6), (7), (11), (12), or (17) are in the form of salts, preferably in the form of pharmaceutically acceptable salts. In some embodiments, the pharmaceutically acceptable salt of the sphingolipid of formula (1), (6), (7), (11), (12), or (17) are salt of sodium, potassium, lithium, calcium, magnesium, zinc, aluminum, triethylamine, diethanolamine, ethanolamine, ethylenediamine, arginine, lysine, histidine, choline, benzathine, chloroprocaine, procaine, or meglumine salt. In some embodiments the lysosphingolipid of formula (2), (9), (10), (13), (14), (15), or (16) is in the form of a salt, preferably in the form of pharmaceutical acceptable salt. In some embodiments, the pharmaceutically acceptable salt of the lysosphingolipid of formula (2), (9), (10), (13), (14), (15), or (16) is a salt of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, polyphosphoric acid, acetic acid, camphor sulfonic acid, p-toluene sulfonic acid, methane sulfonic acid, trifluoromethanesulfonic acid, perchloric acid. Examples The working examples below describe non-limiting embodiments of the invention and are given only to illustrate the invention. General methods and material: LCMS analysis was performed with a Shimadzu ECO 2020 LC system coupled with a Shimadzu LCMS-2020 system equipped with a Merck Ascentis Express RP-Amide column (15cm x 4.6mm, 2.7 µm). For the LCMS analysis the following gradient was applied: 90-98% eluent D in C in 20 min. Eluent C: 2 mM AF, 2 mL FA, 1000 mL water; Eluent D: 2 mM AF, 2 mL FA, 750 mL MeOH, 250 mL MeCN. HPLC analyses were performed on a Dionex Ultimate 3000 HPLC system coupled with a Corona Veo Charged Aerosol Detector using an Accucore aQ (150 mm x 4.6 mm, 2.6 µm) column. Methods are described in Example 10. Fatty acids anhydrides were purchased by established manufacturers or synthesized according to Kargar et al., J Am Oil Chem Soc 2013, 90, 259 – 264. Lactosyl D-erythro-sphingosine was synthesized as described in WO2023118378 A1, or by Vaughan et al., J. Am. Chem. Soc.2006, 128, 6300-6301. N-lyso-GM3 and N-lyso-GD3, or compositions thereof were prepared according to the procedures described in Examples 6 and 7, respectively. Mutant 2,3-trans-sialidase (TcTS, wild-type Q26966, mutations / modifications: Ser263Thr, Arg477His, Val485Leu, Glu559Val, Ser496Lys, N-terminal His-tag, deletion of 7 amino acids Δ636-642) was expressed from Escherichia coli (E. coli) strains following methods described in Paris et al., Glycobiology 2001, 11, 305-311, or in Buschiazzo et al., Molecular Cell 2002, 10, 757-768. The following cell-free extracts were produced from E. coli expression strains: (i) Cell-free extract from E. coli BL21 (DE3) dLacZ genetically engineered for the expression of MtCMK, accession: WP_129368399; (ii) Cell-free extract from E. coli BL21 (DE3) dLacZ genetically engineered for the expression of recombinant NmCSS (wild-type: WP_061726245, modification: N-terminal histidine tag MGHHHHHH); (iii) Cell-free extract from E. coli BL21 (DE3) dLacZ genetically engineered for expression a mutant CSTII (wild-type Q9LAK3, mutation / modifications: Ile53Ser or Ile53Gly, N-terminal-histidine tag, deletion of 32 amino acids at the C-terminus). Cell-free extracts were prepared as described in Example 9. Example 1:General Procedure for the N-acylation of Lysosphingolipids To a methanolic solution containing one or more lysosphingolipid (1 eq.), Na2CO3 (0.5-1.5 eq.) was added. The resulting mixture was stirred for 15 min, followed by the addition of stearic anhydride (1.1 eq.). The mixture was heated to a temperature between 60-50 °C and stirred for another 0.5 – 2 h. The reaction mixture was cooled down to -5 °C, stirred for 15 min and the formed solid was filtered off. The solid was subjected to re-suspensions and heat treatment in MeOH / H2O (95:5, 37oC), followed by re-suspensions and reflux in acetone. Further cycles of re-suspensions and reflux may be applied to obtain an increase in the wt.% of one or more sphingolipids. Following the procedure, a solid comprising between about 70-98 wt.% of one or more sphingolipids was obtained. Final products were characterized via LCMS or HPLC analysis. Example 2: Production of GM3 N-lyso-GM3 (obtained according to Example 4) was subjected to the general procedure of Example 1 to afford GM3. LC-MS analysis: Rt = 9.5 min; MS: [M-H+]- calculated: 1179.7, found: 1180.0. Example 3: Production of Lactosyl Ceramide Lactosyl D-erythro-sphingosine was subjected to the general procedure of Example 1 to afford lactosyl ceramide. LC-MS analysis: Rt = 11.1 min; MS: [M+H+]+calculated: 890.7, found: 890.9. Example 4: Production of a Composition Comprising GM3 N-lyso-GM3 (obtained according to Example 4) was subjected to the general procedure of Example 1 to afford a composition comprising GM3, lactosyl ceramide, and stearic acid, which was characterized via HPLC: Compound wt.% Rt (min) GM3 70-80 11.6 Lactosyl Ceramide 5-10 10.6 Stearic acid 4-10 8.8 Example 5: Production of a Composition Comprising Lactosyl ceramide Lactosyl D-erythro-sphingosine was subjected to the general procedure of example 1 to afford a composition comprising lactosyl ceramide, and stearic acid, which was characterized via HPLC: Compound wt.% Rt (min) Lactosyl Ceramide 5-10 10.6 Stearic acid 4-10 8.8 Example 6. Production of N-lyso-GM3 N-lyso-GM3 was synthesized via the TCTs catalyzed sialylation of lactosyl D-erythro-sphingosine. The reaction was performed in an aqueous solution at a pH between about 6.5 to about 7.0. A typical reaction mixture contained lactosyl D-erythro-sphingosine (1 eq.), 3’-sialyllactose (1.5-2.0 eq.), the 2,3- transialidase (TcTS, 0.4 g / L) and the β-galactosidase (0.5 g / L). During the course of the reaction nanofiltration was applied. The nanofiltration of the reaction mixture was performed applying 300-500 Da membranes, a pressure of 15-20 bar and a temperature of about 30-40oC for about 6-8 hours. The NF retentate (NFR) was heated at a temperature between about 60-95oC for about 10-60 minutes, and then diafiltrated according to example 6. The procedure afforded a composition comprising 70-80 wt% of N-lyso-GM3, and 5-10 wt % of lactosyl D-erythro-sphingosine. Example 7: Production of Mixtures of N-lyso-GD3 and N-lyso-GM3 N-lyso-GD3 was synthesized via the sialyltransferase cycle. The sialyltransferase cycle was performed in an aqueous solution at a pH between about 7.0 to about 7.5, the temperature ranged between about 25oC to about 37oC. A typical reaction mixture contained the glycoside acceptor (1 eq.), N-acetylneuraminic acid (Neu5Ac, 1.2-2.5 eq.), β-cyclodextrin (0.5 eq.), ATP (2.0-3.5 eq.), CMP (0.1-0.3 eq.), MgCl2 (0.5 M), and the following three cell-free extracts: cell-free extract (i) (5-12 g / L), cell-free extract (ii) (1-2 g / L), cell- free extract (iii) (2.5-5 g / L). The sialylation cycle was monitored by LCMS (For method and conditions see example 22). Conversions were typically 10-99%. The rection mixture was then subjected to diafiltration according to example 6. Example 8: Isolation of Lysosphingolipids The diafiltration (DF), was performed by applying 250 kDa spiral-wound membranes having a membrane area of about 0.668 m2, a flow rate of about 10 l / h, a transmembrane pressure of about 8-10 bar, a temperature between about 20-25oC, and around 2-10 DF volumes relative to the volume of the feed solution. During diafiltration, a high flux of about 15.3-18.1 l / m2h was maintained. The DF retentate (DFR), containing the sialylated glycosphingolipid, was spray-dried on a Mobile Minor ® (GEA) spray drier under the following conditions: Inlet flow rate: 45-50 g / min Atomizer speed: 20,000 rpm Inlet temperature: 160oC Outlet temperature: 85oC Following this procedure, a spry-dried powder comprising about 70-90 wt.% of N-lyso-GM3 and / or N- lyso-GD3 was obtained. Example 9. Preparation of the cell-free extract Genes encoding the enzymes are usually ordered as codon-optimized synthetic genes for optimal expression in the E. coli host strain. The synthetic constructs contain overhangs with BsaI restriction sites for golden gate cloning into a pET28a-based expression vector (carrying introduced BsaI restriction sites and a fluorescent drop-out cassette). The resulting plasmids were used for transformation of E. coli BL21 (DE3) dLacZ. A preculture of the expression strain was prepared in 10mL LB medium supplemented with the respective antibiotic and incubated at 37°C shaking overnight. The culture of the expression strain was started by a 1:100-fold dilution of the preculture into TB medium supplemented with the respective antibiotic. The culture was incubated at 37°C until an OD600 of 0.7-1.0 was reached. The culture was cooled to the desired expression temperature, induced with 0.5 mM IPTG, and incubated for the desired expression time. Cells were harvested by centrifugation and resuspended in water. Cell lysis was achieved by sonication. The resulting lysed cell suspension was centrifuged to separate the cell-free extract, comprising soluble enzymes, from the debris. The supernatant, containing the cell-free extract, was freeze-dried to dryness. Example 10. HPLC Analysis The lysosphingolipid content of the composition comprising N-lyso-GM3 was determined under the following conditions: HPLC eluent profile: solvent A: 1 L water + 0.5 mL formic acid + 4 mmol ammonium formate, and solvent B: 1 L MeOH + 1 L acetonitrile + 4 mL formic acid + 4 mmol ammonium formate. A gradient of 50-100% B in A was applied over 13 min, followed by an isocratic of 100% B for 18 min, followed by an isocratic of 50% B in A for 40 min. The lysosphingolipid content of the composition was quantified via peak area analysis using external standards. The lysosphingolipid content of the composition comprising N-lyso-GD3 and N-lyso-GM3 was determined under the following conditions: HPLC eluent profile: solvent A: 1 L water + 2.0 mL formic acid + 2 mmol ammonium formate, and solvent B: 1.5 L MeOH + 0.5 L acetonitrile + 4 mL formic acid + 4 mmol ammonium formate. A gradient of 70-100% B in A was applied over 8 min., followed by an isocratic of 100% B for 11 min., followed by an isocratic of 70% B in A for 25 min. The glycosphingolipid content of the powders was quantified via peak area analysis using external standards. The HPLC analysis of the composition comprising GM3 was performed under the following conditions: HPLC eluent profile: solvent A: 100 mM Ammonium Formate in 1L water + 2mL Formic acid; solvent B: 600 mL MeOH + 400 mL acetonitrile. A gradient of 65-100% B in A was applied over 10 min., followed by an isocratic of 100% B for 13 min., followed by an isocratic of 65% B in B for 30 min. The sphingolipid content of the was quantified via peak area analysis using external standards. The HPLC analysis of the composition comprising lactosyl ceramide performed under the following conditions: HPLC eluent profile: solvent A: 2mM Ammonium Formate in 500 mL MeOH+500mL ACN + 2mL Formic acid; solvent B: 2 mM Ammonium Formate in 1 L water + 2mL formic acid. A gradient of 65-100% A in B was applied over 15 min., followed by an isocratic of 100% A for 35 min., followed by an isocratic of 65% A in B for 40 min. The sphingolipid content of the was quantified via peak area analysis using external standards. The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.

Claims

Claims 1. Method for production of at least one sphingolipid of formula (1), or a composition thereof:wherein, W is hydrogen or a glycosyl moiety, R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6 alkyl, preferably R5is hydrogen, R3is hydrogen, or a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6 acyl, preferably hydrogen, the bond is a double or a single bond when R2is hydrogen, or is a single bond when R2is - OR5, R4is a substituted or unsubstituted C4-31alkyl, preferably a substituted or unsubstituted C15-19alkyl; the method comprising reacting at least one lysosphingolipid of formula (2), or a salt thereof:with a compound of formula (3): R4-(C=O)-O-(C=O)-R4(3), wherein W, R1, R2, R3, and R4are as defined as for the sphingolipid of formula (1),and wherein, said reacting is performed in the presence of a base of formula (4): (X)n─(Y)m(4), wherein n is 1, 2, or 3; m is 1 or 2; X is selected from Na+, K+, NH4+, Mg2+, or Ca2+; Y is selected from OH-, CO32-, HCO3-, PO43-, methoxide, acetate, citrate, or succinate.

2. The method according to claim 1, wherein the base of formula (3) is selected from Na2CO3, NaHCO3, or NaOH, preferably Na2CO3.

3. The method according to claims 1 or 2 wherein, for the at least one sphingolipid of formula (1) and the lysosphingolipid of formula (2), W is a glycosyl moiety, and wherein the glycosyl moiety is selected from the group consisting of Glc1-, Gal1-, Galβ1-4Glc1-, Neu5Acα2-3Galβ1-, Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-8 Neu5Acα2-3Galβ1-4Glc1-, and Neu5Acα2-3Gal1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-.

4. The method according to any one of claims 1 to 3 wherein R4is a C17 alkyl.

5. The method according to any one of claims 1 to 4, wherein the at least one sphingolipid of formula (1) is selected from the group consisting of lactosyl ceramide, GM3, GD3, GT3, GD1a, or a composition thereof.

6. A composition comprising at least one sphingolipid of formula (1):wherein, W is hydrogen or a glycosyl moiety,R1is hydrogen, or aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, or a substituted or unsubstituted C1-6 alkyl, preferably R5is hydrogen, R3is hydrogen, or a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6 acyl, preferably hydrogen, the bond is a double or a single bond when R2is hydrogen, or is a single bond when R2is - OR5, R4is a substituted or unsubstituted C4-31 alkyl, preferably a substituted or unsubstituted C15-19 alkyl; and a compound of formula (5), or a salt thereof:(5), wherein R4is as defined as for the sphingolipid of formula (1).

7. The composition according to claim 6 wherein, for the at least one sphingolipid of formula (1), W is a glycosyl moiety, and wherein the glycosyl moiety is selected from the group consisting of Glc1-, Galβ1-4Glc1-, Neu5Acα2-3Galβ1-, Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2- 3Galβ1-4Glc1-, Neu5Acα2- 8Neu5Acα2-8 Neu5Acα2-3Galβ1-4Glc1-, and Neu5Acα2-3Gal1- 3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-.

8. The composition according to claims 6 or 7, wherein the at least one sphingolipid is a sphingolipid of formula (6), or a salt thereof: (6) wherein R1and R4are defined as for the sphingolipid of formula(1).

9. The composition according to any one of claims 6 to 8, wherein the composition comprising a sphingolipid of formula (7), or salts thereof:wherein R1and R4are defined as for the sphingolipid of formula (1).

10. The composition according to claims 8 or 9, further comprising a sphingolipid of formula (8):(8), wherein R1and R4are defined as for the sphingolipid of formula (1).

11. The composition according to claim 8, wherein the composition comprising at least 70 wt.% of the sphingolipid of formula (6), and at least 4 wt.% of a compound of formula (5), or salts thereof.

12. The composition according to claim 11, wherein the composition further comprising at least 5 wt% of a sphingolipid of formula (8).

13. The composition according to claim 8, wherein the composition comprising 70-80 wt.% of the sphingolipid of formula (6) or the salt thereof, 5-10% wt.% of sphingolipid of formula (8), and 4-10 wt.% of a compound of formula (5).

14. The composition according to any one of claims 6 to 13 wherein R4is a C17alkyl.

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