Production of glucosylceramide

Abstract

The present invention relates to a method of producing a compound of formula (1): (1) from a compound of formula (2): (2), wherein R1 is hydrogen, 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, R2 is hydrogen or -OR5, wherein R5 is selected from hydrogen or a substituted or unsubstituted C1-3 alkyl, the bond - - - may be a double or a single bond when R2 is H, or is a single bond when R2 is -OR5, R3 is H or a substituted or unsubstituted C2-4 alkyl, R4 is hydrogen or -C(=O)R6, wherein R6 is a C15-C33 alkyl, Glc is a glucosyl moiety, Gal is galactosyl moiety, wherein the method comprises a step of reacting the compound of formula (2) with an enzyme having β-galactosidase activity.

Description

[0001] DESCRIPTION

[0002] PRODUCTION OF GLUCOSYLCERAMIDE

[0003] The present invention relates to a method for producing glucosylceramide.

[0004] Background

[0005] In higher eukaryotes, glucosylceramide (GlcCer) is the simplest member and major precursor of complex glycosphingolipids and it is essential for viability of multicellular organisms. GlcCer is the precursor of lactosylceramide (LacCer) and the rest of the neutral and acidic oligoglycosphingolipids. Most of the GlcCer that is synthesized is however not converted to LacCer. There appears to be a distinct pool of GlcCer that is used for LacCer and downstream oligo glycosphingolipid synthesis, which raises the possibility that the cell utilizes GlcCer for different functions.

[0006] GlcCer has been shown to possess important functions in the skin. GlcCer constitutes approximately 4% of the total epidermal lipid mass and is one of the main components of lamellar bodies. Lamellar bodies are lipid-storage organelles that are exocytosed from keratinocytes, which represent the major cell type of the epidermis, the outermost layer of the skin. The skin uses this storage function to respond to the environment quickly by altering the level of ceramides, which have essential roles for maintaining proper water and electrolyte levels and for skin barrier repair. GlcCer is involved in providing the correct ceramides needed for skin barrier function. Other functions of GlcCer in the skin include signal proliferation in keratinocytes and anti-inflammatory effects.

[0007] In addition, prebiotic effects of glucosylceramide administered as food additives have recently been suggested.

[0008] A typical approach to produce glucosylceramides is via direct glycosylation of a ceramide with UDP-GIc and a glycosyltransferase, mainly of mammalian origin. Applying a ceramide as an acceptor for glycosylations is complicated by its poor solubility in aqueous solution as well as difficulties in production or formulation of mammalian enzymes on industrial scale.

[0009] Accordingly, there is a demand for suitable large-scale production technologies of GlcCer.

[0010] Summary of the invention

[0011] In one aspect the present invention relates to a method of producing a compound of formula (1): from a compound of formula (2):

[0012] (2), wherein

[0013] R1is hydrogen, 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,

[0014] R2is hydrogen or -OR5, wherein R5is selected from hydrogen or a substituted or unsubstituted C1-3 alkyl, the bond - may be a double or a single bond when R2is H, or is a single bond when R2is -OR5,

[0015] R3is H or a substituted or unsubstituted C2-4 alkyl,

[0016] R4is hydrogen or -C(=O)R6, wherein R6is a C15-C33 alkyl,

[0017] Glc is a glucosyl moiety,

[0018] Gal is galactosyl moiety, wherein the method comprises a step of reacting the compound of formula (2) with an enzyme having P-galactosidase activity.

[0019] Description of the Figures

[0020] Figure 1 describes amino acid sequences of the invention.

[0021] Figure 2 shows the enzymatic activity of p-galactosidase on lactosylsphingosine (Lac-sph) in a Pichia pastoris cell (B) and as a control a Pichia pastoris cell that is not expressing a p-galactosidase enzyme (A).

[0022] Detailed description of the invention

[0023] The present inventors have established a novel and economically viable method for producing GlcCer on a large-scale. The present inventors have surprisingly found that glycosylated sphingoid bases having lactosyl as a glycosyl moiety can provide efficient substrates for p-galactosidases. The obtained glycosylated sphingoid base having galactosyl as a glycosyl moiety may then further be acylated to receive a ceramide by various techniques.

[0024] As used herein, the term "alkyl" refers to an acyclic straight or branched hydrocarbyl group 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,l-pentenyl, 2-pentenyl, 2-methyl-l-butenyl, 3-methyl-l-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. The term alkyl preferably refers to a straight saturated acyclic hydrocarbyl group having 1-31 carbons, which may be substituted or unsubstituted.

[0025] 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. The term "aryl" preferably refers to a substituted or unsubstituted phenyl.

[0026] 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 may be a saturated or unsaturated C2-32 acyl, which may be substitute or unsubstituted.

[0027] As used herein, the term "substituted" means that the group in question is substituted with a group which may modify 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.

[0028] 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, Ci- 6-alkoxy (i.e. Ci.g-alkyl-oxy), C2-s-alkenyloxy, carboxy, oxo, Ci.g- alkoxycarbonyl, Ci.g-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci-6-alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)aminocarbonyl, amino-Ci.g-alkyl-aminocarbonyl, mono- and di(Ci.g- alkyl)amino-Ci-6-alkyl-aminocarbonyl, Ci.g-alkylcarbonylamino, cyano, guanidino, carbamido, Ci.g-alkyl- sulphonyl-amino, aryl-sulphonyl-amino, heteroaryl-sulphonyl-amino, Ci.g-alkanoyloxy, ci-s-alkyl- sulphonyl, Ci.g-alkyl-sulphinyl, Ci.g-alkylsulphonyloxy, nitro, Ci.g-alkylthio, halogen, where any alkyl, alkoxy, and the like representing substituents may be substituted with hydroxy, Ci.g-alkoxy, C2-6- alkenyloxy, carboxy, Ci.g-alkylcarbonylamino, halogen, Ci.g-alkylthio, Ci.g-alkyl-sulphonyl-amino, or guanidino.

[0029] 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.

[0030] The terms "Glc" and "Gal" refer to glucosyl and galactosyl moiety, respectively.

[0031] A glucosyl moiety may be represented by a moiety of formula (10):

[0032] A galactosyl moiety can be represented by a moiety of formula (11):

[0033] The glucosyl and galactosyl moieties according to the present invention may be illustrated in the following style: Glc- wherein the dash (-) represents the point of attachment of the glycosyl moiety and wherein the glycosyl moiety, is preferably linked via a beta glycosidic bond.

[0034] In the method of the present invention, a compound of formula (2) is reacted with an enzyme having p- galactosidase activity.

[0035] P-galactosidases (EC 3.2.1.23) are enzymes belonging to the glycoside hydrolase family 35 (GH35), which typically catalyse the hydrolysis of terminal non-reducing -D-galactose residues in p-D-galactosides. The term "an enzyme having p-galactosidase activity" may be interchangeably used with the term "P- galactosidase" or "lactase". Functional analogues of p-galactosidases are included in the term.

[0036] The p-galactosidase in its wild-type form may originate from a microorganism such as bacterium, yeast, ascomycete, actinomycete, hyphomycetes, basidiomycotina, and the like. Preferably, the p-galactosidase originates from a bacterium or a yeast, such as Escherichia coli (E. coli), Lactobacillus thermophilus, Bacillus circulans, Leuconostoc citrovorum, Bifidobacterium infantis, Bifidobacterium longum, Aspergillus oryzae (A. oryzae), Kluyveromyces lactis, Kluyveromyces fragilis. The p-galactosidase may originate from any known -galactosidase sequence or from any p-galactosidase sequence which has yet to be determined, p-galactosidases yet to be determined can be identified using sequence databases and sequence alignment algorithms, for example, the publicly available GenBank database and the BLAST alignment algorithm.

[0037] In some embodiments, the enzyme having p-galactosidase activity is a p-galactosidase originating from Aspergillus oryzae, or a functional analogue thereof. The amino acid sequence of the wild-type p- galactosidase originating from Aspergillus oryzae corresponds to the amino acid sequence having Accession No: Q.2UCU3 (https: / / www.uniprot.org / , accession number).

[0038] In some preferred embodiments, the enzyme having p-galactosidase activity is a truncated variant of the wild-type p-galactosidase originating from Aspergillus oryzae.

[0039] The truncated variant of the p-galactosidase, according to the present invention, can be purchased from established manufacturers, e.g. Calza Clemente, or produced by methods known to the skilled person such as that described in M.M. Maksimainen et al., International Journal of Biological Macromolecules 2013, 60, 109-115.

[0040] In connection with the term enzyme the term "functional analogue" refers to a protein wherein the amino acid sequence has a certain percent homology compared to the amino acid sequence of a reference protein (i.e. about 30% homology / identity, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology over a specified region, for example over a region of at least about 25, 50, 75, 100, 150, 200, 250, 500, 1000, or more amino acids, up to the full length sequence, when compared and aligned for maximum correspondence over a comparison window or designated region) and maintains the same functional activity of the reference protein. The percent homology may be determined using e.g. a BLAST sequence comparison algorithm, or by manual alignment and visual inspection (see e.g. NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences may be termed "substantially identical". In some embodiments, the term functional analogue refers to a mutant protein, a truncated variant of the protein, or to a fusion protein which maintains the same functional activity of the reference protein.

[0041] The compounds of formula (1) and (2) comprise a sphingolipid moiety.

[0042] The sphingolipid moiety typically derives from an aliphatic amino alcohol such as a sphingoid base or from a ceramide.

[0043] Sphingoid bases denote in the context of the present invention naturally occurring sphingoid bases, analogues thereof or derivatives thereof. Naturally occurring sphingoid bases are D-erythro-sphingosine (S), 6-Hydroxy-d-erythro-sphingosine (H), d-r / bo-phytosphingosine (P) or dl-erythro-dihydrosphingosine (DS), wherein the number of sphingoid carbons may be expressed in parenthesis following the letters S, H, P, and DS.

[0044] The letters S, H, P, and DS refer to the shorthand nomenclature developed by Motta et al., Biochim Biophys Acta. 1993, 1182:147-151 and expanded by Rabionet, Biochim Biophys Acta 2014, 1841:422-434 and by Masukawa et al., Journal of Lipid Research 2008, 49, 1466-1476. d-Erythro-dihydrosphingosine may also be represented by the letter G according to the INCI nomenclature.

[0045] 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 [CER] 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], The letters in brackets refer to the shorthand nomenclature developed by Motta et al., Biochim Biophys Acta. 1993, 1182, 147-151 and expanded by Rabionet, 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 nonhydroxy 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 represent D-erythro- sphingosine (S), 6-hydroxy-D-erythro-sphingosine (H), D-ribo-phytosphingosine (P), D-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 represents the INCI name for D-erythro-dihydrosphingosine.

[0046] The method of producing the compound of formula (1) may be performed in vitro or in vivo.

[0047] When performed in vitro, a cyclodextrin may be added to the step of reacting the compound of formula (2) with the enzyme having p-galactosidase activity.

[0048] The term "cyclodextrin", in the context of the present invention, refers to a cyclic oligosaccharide consisting of a macrocyclic ring of monosaccharide subunits (e.g., glucose). Cyclodextrins, typically contain 6-, 7- or 8-monosaccharide subunits and may be referred to as a-cyclodextrins, p-cyclodextrins, and y- cyclodextrins, respectively. The cyclodextrin may be modified such that some or all of the primary or secondary hydroxyl groups of the macrocycle, or both, may be alkylated or acylated. Methods of modifying these alcohols are well known to the person skilled in the art and many derivatives are commercially available. Thus, some or all of the hydroxyl groups of the cyclodextrin may be substituted with an -OR11group and / or an O-C(=O)-R12group, wherein R11and R12are independently selected from a saturated or unsaturated Cl-6 alkyl, a saturated or unsaturated Ci.g heteroalkyl, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycloalkyl, an aryl, or a heteroaryl, each of which may be substituted or unsubstituted. In some embodiments, R11and R12are independently selected from the group consisting of 2-hydroxyethyl, 2-hydroxypropyl, and sulfobutylether.

[0049] In some embodiments, the cyclodextrin is a-cyclodextrin, p-cyclodextrin, y-cyclodextrin, or derivatives thereof.

[0050] In some embodiments, the cyclodextrin is selected from the group consisting of p-cyclodextrin, hydroxypropyl-p-cyclodextrin, randomly methylated p-cyclodextrin, or sulfobutylether-p-cyclodextrin. In some preferred embodiments, the cyclodextrin is p-cyclodextrin.

[0051] The cyclodextrin may be used in an amount between about 0.1 equivalents to about 1 equivalent based on the amount of the glycosphingolipid. In some preferred embodiments the cyclodextrin is used in an amount between about 0.1 equivalents to about 0.5 equivalents based on the amount of the glycosphingolipid. Accordingly, in some preferred embodiments, the cyclodextrin is used in an amount of about 0.1, 0.2, 0.3, 0.4, or 0.5 equivalents based on the amount of the glycosphingolipid.

[0052] The use of a cyclodextrin provides several advantages such as high yields, and eliminates the need for the use of a detergent or organic solvent to increase accessibility to the glycosyl moiety of the substrate. However, detergents or organic solvents can also be used in the method of the invention.

[0053] The method of producing the compound of formula (1) may be performed in vivo in a genetically modified microorganism. The genetically modified microorganism is in some embodiments a bacterial cell or a yeast cell. The bacterium may be e.g. selected from the group comprising Escherichia coli (e.g. E. coli), Bacillus spp. (e.g. Bacillus subtilis), Campylobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitidis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp. and Pseudomonas, and the yeast may be e.g. selected from the group comprising Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and Candida albicans. In preferred embodiments, the genetically modified organism is Pichia pastoris.

[0054] The genetically modified microorganism is in some embodiments capable of internalizing the compound of formula (2). The internalization of the compound of formula (2) may be solely or also via a passive transport during which the compound of formula (2) diffuses passively across the plasma membrane of the cell. The flow is directed by the concentration difference in the extra- and intracellular space with respect to the compound of formula (2) to be internalized, which the compound of formula (2) is supposed to pass from the place of higher concentration to the zone of lower concentration tending towards an equilibrium. In some embodiments, the genetically modified microorganism comprises a transporter protein, which internalizes the compound of formula (2) via active transport. Different transporter proteins have specificities for different sugar moieties of the molecules to be internalized. This specificity may be altered by mutation by means of common recombinant DNA techniques. The present inventors have surprisingly found that a compound of formula (2) can efficiently internalize into a genetically modified microorganism, such as a yeast, especially Pichia pastoris.

[0055] The expression "genetically modified" denotes that at least one alteration in the DNA sequence has been performed in the genome of the cell in order to give that cell a specific phenotype. The alteration in the DNA may e.g. be an introduction or a deletion of a DNA fragment in the genome. Genome editing may be performed e.g. by commonly known recombinant nucleic acid techniques as e.g. described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0056] The p-galactosidase may be endogenous to the microorganism, i.e. naturally present in the microorganism, where it is expressed naturally as a part of a genomic nucleic acid sequence, or it may be heterologously expressed, i.e. introduced by recombinant nucleic acid techniques encoding a p- galactosidase. Commonly known recombinant nucleic acid techniques are e.g. described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). A heterologous nucleic acid sequence may be a nucleic acid sequence that originates from a source foreign to the particular host cell, or it may originate from the same source. If derived from the same source, the nucleic sequence may be modified from its original form. Thus, a heterologous nucleic acid sequence in a cell also includes nucleic acid sequences that are endogenous to the particular cell, these may be a non-modified fragment of the original genomic sequence, or a genomic sequence that has been subjected to one or more modifications. Non-limiting examples of endogenous nucleic acid sequences that are subjected one or modifications include constructs comprising an endogenous nucleic acid sequence that operably linked to a promoter and / or another regulatory sequence that is not naturally linked to said sequences in the genome, or the nucleic acid sequences that comprise nucleobase substitutions introduced by site-directed mutagenesis.

[0057] A heterologous nucleic acid sequence can be expressed in the cell transiently, e.g. as plasmid borne, or stably, e.g. from a genome integrated expression cassette. One expression vector can be used for one or several expression cassettes or more than one expression vector can be used for more than one expression cassette. Heterologous nucleic acid sequences according to the present invention can also be inserted into the chromosome of the cell, using methods known to those skilled in the art, including homologous recombination, site-specific recombination or transposon-mediated gene transposition.

[0058] The term "nucleic acid sequence" refers to a DNA fragment, which is either double-stranded or single stranded, or to a product of transcription of said DNA fragment, and / or to an RNA fragment. The nucleic acid sequence encoding the p-galactosidase according to the present invention comprise or consists of a coding DNA sequence, i.e. a gene, a derivative of a gene or a transcription product of a gene, or a synthetic construct substantially identical to a gene. A derivative of a gene includes a nucleic acid sequence that is a fragment of a gene or a nucleic acid sequence that contains one or more mutations and / or deletions as compared to the original gene, or a cDNA; the mutations or deletions must not strongly impair the function of the encoded enzyme. A derivative of a gene is preferably at least 60% identical to a gene, more preferably at least 90% identical to a gene, even more preferably at least 95% identical to a wildtype gene. The value for gene identity may be generated when two or more nucleotide sequences are compared and aligned for maximum correspondence, as measured using one of algorithms accepted for this purpose in the art, e.g., as the following sequence comparison algorithms. A synthetic construct substantially identical to a gene may be produced by synthesis techniques known to the skilled person. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given peptide or protein. For instance, the codons CGU, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded peptide or protein.

[0059] In some embodiments, the compound of formula (1) is a compound of formula (3).

[0060] In some embodiments, the present invention describes a method for the production of a compound of formula (3), or a salt thereof:

[0061] (3), from a compound of formula (2): wherein

[0062] R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted Ci-17 alkyl, more preferably a substituted or unsubstituted C 10-17 alkyl,

[0063] R2is hydrogen or -OR5, wherein R5is selected from hydrogen or a substituted or unsubstituted C1-3 alkyl, the bond - may be a double or a single bond when R2is H, or is a single bond when R2is -OR5, R3is H or a substituted or unsubstituted C2-4 alkyl,

[0064] Glc is a glucosyl moiety,

[0065] Gal is a galactosyl moiety, wherein the method comprises a step of reacting the compound of formula (2) with an enzyme having p- galactosidase activity.

[0066] In some embodiment the method further comprising a nanofiltration step.

[0067] In some embodiments the present invention describes a method for the production of a compound of formula (3), from a compound of formula (2), wherein the method comprises the steps of:

[0068] - reacting the compound of formula (2) with an enzyme having p-galactosidase activity,

[0069] - nanofiltration of the reaction mixture.

[0070] In some embodiments, the compound of formula (1) is a compound of formula (4).

[0071] A compound of formula(4) may also be referred to as glucosyl ceramide.

[0072] In some embodiments, the present invention describes a method for the production of a compound of formula (4):

[0073] (4), from a compound of formula (2): wherein

[0074] R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted Ci-17 alkyl, more preferably a substituted or unsubstituted C 10-17 alkyl,

[0075] R2is hydrogen or -OR5, wherein R5is selected from hydrogen or a substituted or unsubstituted C1-3 alkyl, the bond - may be a double or a single bond when R2is H, or is a single bond when R2is -OR5, R3is H or a substituted or unsubstituted C2-4 alkyl,

[0076] R4is hydrogen or -C(=O)R6, wherein R6is a C15-C33 alkyl,

[0077] Glc is a glucosyl moiety,

[0078] Gal is galactosyl moiety, wherein the method comprising the steps of:

[0079] - reacting the compound of formula (2) with an enzyme having p-galactosidase activity, thereby obtaining a compound of formula (3), or a salt thereof:

[0080] G Icp

[0081] (3), wherein

[0082] R1, R2, R3and Glc are defined as for the compound of formula (1);

[0083] - acylating the compound of formula (3) thereby obtaining the compound of formula (4).

[0084] In some embodiment the method further comprising a nanofiltration step.

[0085] In some embodiments the present invention describes a method for the production of a compound of formula (4), from a compound of formula (2), wherein the method comprises the steps of:

[0086] - reacting the compound of formula (2) with an enzyme having p-galactosidase activity, thereby obtaining a compound of formula (3), or a salt thereof;

[0087] - nanofiltration of the reaction mixture;

[0088] - acylating the compound of formula (3) thereby obtaining the compound of formula (4).

[0089] In some embodiments, the step of reacting the compound of formula (2) with an enzyme having p- galactosidase activity and the nanofiltration step are performed simultaneously.

[0090] In some embodiments, the method further comprising the step of isolating the compound of formula (4).

[0091] In some embodiments the present invention describes a method for the production of a compound of formula (4), from a compound of formula (2), wherein the method comprises the steps of: reacting the compound of formula (2) with an enzyme having p-galactosidase activity, thereby obtaining a compound of formula (3), or a salt thereof; - acylating the compound of formula (3) thereby obtaining the compound of formula (4);

[0092] - isolating the compound of formula (4).

[0093] In some embodiments the present invention describes a method for the production of a compound of formula (4), from a compound of formula (2), wherein the method comprises the steps of:

[0094] - reacting the compound of formula (2) with an enzyme having p-galactosidase activity, thereby obtaining a compound of formula (3), or a salt thereof;

[0095] - nanofiltration of the reaction mixture;

[0096] - acylating the compound of formula (3) thereby obtaining the compound of formula (4);

[0097] - isolating the compound of formula (4).

[0098] The nanofiltration (NF) step may be used to concentrate the reaction, to remove ions, mainly monovalent ions, and / or to remove organic materials having a molecular weight lower than that of the compound of formula (1), such as monosaccharides. Preferably, the nanofiltration step is used to remove galactose from the reaction mixture.

[0099] Typically, the nanofiltration membrane has a molecular weight cut-off (M WCO) that ensures the retention of the compound of interest. As an example, a nanofiltration membrane having a MWCO of about 200- 500 Da, is suitable for retaining the compound of formula (3). In this regard the compound of formula (3) is accumulated in the NF retentate (NFR). The nanofiltration can be combined with diafiltration (DF) with water in order to remove permeable molecules more effectively, e.g., until the conductivity of the permeate showing no or very low presence of salts.

[0100] The NF step according to the present invention is conducted, with or without the optional DF step, at a constant temperature, preferably between about 15-45 °C, more preferably between about 20-35 °C. The NF step, with or without diafiltration, is continued until reaching the desired concentration of the glycosphingolipid in the NFR. Other technical parameters like setting in the flux and pressure is a matter of routine skills.

[0101] In some embodiments, the acylation step of the compound of formula (3) is performed by the addition of an ester of formula (5)

[0102] O

[0103] RIOAR6

[0104] (5), wherein

[0105] R6is a is a C15-C33 alkyl,

[0106] R7is selected from a Ci-C4alkyl, preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl, or ethyl; in the presence of a base.

[0107] The compound of formula (3) and the ester of formula (5) may be reacted in the presence of a base such as an alkoxide, an amine, a carbonate, or a bicarbonate.

[0108] In some embodiments, the compound of formula (3) and the ester of formula (5) are reacted in the presence of an amine, wherein the amine is preferably selected from triethylamine, N,N- diisopropylethylamine, and pyridine.

[0109] In some embodiments, the compound of formula (3) and the ester of formula (5) are reacted in the presence of a carbonate, wherein the carbonate is preferably selected from NajCOs, K2CO3, CaCOs, IJ2CO3, (NH4)2CO3.

[0110] In some embodiments, the compound of formula (3) and the ester of formula (5) are reacted in the presence of a bicarbonate, wherein the bicarbonate is preferably selected from NaHCOs, KHCO3, Ca(HCO3)2, LiHCO3, NH4HCO3.

[0111] In some preferred embodiments, the compound of formula (3) and the ester of formula (5) are reacted in the presence of an alkoxide, and wherein the alkoxide is an alkoxide.

[0112] In some embodiments the alkoxide is selected from sodium methoxide, potassium methoxide, lithium methoxide, ammonium methoxide, sodium ethoxide, potassium ethoxide, lithium ethoxide, or ammonium ethoxide.

[0113] In some more preferred embodiments, the compound of formula (3) and the ester of formula (5) are reacted in the presence of sodium methoxide.

[0114] The base may be used in catalytic amounts, equimolar amounts or in excess.

[0115] In some embodiments, the compound of formula (3) is in the free-base form and the base is used in a catalytic amount from about 0.1 to about 0.5 molar equivalents based on the amount of the compound of formula (3).

[0116] In some embodiments, the compound of formula (3) is in a salt form and the base is used in an amount from about 0.5 to about 1.7 molar equivalents based on the amount of the compound of formula (3).

[0117] The compound of formula (3), the ester of formula (5), and the base may be reacted in a polar solvent such as methanol, ethanol, propanol, isopropanol, butanol, or isobutanol. In some preferred embodiments, 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 the mixture of water and an aliphatic alcohol. In some embodiments, the reaction is performed in acetonitrile.

[0118] In some embodiments, the reaction is performed solvent-free.

[0119] The compound of formula (3), the ester of formula (5), and the base may be reacted at a temperature from about 50 °C to about 125 °C. Preferably at a temperature from about 60 °C to about 65 °C. Accordingly, in some preferred embodiments, the reaction is performed at a temperature of about 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, or 65 °C.

[0120] 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.

[0121] For example, the base may be added to a solution of the compound of formula (3) and the ester of formula (5). As another example, a solvent may be added to a flask containing the lysosphingolipid and the ester, followed by the base.

[0122] The compound of formula (3), the ester of formula (5), and the base, 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.

[0123] In some embodiments, the acylation step of the compound of formula (3) is performed by the addition of a 1,3,5-triazine-based acylating agent of formula (6), or a combination thereof: represents a conjugated system of bonds such that either two or three double bonds are present in the ring;

[0124] R8is selected from C(O-C(=O)R6), or C(=O);

[0125] Zais selected from N, NR10, or N(C(=O)R6); Zbis selected from N, or NR10;

[0126] R9is selected from C(=O), or C(OR10); and provided that: when R8is C(O-C(=O)R6), R9is C(OR10), Zaand Zbare N, and three double bonds are present in the ring, or when R8is C(O-C(=O)R6), R9is C(=O), one of Zaand Zbis N and the other group is NR10, and two double bonds are present in the ring, or when R8is C(=O), R9is C(OR10), Zais N(C(=O)R6), Zbis N, and two double bonds are present in the ring; and wherein

[0127] R6is a substituted or unsubstituted C15-C33 alkyl,

[0128] R10is selected from methyl, ethyl, 2,2,2-trifluoroethyl, and substituted or unsubstituted benzyl.

[0129] In some embodiments, the triazine-based acylating agent of formula (6) is selected from the group consisting of triazine-based acylating agents of formulas (12), (13), (14) and (15): wherein R6and R10of compounds of formulas (12), (13), (14) and (15) are as defined as for the triazinebased acylating agent of formula (6).

[0130] In some embodiments, the triazine-based acylating agent of formula (6) is a combination comprising triazine-based acylating agents of formulas (12), (13), (14) and (15) and wherein each of said triazinebased acylating agent is present, in said combination, in the amount from about 1% to about 99%.

[0131] In some embodiments, the triazine-based acylating agent of formula (6) is a combination comprising triazine-based acylating agents of formulas (12) and (13), and wherein each of said triazines is present, in said combination, in the amount from about 1% to about 99%.

[0132] In some embodiments, the triazine-based acylating agent of formula (6) is a combination comprising triazine-based acylating agents of formulas (14) and (15), and wherein each of said triazines is present, in said combination, in the amount from about 1% to about 99%. Triazine-based acylating agents according to the present invention, or their combination thereof, may be produced by standard methods known to the skilled person. A method for the synthesis of triazine-based acylating agent is e.g. described by Z. J. Kaminski, J. prakt. Chem. 1990, 4, 579-583. Alternatively, triazinebased acylating agents or combinations thereof may be synthesized according to the described in the example below.

[0133] The compound of formula (3) and the triazine-based acylating agent, or a composition thereof may be reacted in a polar solvent such as methanol, ethanol, propanol, isopropanol, butanol, or isobutanol.

[0134] In some preferred embodiment, the reaction is performed in methanol. In some embodiment 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.

[0135] In some embodiments, the compound of formula (3) and the triazine-based acylating agent, or the composition thereof are reacted in the presence of a base such as NaOH, KOH, LiOH, Ca(OH)2, triethylamine, / V, / V-diisopropylethylamine, and pyridine. In some preferred embodiments, the based is selected from NaOH, or KOH.

[0136] The purity of the triazine-based acylating agent according to the present invention, or the combination thereof may vary from a purity of about 99% to a purity of about 70%.

[0137] The reaction between the compound of formula (3) and the triazine-based acylating agent, or the composition thereof may be performed at temperature from about 25 °C to about 65 °C. Accordingly, in some embodiments, the reaction is performed at a temperature of about 25 °C, 26 °C , 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, or 65 °C. Preferably, the reaction between the compound of formula (3) and the triazinebased acylating agent, or a composition thereof is performed at temperature from about 45 °C to about 55 °C. Accordingly, in some preferred embodiments the reaction is performed at a temperature of 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, or 55 °C.

[0138] 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.

[0139] For example, the compound of formula (3) may be added to a solution of the triazine-based acylating agent, or the composition thereof. As another example the triazine-based acylating agent, or the composition thereof may be added to a solution of the compound of formula (3). As yet another example, a solvent may be added to a flask containing the compound of formula (3) and the triazine-based acylating agent, or the composition thereof. The compound of formula (3) and the triazine-based acylating agent, or the combination thereof, 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.

[0140] The glucosylceramide of formula (4) may be isolated by standard methods known to the skilled person. A preferred method for the isolation of the glucosylceramide of formula (4) is via precipitation from the reaction mixture. Precipitation may be achieved for example via partial removal of the reaction solvent by evaporation, i.e. concentrating the rection mixture, or via the addition of another solvent to the reaction mixture, or via changes of temperature or pressure, or via addition of other solutes, or combinations of these.

[0141] In some embodiment, the glucosylceramide of formula (4) is isolated via precipitation from the reaction mixture, wherein the precipitation is achieved via addition of another solvent to the reaction mixture, preferably via the addition of water.

[0142] The glucosylceramide of formula (4) may be isolated in different polymorphic forms. Polymorphic forms as referred to herein can include crystalline and amorphous forms as well as solvate and hydrate forms, which can be further characterized as follows:

[0143] Crystalline forms have different arrangements and / or conformations of the molecules in the crystal lattice.

[0144] Amorphous forms consist of disordered arrangements of molecules that do not possess a distinguishable crystal lattice.

[0145] Solvates are crystal forms containing either stoichiometric or non-stoichiometric amounts of a solvent. If the incorporated solvent is water, the solvate is commonly known as a hydrate.

[0146] In some embodiments, the glucosylceramide of formula (4) may be isolated as a solvate.

[0147] In some embodiments, the glucosylceramide of formula (4) may be isolated as hydrates, such as in the form of monohydrates, dihydrates or trihydrates.

[0148] In some embodiments, the glucosylceramide of formula (4) may be isolated in a crystalline form.

[0149] In some embodiments, the glucosylceramide of formula (4) may be isolated in an amorphous form.

[0150] In those embodiments, where the glucosylceramide of formula (4) is isolated in a crystalline form, the step of isolating the glucosylceramide of formula (4) may also be referred to as crystallization.

[0151] In some embodiments, the compound of formula (2) is produced by reacting a glycosyl donor of formula (7):

[0152] J-B (7), wherein

[0153] J is a lactosyl moiety,

[0154] B is selected from a fluoride, chloride, bromide, azide, formate, iodide. with a sphingolipid acceptor of formula (8), or a salt thereof: wherein

[0155] R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted Ci-17 alkyl, more preferably a substituted or unsubstituted C 10-17 alkyl,

[0156] R2is hydrogen or -OR5, wherein R5is selected from hydrogen or a substituted or unsubstituted C1-3 alkyl, the bond - may be a double or a single bond when R2is H, or is a single bond when R2is -OR5,

[0157] R3is H or a substituted or unsubstituted C2-4 alkyl, in the presence of a glycosynthase.

[0158] In a preferred embodiment, the glycosynthase comprises an amino acid motif of formula (9):

[0159] Xx-X2-X3-X4-X5-X6

[0160] 0), wherein

[0161] X1is an amino acid residue selected from I, M, L, V, A, F or W;

[0162] X2is an amino acid residue selected from L, M, I, V or A;

[0163] X3is an amino acid residue A, L or M;

[0164] X4is an amino acid residue selected from G, A, S, N, Q, C, T, I, V, L or M;

[0165] X5is an amino acid residue selected from F, T, M, L or S;

[0166] X6is an amino acid residue selected from G, L or F;

[0167] Amino acid residues or sequences are herein defined by the commonly used one-letter code or by their three-letter code, as summarized in Table 1.

[0168] Table 1 Amino acid codes:

[0169] Preferred glycosynthases are endoglycoceramide synthases (EGC synthases). Endoglycoceramide synthases are mutant endoglycoceramidases, in which the nucleophilic region has been mutated, especially the conserved glutamate or aspartate has been exchanged to a non-nucleophilic residue, thereby having their hydrolytic activity reduced, while preserving the synthetic activity. In a preferred embodiment, the endoglycoceramide synthase originates from Rhodococcus sp. AQ5-07 (accession number WP_113682895), such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, that are described in Table 2.

[0170] Table 2: Overview of glycosynthases EXAMPLES

[0171] The working examples below describe non-limiting embodiments of the invention and are given only to illustrate the invention.

[0172] Example 1: Strain design and growth conditions LacZ gene from E. coli encoding a p-galactosidase was cloned into a plasmid according to standard molecular cloning techniques and transformed into a P. pastoris host strain. Strain growth was performed in minimal medium using standard conditions. The medium was supplemented with lactosyl D-erythro- sphingosine (LacSph) to a final concentration of 2.5 g / L. Growth was recorded every 24 h and samples from supernatant (extracelllar) and in the cells (intracellular) were collected for further analysis. From these, the concentrations of Lac-sph and Glc-sph were measured (Fig. 2).

[0173] Example 2: General procedure for the enzymatic production of galactosyl-sphingoid bases with p- galactosidase in vitro

[0174] Enzymatic reactions were performed in 1 M NaOAc buffer (pH set to 5.0). The reaction mixture contained 50-100 g / L of a lactosyl sphingoid base, such as lactosyl sphingosine (LacS), lactosyl dihydrosphingosine (LacG), or lactosyl phytosphingosine (LacP), 5 g / L of R-cyclodextrin, 5 g / L of R-galactosidase. The enzymatic reaction was followed by LCMS analysis.

[0175] Example 3: Analytic methods for the enzymatic in vitro production of galactosyl-sphingoid bases with P-galactosidase.

[0176] Samples (25 pL) were taken from reaction mixtures, mixed with DMSO (950 pL) and subjected to centrifugation (16.000 rpm, 5 min) and analyzed with a Shimadzu ECO 2020 LC system coupled with a Shimadzu LCMS-2020 system. The HPLC analysis was performed using a Merck Ascentis Express RP-Amide column (15cm x 4.6mm, 2.7 pm). For analysis of compounds the eluent consisted of solvent D (2 mM ammonium formate, 2 mL formic acid, 75% v / v MeOH, 25% v / v ACN) - solvent C (2 mM formic acid in filtered ddHjO), and the following gradient was applied as stated in the table below. The flow rate was 1 mL / min. The MS analysis was performed under the following conditions: ESI positive ionization, vaporizer temperature 300 °C; LC-MS mode, 1:1 split of flow; ESI negative ionization, vaporizer temperature 300 °C; LC-MS mode, 1:1 split of flow.

[0177] Table 3. LC-MS analysis for the production of galactosyl-sphingoid bases with p-galactosidase.

[0178] Example 4: In-vitro enzymatic glycosylation of phytosphingosine, dihydrosphingosine and sphingosine

[0179] Reaction contained: 200-600 mM of lactosyl fluoride donor, 12-18 mg / ml EGC synthase protein, 1 M NaOAc buffer at pH 6.0, 100-600 mM sphingolipid acceptor. Reaction was executed at 37°C until desired conversion was reached. Product analysis was done by ELSD (% rel. conversion) and LC-MS using standard conditions.

[0180] Example 5: Analytic methods for the in-vitro enzymatic glycosylation of sphingolipids

[0181] LC / MS analysis was performed with a Shimadzu ECO 2020 LC system coupled with a Shimadzu LCMS-2020 system. LC analysis was performed using a Merck Ascentis Express RP-Amide column (15cm x 4.6mm, 2.7 pm). The eluent consisted of solvent D (2 mM ammonium formate, 2 mL formic acid, 75% v / v MeOH, 25% v / v ACN) - solvent C (2 mM formic acid in filtered ddHjO), and the following gradient was applied as stated in the table below. The flow rate was 1 mL / min.

[0182] Table 4: LC-MS analysis for the in-vitro enzymatic glycosylation of sphingolipids with glycosyl fluoride donors a = main mass ion detected was [M+Na]+; b = main mass ion detected was [M+Formate] Example 6: General procedure for the synthesis of triazine-based acylating agents

[0183] A fatty acid (1 eq.) was suspended in n-heptane, acetone, or a mixture of ethanol / methanol. 2-chloro-4,6- dimethoxy-l,3,5-triazine (1 eq.), and 4-methylmorpholine (1 eq.) were added to the suspension / solution. The reaction mixture was stirred at a temperature between 20 °C to 50 °C until a TLC-analysis showed complete consumption of the starting material.

[0184] The reaction mixture was cooled down to a temperature between 5 °C to 30 °C. When acetone was used as solvent, water was added to the reaction mixture prior cooling. A solid precipitated, which was filtered and dried in vacuum to obtain the final product.

[0185] Example 7: Synthesis of 4,6-dimethoxy-l,3,5-triazyn-2-yl stearate (16)

[0186] Compound (16) was synthesised from stearic acid and 2-chloro-4,6-dimethoxy-l,3,5-triazine following the general procedure described in Example 1.

[0187] XH NMR (500 MHz, CDCI3) 6 4.06 (s, 6H), 2.60 (t, J = 7.5 Hz, 2H), 1.73 (m, 2H), 1.77-1.68 (m, 28H), 0.88 (t, J = 6.9 Hz, 3H).

[0188] Example 8: Synthesis of 6-methoxy-5-methyl-4-oxo-4,5-dihydro-l,3,5-triazyn-2-yl stearate (17):

[0189] Compound (17) was synthesised from stearic acid and 2-chloro-4,6-dimethoxy-l,3,5-triazine following the general procedure described in Example 1.

[0190] XH NMR (400 MHz, CDCI3) 6 4.10 (s, 3H), 3.35 (s, 3H), 2.84 (t, J = 7.4 Hz, 2H), 1.78-1.67 (m, 2H), 1.43-1.19 (m, 28H), 0.87 (t, J = 6.7 Hz, 3H).

[0191] 13C NMR (101 MHz, CDCI3) 6 174.66, 160.13, 152.21, 148.83, 57.51, 40.30, 31.93, 29.70, 29.67, 29.66, 29.64, 29.58, 29.41, 29.36, 29.25, 28.81, 28.63, 23.61, 22.69, 14.11. Example 9: Synthesis of a combination of 6-methoxy-5-methyl-4-oxo-4,5-dihydro-l,3,5-triazyn-2-yl stearate (17) and 4-methoxy-l-methyl-6-oxo-l,6-dihydro-l,3,5-triazin-2-yl stearate (18): ( ).

[0192] The combination of compounds (17) and (18) was synthesised from stearic acid and 2-chloro-4,6- dimethoxy-l,3,5-triazine following the general procedure described in Example 1. The amounts of compounds (17) and (18) may vary over time due to interconversion between the two isomeric structures.

[0193] XH NMR (400 MHz, CDCI3): 64.10 (s), 4.05 (s), 3.35 (s), 3.34 (s), 2.85 (m), 1.78-1.67 (m), 1.43-1.19 (m), 0.88 (m).

[0194] Example 10: Synthesis of a combination of 4,6-dimethoxy-l,3,5-triazyn-2-yl stearate (16) and 4,6- dimethoxy-l-stearoyl-l,3,5-triazin-2-one (19):

[0195] The combination of compounds (16) and (19) was synthesised from stearic acid and 2-chloro-4,6- dimethoxy-l,3,5-triazine following the general procedure described in Example 1. The amounts of compounds (16) and (19) may vary over time due to interconversion between the two isomeric structures.

[0196] XH NMR (500 MHz, CDCI3) 6 4.06 (s), 4.08 (s), 4.02 (s), 2.88 (t), 2.60 (t), 1.77-1.68 (m), 1.73 (m), 1.43-1.20 (m), 0.88 (t). Example 11: Synthesis of p-D-glucopyranosyl-(l-> )- / V-stearoyl-D-eryt / jro-sphingosine (20) p-D-glucopyranosyl-(l^l')-D-erythro-sphingosine (1 eq) was dissolved in methanol, the triazine-based acylating agent of formula (13) (1 eq), or the triazine-based acylating agent of formula (14) (1 eq), or the combination of the triazine-based acylating agents of formulas (13) and (16) (1 eq), or the combination of the triazine-based acylating agents of formulas (14) and (15) (1 eq) was added. The resulting suspension was stirred at about 50 °C until a TLC-analysis showed complete consumption of the starting material. Water was added, and the suspension was subsequently heated to reflux, and then cooled down the to a temperature between about 5 °C to about 10 °C. A solid precipitated, which was filtered, washed with a methanol / water (5 / 1) mixture, and dried in vacuum to obtain the final product.

[0197] MS spectrum: [M+H]+728, [M+Na]+750

Claims

ClaimsMethod of producing a compound of formula (1):from a compound of formula (2):NH2R2Gaipi-GIcpi— O X X<^R1OR3(2), whereinR1is hydrogen, 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 Ci- 3 alkyl, the bond - may be a double or a single bond when R2is H, or is a single bond when R2is -OR5,R3is H or a substituted or unsubstituted C2-4 alkyl,R4is hydrogen or -C(=O)R6, wherein R6is a C15-C33 alkyl,Glc is a glucosyl moiety,Gal is galactosyl moiety, wherein the method comprises a step of reacting the compound of formula (2) with an enzyme having p-galactosidase activity.

2. The method according to claim 1, wherein the enzyme with p-galactosidase activity is from Aspergillus oryzae or Escherichia coll.

3. The method according to claims 1 or 2, wherein the step of reacting the compound of formula (2) with the enzyme having p-galactosidase activity is performed in the presence of a cyclodextrin.

4. The method according to any one of claims 1 to 3, wherein the cyclodextrin is p-cyclodextrin.

5. The method according to any one of claims 1 to 4, wherein for the compounds of formula (1) and(2) R1is -C13H27, R2and R3are hydrogen, and the bond - is a double bond.

6. The method according to any one of claims 1 to 5, wherein the compound of formula (1) is a compound of formula (3), or a salt thereof:G Icp(3), whereinR1, R2, R3and Glc are defined as for the compound of formula (1).

7. The method according to any one of claims 1 to 5, wherein the compound of formula (1) is a compound of formula (4):G Icp(4), whereinR1, R2, R3, R6and Glc are defined as for the compound of formula (1), and wherein the method comprising the steps of:- reacting the compound of formula (2) with an enzyme having p-galactosidase activity, thereby obtaining a compound of formula (3), or a salt thereof:NH2R2G Icp O^^AAA^ R1OR3(3), whereinR1, R2, R3and Glc are defined as for the compound of formula (1),- acylating the compound of formula (3) thereby obtaining the compound of formula (4).

8. The method according to claims 7, wherein the step of acylating the compound of formula (3) is performed: a) by the addition of an ester of formula (5):(5), whereinR6is a is a C15-C33 alkyl,R7is selected from a C1-C4 alkyl, preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl, or ethyl; in the presence of a base, or b) by the addition of a 1,3,5-triazine-based acylating agent of formula (6), or a combination thereof:whereinrepresents a conjugated system of bonds such that either two or three double bonds are present in the ring;R8is selected from C(O-C(=O)R6), or C(=O);Zais selected from N, NR10, or N(C(=O)R6);Zbis selected from N, or NR10;R9is selected from C(=O), or C(OR10); and provided that:when R8is C(O-C(=O)R6), R9is C(OR10), Zaand Zbare N, and three double bonds are present in the ring, or when R8is C(O-C(=O)R6), R9is C(=O), one of Zaand Zbis N and the other group is NR10, and two double bonds are present in the ring, or when R8is C(=O), R9is C(OR10), Zais N(C(=O)R6), Zbis N, and two double bonds are present in the ring; and whereinR6is a substituted or unsubstituted C15-C33 alkyl,R10is selected from methyl, ethyl, 2,2,2-trifluoroethyl, and substituted or unsubstituted benzyl.

9. The method according to any one of claims 1 to 8, wherein the method further comprises a step of isolating the compound of formula (1), (3), or (4).

10. The method according to any one of claims 6 to 9, wherein the compound of formula (3) is produced in vitro.

11. The method according to claim 10, wherein the compound of formula (3) is produced in vivo in a genetically modified microorganism.

12. The method according to claim 11, wherein the genetically modified microorganism is a bacterial cell or a yeast cell.

13. The method according to any one of claims 10 to 12, wherein the microorganism is capable of internalizing the compound of formula (2).

14. The method according to any one of claims 1 or 13, further comprising a step of producing the compound of formula (2) by reacting a glycosyl donor of formula (7):J-B(7), whereinJ is Gaipi-GIcl-, wherein Glc is a glucosyl moiety, Gal is a galactosyl,B is selected from a fluoride, chloride, bromide, azide, formate, iodide, with a sphingolipid acceptor of formula (8), or a salt thereof:(8), whereinR1is hydrogen, 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 Ci- 3 alkyl, the bond - may be a double or a single bond when R2is H, or is a single bond when R2is -OR5,R3is H or a substituted or unsubstituted C2-4 alkyl, in the presence of a glycosynthase.

15. The method according to claim 14, wherein the glycosynthase is an endoglycoceramide synthase.

16. The method according to claims 14 or 15, wherein the glycosynthase comprises an amino acid motif of formula (9):Xx-X2-X3-X4-X5-X60), whereinX1is an amino acid residue selected from I, M, L, V, A, F or W;X2is an amino acid residue selected from L, M, I, V or A;X3is an amino acid residue A, L or M;X4is an amino acid residue selected from G, A, S, N, Q, C, T, I, V, L or M;X5is an amino acid residue selected from F, T, M, L or S;X6is an amino acid residue selected from G, L or F.

17. The method according to any one of claims 14 to 16, wherein the glycosynthase is any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a functional analogue of any of them.

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