Sialylation of glycosphingolipids

Abstract

The present invention relates to a method for producing a sialylated glycosphingolipid, comprising the steps of:-providing a glycosphingolipid, a sialic acid donor and an enzyme having transsialidase activity,-mixing the glycosphingolipid with the sialic acid donor in the presence of the enzyme having transsialidase activity, the sialylated glycosphingolipid is a sialylated glycosphingolipid, and wherein the sialic acid donor is 3 '-sialylated lactose.

Description

Technical Field

[0001] This invention relates to a novel and efficient method for sialylation of glycosphingolipids. Background Technology

[0002] Glycosphingolipids (GSLs) are glycoconjugates in which the sugar chain is linked via a glycosidic bond to a 1-hydroxy group of a ceramide or sphingoid base.

[0003] GSLs participate in a variety of biological processes and play important structural and functional roles such as cell recognition, communication, and cell adhesion. In particular, sialylated glycosphingolipids, such as gangliosides, are found in the brain and may play a role in neurological diseases, especially Alzheimer's, Parkinson's, and Huntington's diseases. Furthermore, some gangliosides are found in the intestinal mucosa, promoting gut health and acting as anti-infective agents.

[0004] Sialidized glycosphingolipids, such as gangliosides, hold great potential as therapeutic agents and food ingredients; however, they are difficult to obtain for use in basic and clinical research. In fact, their high structural complexity makes their preparation extremely challenging.

[0005] There are methods for preparing sialylated glycosphingolipids based on extraction from natural sources, chemical synthesis, and / or enzymatic synthesis.

[0006] Sialinated glycosphingolipids can be extracted from animal brain or epidermal tissue (EP 3095451 A1, US5532141 A). However, the extraction and isolation of sialylated glycosphingolipids from animal sources is a time-consuming, labor-intensive, and costly process, often yielding small quantities of the desired compound with low purity. Furthermore, the resulting sphingolipids may pose potential safety risks due to the possible presence of harmful biological contaminants.

[0007] Alternatively, sialylated glycosphingolipids can be obtained via chemical synthesis (JA Morales-Serna, Carbohydr. Res. 2007), typically by first synthesizing the glycan moiety and then coupling it with a ceramide or sphingosine base. Disadvantages of this method include difficulty in controlling stereochemistry and regiochemistry, the need for multiple protecting group operations, and challenges in purification and scale-up.

[0008] The enzymatic synthesis of sialylated glycosphingolipids has also been described, involving the use of sialyltransferase to transfer sialic acid from CMP-sialic acid to glycosphingolipid receptors (Yu H. et al.). Org Biomol Chem(2018;16, 4076-4080; WO 9928491). Disadvantages of this method include the use of expensive nucleotide donors and / or reagents, and low conversion rates.

[0009] Therefore, there is a need to develop novel methods that are technically feasible and low-cost in order to achieve efficient and large-scale production of sialylated glycosphingolipids. Summary of the Invention

[0010] The first aspect of this invention relates to a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, as well as enzymes with trans-sialidase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. in Glycosphingolipids are compounds of formula (1): in W is a glycosyl moiety consisting of Galβ1- or having one or more terminal β-galactopyranyl units. R 1 It is hydrogen, aryl, or substituted or unsubstituted C 1-50 Alkyl groups, preferably substituted or unsubstituted C4 groups 1-17 Alkyl groups, more preferably substituted or unsubstituted C4 groups. 10-17 alkyl, R 2 For hydrogen or -OR 5 , where R 5 Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 2-6 Acyl group, preferably R 5 It is hydrogen. key In R 2 When it is hydrogen, it can be a double bond or a single bond, or in R 2 For -OR 5 It is a single key. R 3 Hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 1-6 Acyl group, preferably hydrogen, R 4 Selected from hydrogen, substituted or unsubstituted aryl, heteroalkyl, substituted or unsubstituted C 2-32 Acyl group, And the sialic acid donor is 3'-sialyl lactose. Attached Figure Description

[0011] Figure 1 A schematic diagram of the sialyltransferase cycle is shown, in which CMP-Neu5Ac is generated / regenerated.

[0012] Figure 2 A schematic diagram of the sialyltransferase cycle is shown, in which CMP-Neu5Ac is generated / regenerated and ATP is regenerated. Detailed Implementation

[0013] Surprisingly, the inventors have discovered that sialylated glycosphingolipids can be prepared in vitro using readily available and inexpensive 3'-sialyllactose as a sialic acid donor via sialylation catalyzed by transsialylase of a glycosphingolipid receptor, wherein the glycosphingolipid receptor is preferably obtained via synthetic and / or biotechnological methods. The transsialylase-catalyzed sialylation can be carried out in the presence of β-galactosidase and may include a nanofiltration step. Furthermore, a further sialylation step catalyzed by sialate transferase can be performed after the transsialylase-catalyzed sialylation, wherein an expensive nucleotide donor is generated in situ and regenerated in a sialate transferase cycle. This method is characterized by high yield, high selectivity, and high purity of the desired product. Therefore, this method is suitable for large-scale production of sialylated glycosphingolipids, such as gangliosides, and includes the following steps: - Provides glycosphingolipid and sialic acid donors, as well as enzymes with transsialylase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. in Glycosphingolipids are compounds of formula (1): in W is a glycosyl moiety consisting of Galβ1- or having one or more terminal β-galactopyranyl units. R 1 It is hydrogen, aryl, or substituted or unsubstituted C 1-50 Alkyl groups, preferably substituted or unsubstituted C4 groups 1-17 Alkyl groups, more preferably substituted or unsubstituted C4 groups. 10-17 alkyl, R 2 For hydrogen or -OR 5 , where R 5 Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 2-6 Acyl group, preferably R 5 It is hydrogen. key In R 2 When it is hydrogen, it can be a double bond or a single bond, or in R 2 For -OR 5 It is a single key. R 3 Hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 1-6 Acyl group, preferably hydrogen, R 4 Selected from hydrogen, substituted or unsubstituted aryl, heteroalkyl, substituted or unsubstituted C 2-32 Acyl group, And the sialic acid donor is 3'-sialyl lactose.

[0014] The following describes non-limiting embodiments of different aspects of the present invention and illustrates them by way of non-limiting examples.

[0015] The terms, definitions, and implementation methods described throughout this specification relate to all aspects and implementations of the invention.

[0016] Grammatically, the term "a" is singular, but it can also refer to the plural, such as to the intended compound. For example, a person skilled in the art will understand that the expression "glycosphingolipid" does not refer to providing only one type of glycosphingolipid, but rather to providing a variety of similar glycosphingolipids.

[0017] As used herein, the term "alkyl" refers to a non-cyclic straight-chain or branched hydrocarbon group having 1 to 50 carbon atoms, which may be saturated or contain one or more double and / or triple bonds (e.g. to form an alkenyl or alkynyl group), and / or may be substituted or unsubstituted, as further described herein. Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neopentyl, n-hexyl, vinyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, methylpentenyl, dimethylbutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, pentyynyl, and hexynyl, each of which may be substituted or unsubstituted. Generally, the term alkyl refers to a straight-chain saturated acyclic hydrocarbon group having 1-31 carbon atoms, which may be substituted or unsubstituted.

[0018] As used herein, the term "aryl" refers to an aromatic cyclic hydrocarbon group having 5-14 ring carbon atoms, which may be monocyclic or polycyclic, may contain fused rings, preferably 1 to 3 fused or non-fused rings, and may contain one or more heteroatoms, and / or may be substituted or unsubstituted, as further described herein. Examples of "aryl" include, but are not limited to, phenyl, naphthyl, anthracene, phenantryl, pyrrole, imidazolyl, thiophene, furanyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazine, and benzofuranyl, each of which may be substituted or unsubstituted. Generally, the term "aryl" refers to a substituted or unsubstituted phenyl group.

[0019] As used herein, the term "acyl" refers to a group derived by removing one or more hydroxyl groups from an oxyacid (preferably a carboxylic acid). Acyl groups according to the invention are typically saturated or unsaturated C4 groups. 2-32 Acyl groups, which can be substituted or unsubstituted.

[0020] As used herein, the term "substitution" means replacing a group with a group that typically alters the general chemical properties of the group. Substituents can be used to modify molecular properties such as molecular stability, molecular solubility, and molecular crystallinity. Those skilled in the art will recognize other suitable substituents with similar size and charge properties that can be used as alternative substituents in a given situation.

[0021] With regard to the terms "alkyl", "aryl", and "acyl", the term substitution means that the group is selected from hydroxyl (which, when attached to an unsaturated carbon atom, may exist as a tautomer ketone), oxo, C... 1-6 -alkoxy group (i.e., C) 1-6 -alkyl-oxygen), C 2-6 -Alkenyl group, carboxyl group, oxo group, C 1-6 -alkoxycarbonyl, C 1-6 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy group, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy group, heteroarylcarbonyl, amino, mono- and di(C 1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl)aminocarbonyl, amino-C 1-6 -alkyl-aminocarbonyl, mono- and di(C 1-6 -alkyl)amino-C 1-6 -alkyl-aminocarbonyl, C 1-6 -alkylcarbonylamino, cyano, guanidinyl, urea (carbamido), C 1-6 -alkyl-sulfonyl-amino, aryl-sulfonyl-amino, heteroaryl-sulfonyl-amino, C 1-6 -alkyl acyloxy, C 1-6-alkyl-sulfonyl, C 1-6 -alkyl-sulfinyl group, C 1-6 -alkylsulfonyloxy, nitro, C 1-6 -The alkyl thio or halogen group is substituted once or multiple times, preferably 1 to 3 times, wherein any alkyl, alkoxy, or other representative substituent can be replaced by a hydroxyl, C 1-6 -alkoxy group, C 2-6 -Alkenyl group, carboxyl group, C 1-6 -alkylcarbonylamino, halogen, C 1-6 -alkylthio, C 1-6 -alkyl-sulfonyl-amino or guanidine-substituted.

[0022] With regard to the term "alkyl", the term "substitution" preferably means that the group is substituted once or more, preferably once to three times, by a group selected from hydroxyl, alkoxy, acyloxy, amide, thiol, thioether or phosphorus-containing functional groups.

[0023] With regard to the term enzyme, the term "functional analog" refers to a protein whose amino acid sequence has a certain percentage of homology with that of a reference protein (i.e., when performing maximum correspondence comparisons and alignments within a comparison window or designated region, it has approximately 30% homology in a specific region, such as in 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, preferably about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology), and maintains the same functional activity as the reference protein or polypeptide. The percentage of homology can be determined using, for example, the BLAST sequence comparison algorithm or by manual alignment and visual inspection (see, for example, the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / etc.). Such sequences can be described as "substantially identical". Generally, the term functional analog refers to a mutant protein, a truncated variant of a protein, a recombinant protein, or a fusion protein that retains the same functional activity as the reference protein.

[0024] The amino acid sequences in this paper are usually represented by commonly used single-letter codes or their three-letter codes, as summarized in Table 1.

[0025] Table 1 Amino acid codes: Those skilled in the art will understand that in the chemical formulas showing a particular compound, such as formulas (1), (2) and (3), unless the chemical formula explicitly describes a carbon atom having a particular stereochemical configuration, the chemical formula is intended to cover compounds in which the stereocenter has an R or S configuration or in which the double bond has a cis or trans configuration.

[0026] Those skilled in the art will understand that when referring to positions such as C-1, C-2, C-3, C-4, C-5, etc., this text always refers to the corresponding carbon atom in glycosphingolipids (such as those shown in formulas (1), (2) and (3)).

[0027] In the context of this invention, the terms “about,” “approximately,” or “around” are used interchangeably to indicate a specific value (e.g., “pH is about 4.5,” “pH is approximately 4.5,” or “pH is around 4.5”) or range (e.g., “amount from about 1% to about 99%,” “amount from approximately 1% to approximately 99%,” or “amount from approximately 1% to approximately 99%)”) to indicate a deviation from that specific value or range of 0.1% to 10%.

[0028] In the context of this invention, the term "separation" refers to a procedural step or procedure for separating a desired compound from a mixture containing other compounds. In this context, other compounds in the mixture are considered contaminants. The terms "separation" and "isolation" are used interchangeably.

[0029] As used herein, the term "glycosphingolipid" refers to a compound or analogue that is structurally composed of a glycosyl moiety and a sphingolipid moiety. The glycosyl moiety is typically linked to the sphingolipid moiety via a glycosidic bond between the terminal carbon at the reduced end of the glycosyl moiety and the hydroxyl group at the C-1 position of the sphingolipid.

[0030] According to the present invention, the glycosyl moiety of a glycosphingolipid can be derived from a monosaccharide or an oligosaccharide (more than one monosaccharide unit), wherein the terminal carbon of the monosaccharide or the terminal carbon at the reducing end of the oligosaccharide is linked to another chemical entity (e.g., a sphingolipid) by a glycosidic bond, and, unless otherwise stated, the bond can be an α or β glycosidic bond. The glycosyl moiety having more than one monosaccharide unit can exhibit a linear or branched structure.

[0031] The monosaccharide unit is preferably any sugar with 5-9 carbon atoms, including aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxyglucoses (e.g., L-rhamnose, L-fucose, etc.), deoxyaminoglycans (e.g., N-acetylglucosamine, N-acetymannosamine, N-acetylgalactosamine, etc.), uronic acids, and keturonic 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. In some embodiments, the glycosyl moiety is derived from a monosaccharide, wherein the monosaccharide is a β-galactoside. In some embodiments, the glycosyl moiety is derived from an oligosaccharide, wherein the oligosaccharide has one or more terminal β-galactopyranosyl units.

[0032] The glycosyl portion according to the invention can be shown as Galβ1-4Glc1-, wherein the short horizontal line (-) indicates the connection point of the glycosyl portion, and wherein the glycosyl portion can be connected via α or β glycosidic bonds, preferably via β glycosidic bonds.

[0033] The sphingolipid portion of the glycosphingolipids of this invention is typically derived from aliphatic amino alcohols, such as sphingosine bases or ceramides.

[0034] In the context of this invention, sphingosine bases refer to naturally occurring sphingosine bases, their analogues, or their derivatives.

[0035] Naturally occurring sphingosine bases are D- red style Sphingosine (S), 6-hydroxy-D- red style Sphingosine (H), D-ribose-phytosphingosine (P), or DL- red style Dihydrosphingosine (DS), where the number of carbon atoms in sphingosine can be indicated in parentheses after the letters S, H, P, and DS.

[0036] The letters S, H, P, and DS refer to those developed by Motta et al. (1993) Biochim Biophys Acta. 1182:147-151, and by Rabionet (2014) Biochim Biophys Acta. 1841:422-434 and Masukawa et al. Journal of Lipid Research , 2008, 49, 1466-1476 Extended abbreviation naming convention. According to INCI nomenclature, D- red style -Dihydrosphingosine can also be represented by the letter G.

[0037] In the context of this invention, ceramide refers to naturally occurring ceramides, their analogues, or derivatives thereof. Preferred ceramides are those naturally occurring in the human body. 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 parentheses refer to those developed by Motta et al. (1993) Biochim Biophys Acta. 1182:147-151 and by Rabionet (2014) Biochim Biophys Acta. 1841:422-434 and Masukawa et al. Journal of Lipid Research , 2008, 49, 1466-1476 Extended abbreviation nomenclature. Specifically, the letters N, A, and EO represent non-hydroxy fatty acids (N), α-hydroxy fatty acids (A), and ω-linolenic acid fatty acids (EO), respectively, where the number of carbon atoms and the degree of unsaturation of the fatty acid can be indicated in parentheses after the letters N, A, E, and O. The letters S, H, P, and DS represent D- red style Sphingosine (S), 6-hydroxy-D- red style Sphingosine (H), D-ribose-phytosphingosine (P), D- red style Dihydrosphingosine (DS), where the number of sphingosine carbons can be indicated in parentheses after the letters S, H, P, and DS. Ceramides, CER[NDS], CER[ADS], or CER[EODS], can also be called CER[NG], CER[AG], or CER[EOG], where the letter G represents D- red style The INCI name for dihydrosphingosine.

[0038] Glycosphingolipids lacking an amide-linked fatty acyl group can also be called lysosphingolipids.

[0039] The glycosphingolipids according to the present invention are generally represented by glycosphingolipids of formula (1): in W is a glycosyl moiety consisting of Galβ1- or having one or more terminal β-galactopyranyl units. R 1 It is hydrogen, aryl, or substituted or unsubstituted C 1-50 Alkyl groups, preferably substituted or unsubstituted C4 groups 1-17 Alkyl groups, more preferably substituted or unsubstituted C4 groups. 10-17 alkyl, R2 For hydrogen or -OR 5 , where R 5 Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 2-6 Acyl group, preferably R 5 It is hydrogen. key In R 2 When it is hydrogen, it can be a double bond or a single bond, or in R 2 For -OR 5 It is a single key. R 3 Hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 1-6 Acyl group, preferably hydrogen, R 4 Selected from hydrogen, substituted or unsubstituted aryl, heteroalkyl, substituted or unsubstituted C 2-32 Acyl group, In some embodiments, for the glycosphingolipids of formula (1), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0040] In some embodiments, for the glycosphingolipids of formula (1), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 OR 5 , where R 5 For hydrogen, R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0041] In some embodiments, for the glycosphingolipids of formula (1), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0042] In some embodiments, for the glycosphingolipids of formula (1), R 1 C 10 -C 17 1-Hydroxyalkyl, R 2R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0043] In some embodiments, the glycosphingolipid of formula (1) is a glycosphingolipid selected from those of formulas (3), (4), (5), and (6): .

[0044] In some embodiments, for the glycosphingolipids of formula (1), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a double bond.

[0045] In some embodiments, for the glycosphingolipids of formula (1), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 OR 5 , where R 5 For hydrogen, R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0046] In some embodiments, for the glycosphingolipids of formula (1), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0047] In some embodiments, for the glycosphingolipids of formula (1), R 1 C 10 -C 17 1-Hydroxyalkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a double bond.

[0048] In some embodiments, the glycosphingolipid of formula (1) is a glycosphingolipid selected from those of formulas (7), (8), (9), and (10): .

[0049] In some embodiments, the W of the glycosphingolipids of formulas (1) and (3)-(10) is a glycosyl moiety selected from the following glycosyl moieties, or a salt thereof: In some embodiments, the W of the glycosphingolipids of formula (1) and formulas (3)-(10) is a glycosyl moiety selected from the following glycosyl moieties, or a salt thereof: In some embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (3), and wherein the W of the glycosphingolipid of formula (3) is Galβ1-. Therefore, in some embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (3), and wherein the glycosphingolipid of formula (3) is sphingosine galactoside.

[0050] In some preferred embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (3), and wherein the W of the glycosphingolipid of formula (3) is Galβ1-4Glcβ1-. Therefore, in some preferred embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (3), and wherein the glycosphingolipid of formula (3) is lactosyl D- red style Sphingosine.

[0051] In some embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (3), and wherein the W of the glycosphingolipid of formula (3) is Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-. Therefore, in some embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (3), and wherein the glycosphingolipid of formula (3) is... N -lyso-GM1a.

[0052] In some embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (7), and wherein the W of the glycosphingolipid of formula (7) is Galβ1-.

[0053] In some embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (7), and wherein the W of the glycosphingolipid of formula (7) is Galβ1-4Glcβ1-.

[0054] In some preferred embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (7), and wherein the W of the glycosphingolipid of formula (7) is Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-. Therefore, in some preferred embodiments, the glycosphingolipid of formula (1) is the glycosphingolipid of formula (7), and wherein the glycosphingolipid of formula (7) is GM1a.

[0055] The glycosphingolipids according to the present invention can be produced by methods known to those skilled in the art. Methods for synthesizing glycosphingolipids are described, for example, in WO2023118378A1 or by Vaughan et al. J. Am. Chem . Soc 2006, 128, 6300-6301, wherein glycosyl fluorides (e.g., lactose-based fluorides) are coupled to sphingolipids using endoglycoceramidase glycosynthases (EGCase). Glycosphingolipids with complex oligosaccharide moieties can be produced by biotechnological methods, such as those described in WO 2021170620 (A1).

[0056] As used herein, the term "sialic acid donor" refers to a compound containing a sialic acid unit that can be transferred to a suitable receptor (e.g., glycosphingolipid). Sialic acid donors in the context of this invention are typically α-sialylated compounds that can be derived from natural sources or chemically synthesized. Naturally derived α-sialylated compounds include, for example, 3'-sialyl lactose, sialic acid-rich proteins, and colominic acid. Chemically synthesized α-sialylated compounds include, but are not limited to, p-nitrophenyl... N - Acetylneuraminic acid (Neu5AcαpNP), methylumbelliferyl N - Acetylneuraminic acid (Neu5AcαMU) and its derivatives.

[0057] In a preferred embodiment, the sialic acid donor is 3'-sialyl lactose.

[0058] The term "enzyme with transsialyzed activity" is used interchangeably with the term "transsialyzase," and in the context of this invention, refers to enzymes belonging to the glycosidic hydrolase family 33 (GH33) that typically catalyze the reversible transfer of glycosidically linked sialic acid from sialic acid donors (e.g., oligosaccharides, glycoproteins, glycolipids, and polyacetylneuraminic acid) to acceptor molecules containing terminal β-galactopyranoside units. In the absence of suitable acceptor molecules, these enzymes can act as sialylases, transferring glycosidically linked sialic acid to water molecules. However, their hydrolytic activity is generally low.

[0059] Wild-type transsialyzedase can be derived from parasitic euglena, such as Trypanosoma cruzi (…). Trypanosoma cruzi Congo trypanosome ( Trypanosoma congolense ), or Trypanosoma brucei ( Trypanosome brucei ).

[0060] Wild-type transsialyzase can be derived from microorganisms with a vector that has been linked to or introduced with a gene encoding wild-type transsialyzase.

[0061] Wild-type transsialotases can originate from any known transsialotase sequence or from any unidentified transsialotase sequence. Unidentified transsialotases can be identified using sequence databases and sequence alignment algorithms, such as the publicly available GenBank database and the BLAST alignment algorithm.

[0062] In some embodiments, the enzyme having transsialyzed activity is a wild-type transsialyzed enzyme derived from Trypanosoma cruzi, Trypanosoma congo, or Trypanosoma brevicornu, or a functional analog thereof.

[0063] In some embodiments, the enzyme possessing transsialyzedase activity is a wild-type transsialyzedase derived from Trypanosoma cruzi. The amino acid sequence of the wild-type transsialyzedase derived from Trypanosoma cruzi can be found in... https: / / www.uniprot.org / Found it online, login number: Q26966.

[0064] Transsialidase derived from Trypanosoma cruzi can also be called TcTS.

[0065] In some embodiments, the enzyme having transsialidase activity is a mutant of wild-type transsialidase (Q26966) derived from Trypanosoma cruzi.

[0066] In some embodiments, the mutant transsialidase has at least five mutations at amino acid positions selected from the following (numbered corresponding to the amino acid sequence alignment with the Q26966 amino acid sequence): S263T, R477H, V485L, E559V, N59F, S496K, V497G, E521K, D594G, I598D, and H600R.

[0067] In some embodiments, the mutant transsialidase has mutations at the following amino acid positions (numbered to correspond to the amino acid sequence alignment with Q26966): S263T, R477H, V485L, E559V, N59F, S496K, V497G, E521K, D594G, I598D, and H600R.

[0068] In some embodiments, the mutant transsialidase has mutations at the following amino acid positions (numbered corresponding to the amino acid sequence alignment with Q26966): N59F, S496K, V497G, E521K, D594G, I598D, and H600R, as described by Amaya et al. Structure As described in 2004, 12, 775-784.

[0069] In some embodiments, the mutant transsialidase has mutations at the following amino acid positions (numbered to correspond to the amino acid sequence alignment with Q26966): N59F, V497G, S496K, E521K, and E559V.

[0070] In some embodiments, the mutant transsialidase has mutations at the following amino acid positions (numbered to correspond to the amino acid sequence alignment with Q26966): S263T, R477H, V485L, E559V, and S496K.

[0071] In some embodiments, the mutant transsialidase has mutations at the following amino acid positions (numbered to correspond to the amino acid sequence alignment with Q26966): S496K, V497G, D594G, I598D, and H600R.

[0072] In some embodiments, the mutant transsialidase also includes the deletion of the N-terminal amino acid M1 (numbered to correspond to the amino acid sequence alignment with the Q26966 amino acid sequence) and the insertion of the N-terminal 14 amino acid histidine tag MGGSHHHHHGMAS.

[0073] In some embodiments, mutant transsialidase also includes the deletion of the C-terminal amino acid. 636-642 (numbered to correspond to the alignment of amino acid sequence with amino acid sequence Q26966).

[0074] In some implementations, the amino acid sequence of the mutant transsialidase corresponds to the World Protein Database (WPD). https: / / www.rcsb.org / structure / 1MS0 The amino acid sequence ID NO: 1MSO, where the reference amino acid sequence contains the following mutations / modifications compared to wild type: S263T, R477H, V485L, E559V, N59F, S496K, V497G, E521K, D594G, I598D, H600R, N -Terminal His-tag, C-terminal deletion of 7 amino acids 636-642.

[0075] In some embodiments, the amino acid sequence of the mutant transsialidase comprises or consists of the amino acid sequence of SEQ ID NO: 1, wherein the mutant, compared to the wild-type amino acid sequence Q26966, contains the following mutations / modifications: S263T, R477H, V485L, E559V, S496K. N -Terminal His-tag, 7 amino acids missing 636-642.

[0076] The mutant transsialyzedase according to the present invention can be produced by methods known to those skilled in the art. Methods for expressing and purifying the mutant transsialyzedase are described, for example, by Paris et al. Glycobiology 2001, 11, 305-311 or Buschiazzo et al., Molecular Cell 2002, 10, 757-768.

[0077] The method according to the invention includes the step of mixing glycosphingolipids and sialic acid donors in the presence of an enzyme having transsialylase activity to produce sialylated glycosphingolipids.

[0078] The sialylated glycosphingolipids produced by the above method are usually represented by the sialylated glycosphingolipids of formula (2): in Y is a glycosyl moiety containing at least one sialic acid unit. R 1 R 2 R 3 R 4 s and keys As defined for glycosphingolipids in equation (1).

[0079] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0080] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 OR 5 , where R 5 For hydrogen, R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0081] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0082] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 C 10 -C 17 1-Hydroxyalkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0083] In some embodiments, the sialylated glycosphingolipid of formula (2) is a glycosphingolipid selected from those of formulas (11), (12), (13), and (14): .

[0084] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a double bond.

[0085] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 OR 5 , where R 5 For hydrogen, R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0086] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0087] In some embodiments, for the sialylated glycosphingolipid of formula (2), R 1 C 10 -C 17 1-Hydroxyalkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a double bond.

[0088] In some embodiments, the sialylated glycosphingolipid of formula (2) is a sialylated glycosphingolipid selected from formulas (15), (16), (17), and (18): .

[0089] In some embodiments, the Y of the sialylated glycosphingolipids of formulas (2) and (11)-(18) is a glycosyl moiety selected from the following glycosyl moieties, or a salt thereof: In some embodiments, the Y in the sialylated glycosphingolipids of formula (2) and the sialylated glycosphingolipids of formulas (11)-(18) is a glycosyl moiety selected from the following glycosyl moieties, or a salt thereof: In some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (11), and wherein the Y of the sialylated glycosphingolipid of formula (11) is Neu5Acα2-3Galβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (11), and wherein the sialylated glycosphingolipid of formula (11) is... N -lyso-GM4.

[0090] In some preferred embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (11), and wherein the Y of the sialylated glycosphingolipid of formula (11) is Neu5Acα2-3Galβ1-4Glcβ1-. Therefore, in some preferred embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (11), and wherein the sialylated glycosphingolipid of formula (11) is... N -lyso-GM3.

[0091] In some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (11), and wherein the Y of the sialylated glycosphingolipid of formula (11) is Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (11), and wherein the sialylated glycosphingolipid of formula (11) is... N -lyso-GD1a.

[0092] In some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (15), and wherein the Y of the sialylated glycosphingolipid of formula (15) is Neu5Acα2-3Galβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (15), and wherein the sialylated glycosphingolipid of formula (15) is GM4.

[0093] In some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (15), and wherein the Y of the sialylated glycosphingolipid of formula (15) is Neu5Acα2-3Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (15), and wherein the sialylated glycosphingolipid of formula (15) is GM3.

[0094] In some preferred embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (15), and wherein the Y of the sialylated glycosphingolipid of formula (15) is Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-. Therefore, in some preferred embodiments, the sialylated glycosphingolipid of formula (2) is the sialylated glycosphingolipid of formula (15), and wherein the sialylated glycosphingolipid of formula (15) is GD1a.

[0095] In some embodiments, the method further includes the step of adding an enzyme having β-galactosidase activity.

[0096] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. Furthermore, the sialic acid donor is 3'-sialyllactose.

[0097] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme having transsialyzedase activity and the enzyme having β-galactosidase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. Furthermore, the sialic acid donor is 3'-sialyllactose.

[0098] The term “enzyme with β-galactosidase activity” may be used interchangeably with the term “β-galactosidase”, which in the context of this invention refers to an enzyme belonging to the glycoside hydrolase family 35 (GH35) that typically catalyzes the hydrolysis of the terminal non-reducing β-D-galactose residue in β-D-galactosides.

[0099] In the context of this invention, β-galactosidase may also be referred to as lactase.

[0100] Wild-type β-galactosidases can be derived from microorganisms such as bacteria, yeast, ascomycetes, actinomycetes, filamentous fungi, and basidiomycetes.

[0101] Wild-type β-galactosidase can be derived from Aspergillus oryzae ( Aspergillus oryzae ).

[0102] Wild-type β-galactosidase can be derived from microorganisms with a vector that has been linked to or introduced with a gene encoding wild-type β-galactosidase.

[0103] Wild-type β-galactosidases can originate from any known β-galactosidase sequence or from any unidentified β-galactosidase sequence. Unidentified β-galactosidase sequences can be identified using sequence databases and sequence alignment algorithms, such as the publicly available GenBank database and the BLAST alignment algorithm.

[0104] In some embodiments, the enzyme having β-galactosidase activity is a wild-type β-galactosidase derived from Aspergillus oryzae, or a functional analog thereof. The amino acid sequence of the wild-type β-galactosidase derived from Aspergillus oryzae can be found at https: / / www.uniprot.org / , accession number: Q2UCU3.

[0105] In some preferred embodiments, the enzyme having β-galactosidase activity is a truncated variant of wild-type β-galactosidase (Q2UCU3) derived from Aspergillus oryzae.

[0106] According to the present invention, the truncated variant of this β-galactosidase can be purchased from existing manufacturers (e.g., Calza Clemente) or produced by methods known to those skilled in the art, for example as described in MM Maksimainen et al., International Journal of Biological Macromolecules 2013, 60, 109-115.

[0107] The step of adding an enzyme with β-galactosidase activity can be advantageously used to hydrolyze lactose formed during the sialylation of glycosphingolipids in formulas (1) or (3)-(10) catalyzed by sialyltransferase. Depending on the sialic acid donor used, the sialylation catalyzed by transsialylferase may be a reversible process. When the sialic acid donor is 3'-sialylactose, lactose is formed during the transfer and can act as an acceptor for transsialylferase, thus reaching equilibrium. When β-galactosidase is used, the lactose formed during the transfer is hydrolyzed into galactose and glucose, which generally do not act as an acceptor for transsialylferase, thus allowing the reaction to tend towards completion.

[0108] In some embodiments, an enzyme with β-galactosidase activity is added after 3'-sialyl lactose has reached a certain conversion rate. Preferably, the step of adding the enzyme with β-galactosidase activity is performed when the conversion rate of 3'-sialyl lactose is at least about 50%; more preferably, the step of adding the enzyme with β-galactosidase activity is performed when the conversion rate of 3'-sialyl lactose reaches at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. The conversion rate of 3'-sialyl lactose can be determined by standard techniques known to those skilled in the art. Typically, the conversion rate of 3'-sialyl lactose is determined by HPLC and expressed as a molar percentage (mol.%) or weight percentage (%). wt .% is given.

[0109] In some embodiments, the sialylated glycosphingolipids according to the invention are produced in the presence of cyclodextrin.

[0110] The use of cyclodextrins offers several advantages, such as high yields and improved accessibility of the glycosyl moieties of glycosphingolipids without the need for detergents or organic solvents. However, detergents or organic solvents may also be used in the method of the present invention.

[0111] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialyzedase activity, and cyclodextrin. - In the presence of the enzyme with transsialyzedase activity and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. Furthermore, the sialic acid donor is 3'-sialyllactose.

[0112] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialylase activity, cyclodextrins, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. Furthermore, the sialic acid donor is 3'-sialyllactose.

[0113] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialylase activity, cyclodextrins, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the enzyme with β-galactosidase activity, and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. Furthermore, the sialic acid donor is 3'-sialyllactose.

[0114] In the context of this invention, the term "cyclodextrin" refers to a cyclic oligosaccharide composed of a macrocycle of monosaccharide subunits (e.g., glucose). Cyclodextrins typically contain 6, 7, or 8 monosaccharide subunits and may be referred to as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, respectively. Cyclodextrins can be modified by alkylating or acylating some or all of the primary or secondary hydroxyl groups (or both) of their macrocycle. Methods for modifying these alcohols are well known to those skilled in the art, and many derivatives are commercially available. Thus, some or all of the hydroxyl groups of a cyclodextrin can be -OR 6 Groups and / or OC(=O)-R 7 Group substitution, wherein R 6 and R 7 Independently selected from saturated or unsaturated C 1-6 Alkyl, saturated or unsaturated C 1-6Heteroalkyl, saturated or unsaturated cycloalkyl, saturated or unsaturated heterocycloalkyl, aryl or heteroaryl, each group may be substituted or unsubstituted. In some embodiments, R 6 and R 7 It is independently selected from 2-hydroxyethyl, 2-hydroxypropyl and sulfobutyl ether.

[0115] In some embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a derivative thereof.

[0116] In some embodiments, the cyclodextrin is selected from β-cyclodextrin, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin. In some preferred embodiments, the cyclodextrin is β-cyclodextrin.

[0117] Cyclodextrin is typically used in amounts from about 0.1 equivalents to about 1 equivalent based on the amount of glycosphingolipids. In some preferred embodiments, cyclodextrin is used in amounts from about 0.1 equivalents to about 0.5 equivalents based on the amount of glycosphingolipids. Therefore, in some preferred embodiments, cyclodextrin is used in amounts from about 0.1, 0.2, 0.3, 0.4, or 0.5 equivalents based on the amount of glycosphingolipids.

[0118] In some embodiments, the present invention describes a method for producing sialylated glycosphingolipids, wherein the method described herein further includes a nanofiltration step.

[0119] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. - Nanofiltration of the reaction mixture Furthermore, the sialic acid donor is 3'-sialyllactose.

[0120] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme having transsialyzedase activity and the enzyme having β-galactosidase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. - Nanofiltration of the reaction mixture Furthermore, the sialic acid donor is 3'-sialyllactose.

[0121] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialylase activity, cyclodextrins, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. - Nanofiltration of the reaction mixture Furthermore, the sialic acid donor is 3'-sialyllactose.

[0122] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialylase activity, enzymes with β-galactosidase activity, and cyclodextrin; - In the presence of the enzyme with transsialyzedase activity, the enzyme with β-galactosidase activity, and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. - Nanofiltration of the reaction mixture Furthermore, the sialic acid donor is 3'-sialyllactose.

[0123] Nanofiltration (NF) can be used to concentrate mixtures containing sialylated glycosphingolipids, remove ions (primarily monovalent ions), and / or remove organic materials (e.g., monosaccharides) with molecular weights lower than that of sialylated glycosphingolipids. In a preferred embodiment, the nanofiltration step is used to remove galactose and glucose from the mixture containing sialylated glycosphingolipids.

[0124] Typically, nanofiltration membranes have a molecular weight cutoff (MWCO) that ensures the retention of target sialylated glycosphingolipids. As an example, nanofiltration membranes with an MWCO of approximately 200-500 Da are suitable for retaining sialylated glycosphingolipids. In this case, sialylated glycosphingolipids accumulate in the NF-retentive (NFR). Nanofiltration can be combined with diafiltration (DF) using water to more effectively remove permeable molecules, for example, until the conductivity of the permeate indicates the absence or only very low levels of salt.

[0125] According to the invention, the NF step, whether or not the optional DF step is performed, is carried out at a constant temperature, preferably between about 15-45°C, more preferably between about 20-35°C. The NF step (with or without percolation) continues until the desired sialylated glycosphingolipid concentration is reached in the NFR. Other technical parameters, such as flow rate and pressure settings, are standard technical matters.

[0126] The sialylation method according to the present invention includes the step of mixing glycosphingolipids with a sialic acid donor in the presence of an enzyme having transsialylase activity. In some embodiments, the sialylation method according to the present invention further includes the step of adding β-galactosidase to the mixture of the sialic acid donor, glycosphingolipids, and transsialylase.

[0127] In some embodiments, sialylation is carried out in the presence of cyclodextrin.

[0128] Enzymes, substrates, and cyclodextrins in some embodiments can be added in any order, and it should be understood that the order of reactant combination can be adjusted as needed.

[0129] For example, a sialic acid donor can be added to a glycosphingolipid solution, followed by the addition of transsialylase. As another example, a sialic acid donor can be added to a glycosphingolipid solution, followed by the addition of transsialylase and β-galactosidase.

[0130] Sialic acid donors, glycosphingolipids, transsialidase and β-galactosidase, and any other components used in the sialylation reaction may be added to the reaction mixture as solids or dissolved in a solvent, and in any amount and manner effective for the desired results of the process.

[0131] The temperature range for the above process can range from slightly above freezing point to the temperature at which the most sensitive enzyme denaturation occurs. This temperature range is preferably from about 0°C to about 45°C, and more preferably from about 20°C to 37°C.

[0132] In the presence of an enzyme, and in some embodiments in the presence of cyclodextrin, glycosphingolipids and sialic acid donors are reacted for a reaction time sufficient to obtain the desired high yield of the desired sialylated glycosphingolipids.

[0133] Typically, the reaction can proceed for about 1 to about 24 hours, preferably about 5 to about 10 hours. In some embodiments, the reaction can proceed for about 5, 6, 7, 8, 9, or 10 hours.

[0134] Glycosphingolipids, enzymes, and, in some embodiments, cyclodextrins, can be mixed into an aqueous reaction medium. The pH of this medium is typically from about 5 to about 7.5. The choice of medium is based on its ability to maintain the desired pH level. Therefore, in some embodiments, the medium is buffered to a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some preferred embodiments, the medium is buffered to a pH of about 5.5 to 6.5. Therefore, in some preferred embodiments, the medium is buffered to a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0135] Suitable buffers include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, MOPS, HEPES, PBS, sodium acetate buffer, and sodium citrate buffer. Sodium acetate buffer is preferred. If no buffer is used, the pH of the medium should be maintained at approximately 5 to approximately 7.5 using a base or acid. A suitable base is NaOH, and a suitable acid is HCl.

[0136] Sialidized glycosphingolipids prepared by the above methods can be used without purification. However, sialylated glycosphingolipids can be purified through a separation step.

[0137] Therefore, in some embodiments, the present invention describes a method for producing sialylated glycosphingolipids, wherein the method further includes the step of separating sialylated glycosphingolipids from the reaction mixture.

[0138] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. - Perform nanofiltration on the mixture from the preceding steps. - Separate sialylated glycosphingolipids; Furthermore, the sialic acid donor is 3'-sialyllactose.

[0139] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme having transsialyzedase activity and the enzyme having β-galactosidase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. - Perform nanofiltration on the mixture from the preceding steps. - Separate the sialylated glycosphingolipids produced in the aforementioned steps. Furthermore, the sialic acid donor is 3'-sialyllactose.

[0140] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialylase activity, cyclodextrins, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the enzyme with β-galactosidase activity, and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid. - Perform nanofiltration on the mixture from the preceding steps. - Separate the sialylated glycosphingolipids produced in the aforementioned steps.

[0141] Furthermore, the sialic acid donor is 3'-sialyllactose.

[0142] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipids, sialic acid donors, enzymes with transsialylase activity, cyclodextrins, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity and the cyclodextrin, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. - Perform nanofiltration on the mixture from the preceding steps. - Separate the sialylated glycosphingolipids produced in the aforementioned steps.

[0143] Furthermore, the sialic acid donor is 3'-sialyllactose.

[0144] The separation of sialylated glycosphingolipids can be performed using standard methods known to those skilled in the art, such as organic solvent extraction, chromatography, and / or ion exchange chromatography.

[0145] Preferred methods for separation include percolation (DF) of the reaction mixture, where DF is used to remove ions (primarily monovalent ions) and / or organic material (e.g., proteins and organic molecules). In a preferred embodiment, percolation is performed using a membrane with a molecular weight displacement (MWCO) of about 100-300 kDa, preferably about 200-300 kDa. In some embodiments, the MWCO of the percolation membrane is 100-150 kDa, 150-200 kDa, 200-250 kDa, or 250-300 kDa.

[0146] It is noteworthy that even though the molecular weight (MWCO) of 100-300 kDa is much higher than that of sialylated glycosphingolipids, sialylated glycosphingolipids still accumulate in the DF retrieval fluid (DFR). Ganglioside GM1 has been described as forming micellar aggregates in aqueous solutions with molecular weights between approximately 250 kDa and 450 kDa, where the size of the micellar aggregates depends on the length of the fatty acid chains in the constituent molecules (DB Gammak, Biochem J 1963, 88, 373). This property can prevent gangliosides (e.g., GM1) from permeating ultrafiltration membranes with a MWCO higher than that of GM1.

[0147] Surprisingly, the inventors have discovered that gangliosides lacking fatty acid chains... N -hemolysis ( N Neither the lyso form nor glycosylated sphingosine bases can permeate through a filtration membrane with a MWCO higher than that of lysphingolipids. Therefore, unbound by theory, micellar aggregates can surprisingly occur regardless of the presence of fatty acid chains in the molecule. Those skilled in the art will understand that, according to the method of the present invention, the DF step is capable of removing any contaminants present in an aqueous medium that could permeate through a filtration membrane.

[0148] According to the present invention, the DF step is carried out at a constant temperature, preferably between about 15-45°C, more preferably between about 20-35°C. The DF step continues until the desired sialylated glycosphingolipid concentration is reached in the DFR. Other technical parameters, such as the setting of flow rate and pressure, are standard technical matters.

[0149] A concentration step can be optionally performed after the DF step.

[0150] In some embodiments, the method further includes a step of concentrating DFR, wherein the step of concentrating DFR is performed.

[0151] DFR concentration is typically carried out using the same membrane used in the percolation step and continues for the required period of time to reduce the volume of the DFR to the desired final volume.

[0152] DFRs rich in sialylated glycosphingolipids are subsequently spray-dried or spray-granulated.

[0153] In some preferred embodiments, the DFR containing the glycosphingolipid of formula (1) is spray-dried.

[0154] According to the invention, the spray drying step is carried out using a high-speed rotating disc or nozzle to generate fine particles. These particles then fall to the bottom of the spray drying tower under gravity. A fluidized bed can be provided here, which can use hot air to influence the drying (suitable temperature is about 80°C to about 95°C). Here, agglomeration can occur and the particles can adhere together. Subsequently, the agglomerated (granular) particles are dried, for example, in a belt drying bed or a sub-fluidized bed.

[0155] Another technique is the use of fluidized bed agglomeration. Here, the powder is fluidized in an airflow. An aqueous fluid is sprayed into the particle bed, wetting the powder and promoting agglomeration. This combination of spray drying and a fluidized bed following a dryer is suitable for agglomerating a variety of different types of solutions.

[0156] Drying can be carried out in air or an inert gas (such as nitrogen). In fluidized bed and sub-fluidized bed drying, the bed temperature can be adjusted to a preset value. These values ​​can range widely, for example, 35°C to 120°C, 50°C to 90°C, or 60°C to 80°C.

[0157] Spray drying of the DFR retentate will result in a spray-dried powder containing sialylated glycosphingolipids.

[0158] The median particle size of the spray-dried powder obtained according to the method of the present invention will typically be between about 15 μm and about 30 μm.

[0159] The span of the particles is typically less than about 3, preferably less than about 2.

[0160] The span of particles is a dimensionless parameter that indicates the uniformity of particle size distribution. It is defined as: [D(0.9)-D(0.1)] / D(0.5), where D(0.9), D(0.1) and D(0.5) represent the cutoff size, below which 10%, 50% and 90% (by volume) of the particles are distributed.

[0161] Typically, a lower span (i.e., less than 3) is characterized by a narrower particle size distribution, resulting in improved flow properties of the spray-dried powder.

[0162] The specific volume of the spray-dried powder obtained according to the method of the present invention will generally be less than about 4 mL / g, preferably less than about 3 mL / g.

[0163] Spray-dried powders with such low specific volume (i.e., less than 4 mL / g) are generally preferred because they have improved flow properties.

[0164] The glycosphingolipid content of the spray-dried powder obtained according to the method of the present invention will generally be at least about 65 wt.%, generally at least about 70 wt.%, preferably at least about 75 wt.%, and more preferably at least about 85 wt.%.

[0165] In some embodiments, the spray-dried powder contains at least about 70 wt.% of N -lyso-GM3, or at least approximately 75 wt.% N -lyso-GM3, or at least approximately 80 wt.% N -lyso-GM3.

[0166] In some embodiments, the spray-dried powder contains about 75-80 wt.% of N -lyso-GM3, and the spray-dried powder therein also contains approximately 7-9 wt.% lactose-based D- red style Sphingosine and approximately 0.1-1.0 wt.% glucosyl D- red Mode Sphingosine.

[0167] In some embodiments, the sialylated glycosphingolipids produced by the above method can be used as acceptor substrates for the second enzymatic sialylation step. Typically, the second enzymatic sialylation step is carried out as part of a sialate transferase cycle that includes a CMP-sialic acid recycling system in which CMP-sialic acid is generated / regenerated from sialic acid and CMP using a variety of enzymes. CMP-sialic acid is a relatively expensive glyconucleotide, therefore in-situ generation and regeneration of the sialic acid donor is economically advantageous and enables process scale-up.

[0168] The sialyl transferase cycle described in this invention typically comprises sialic acid, cytidine monophosphate (CMP), nucleoside triphosphate, and at least five enzymes, wherein the at least five enzymes include at least one enzyme with sialyl transferase activity, at least one enzyme with… N An enzyme with α-acylneuraminic acid cytidine transferase activity, at least one enzyme with inorganic diphosphatase activity, and at least two enzymes with kinase activity.

[0169] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: It provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. - Perform nanofiltration on the mixture from the preceding steps. - Separate the sialylated glycosphingolipids produced in the aforementioned steps, and - The resulting sialylated glycosphingolipids are reacted with a mixture containing sialic acid, cytidine monophosphate (CMP), nucleoside triphosphate, and at least five enzymes, wherein the at least five enzymes include at least one enzyme with sialyl transferase activity, at least one enzyme with... N An enzyme with α-acylneuraminic acid cytidine transferase activity, at least one enzyme with inorganic diphosphatase activity, and at least two enzymes with kinase activity.

[0170] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme having transsialyzedase activity and the enzyme having β-galactosidase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and - Perform nanofiltration on the mixture from the preceding steps. - Separate the sialylated glycosphingolipids produced in the aforementioned steps, and - The resulting sialylated glycosphingolipids are reacted with a mixture containing sialic acid, cytidine monophosphate (CMP), nucleoside triphosphate, and at least five enzymes, wherein the at least five enzymes include at least one enzyme with sialyl transferase activity, at least one enzyme with... N An enzyme with α-acylneuraminic acid cytidine transferase activity, at least one enzyme with inorganic diphosphatase activity, and at least two enzymes with kinase activity.

[0171] The term "sialic acid" refers to any member of the nonacarbon carboxylic acid family. The most common member of the sialic acid family is... N - Acetylneuraminic acid (usually abbreviated as Neu5Ac, NeuAc, or NANA). The second member of this family is... N -Hydroxyacetylneuraminic acid (Neu5Gc or NeuGc), of which Neu5Ac N- The acetyl group is hydroxylated. The third member of the sialic acid family is 2-keto-3-deoxynonyl glycosylate (KDN). Also included are 9-substituted sialic acids, such as 9-O-C1-C6 acyl-Neu5Ac, like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, and 9-azido-9-deoxy-Neu5Ac. Preferably, in the context of this invention, the term sialic acid refers to... N - Acetylneuraminic acid (Neu5Ac). N - Acetylneuraminic acid can be synthesized by methods known to those skilled in the art, such as those described in US2011165626 (A1).

[0172] The nucleoside triphosphates applicable to the context of this invention are adenosine-5'-triphosphate (ATP), uridine-5'-triphosphate (UTP), guanosine-5'-triphosphate (GTP), inosine triphosphate (ITP), and thymidine-5'-triphosphate (TTP). Adenosine-5'-triphosphate (ATP) is a preferred nucleoside triphosphate.

[0173] In some preferred embodiments, the nucleoside triphosphate is adenosine-5'-triphosphate (ATP), and the at least five enzymes include the following enzymes: - Enzymes with sialyl transferase activity, - Enzymes with cytidine monophosphate kinase activity, - Enzymes with nucleoside diphosphate kinase activity, - Enzymes with N-acylneuraminic acid cytidine transferase activity, and - Enzymes with inorganic diphosphatase activity.

[0174] Therefore, in some preferred embodiments, the sialyl transferase cycle includes: N - Acetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), an enzyme with cytidine monophosphate kinase activity (CMK) (for CMP phosphorylation), an enzyme with nucleoside diphosphate kinase activity (NDK) (for CDP phosphorylation), an enzyme with N-acylneuraminic acid cytidine diphosphate transferase activity (CSS) (for transferring CMP from CTP to Neu5Ac), an enzyme with sialyl transferase activity (for transferring Neu5Ac from CMP-Neu5Ac to the acceptor substrate), and an enzyme with inorganic diphosphate kinase activity (PPase) (for degrading inorganic pyrophosphate (PPi) formed as a byproduct during the cycle). The sialyl transferase cycle described in this preferred embodiment is as follows: Figure 1 As shown.

[0175] In some implementations, the sialyltransferase cycle also includes the regeneration of ATP, which is achieved by using a phosphate source and an enzyme with kinase activity.

[0176] Phosphate sources that can be used for ATP regeneration include, but are not limited to, polyphosphates, phosphoenolpyruvate, and acetyl phosphate. The choice of a specific kinase for ATP regeneration depends on the phosphate source used.

[0177] In some implementations, ATP regeneration is achieved by using polyphosphate as a phosphate source and an enzyme with polyphosphatase kinase activity.

[0178] Therefore, in some embodiments, the sialyl transferase cycle includes: N - Acetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), polyphosphate, enzyme with cytidine monophosphate kinase activity (CMK) (for CMP phosphorylation), enzyme with nucleoside diphosphate kinase activity (NDK) (for CDP phosphorylation), enzyme with N-acylneuraminic acid cytyltransferase activity (CSS) (for transferring CMP from CTP to Neu5Ac), enzyme with sialyl transferase activity (for transferring Neu5Ac from CMP-Neu5Ac to the acceptor substrate), enzyme with polyphosphatase kinase activity (PPK) (for ADP phosphorylation), and enzyme with inorganic diphosphate kinase activity (PPase) (for degrading inorganic pyrophosphate (PPi) formed as a byproduct during the cycle). The sialyl transferase cycle described in this embodiment is as follows: Figure 2 As shown.

[0179] The term “enzyme with sialyltransferase activity” is used interchangeably with the term “sialyltransferase”. In the context of this invention, it refers to an enzyme belonging to the glycosyltransferase 29 family (GT29) or the glycosyltransferase 42 family (GT42) that typically catalyzes the transfer of sialic acid from CMP-sialic acid to a sugar receptor.

[0180] Sialotransferases applicable to the context of this invention are those capable of catalyzing the addition of sialic acid residues to α-2-3 linked sialic acid residues in sugar acceptors. O -8 sialyltransferase.

[0181] Wild-type sialyltransferases can be derived from microorganisms such as bacteria, yeast, ascomycetes, actinomycetes, filamentous fungi, basidiomycetes, or mammals.

[0182] Wild-type sialyl transferases can be derived from Campylobacter jejuni ( Campilobacter jejuni ).

[0183] Wild-type sialyltransferases can be derived from microorganisms with vectors that have been linked to or introduced with genes encoding wild-type sialyltransferases.

[0184] Wild-type sialyltransferases can be derived from any known sialyltransferase sequence or from any unidentified sialyltransferase sequence. Unidentified sialyltransferases can be identified using sequence databases and sequence alignment algorithms, such as the publicly available GenBank database and the BLAST alignment algorithm.

[0185] In some embodiments, the enzyme possessing sialyltransferase activity is a wild-type α-2,3 / α-2,8-sialyltransferase derived from Campylobacter jejuni strain OX=197, or a functional analog thereof. The amino acid sequence of the wild-type α-2,3 / α-2,8-sialyltransferase derived from Campylobacter jejuni strain OX=19 corresponds to the amino acid sequence of accession number Q9LAK3 (https: / / www.ncbi.nlm.nih.gov / protein / ).

[0186] In some embodiments, the enzyme with sialyltransferase activity is a mutant of wild-type α-2,3 / α-2,8-sialyltransferase (Q9LAK3) derived from Campylobacter jejuni strain OX=197.

[0187] In some embodiments, the mutant α-2,3 / α-2,8-sialyltransferase has the I53G mutation (this number corresponds to the alignment of the mutant amino acid sequence with the Q9LAK3 amino acid sequence), as described by Gilbert et al. Biological Chemistry As stated in 2002, 277, 327-337.

[0188] In some embodiments, the mutant α-2,3 / α-2,8-sialyltransferase is a mutant derived from wild-type CST-IIQ9LAK3, wherein the mutant contains the following mutations / modifications compared to the wild-type: N -Terminal histidine tag MGHHHHHHH.

[0189] In some embodiments, the mutant α-2,3 / α-2,8-sialyltransferase is a mutant derived from wild-type CST-IIQ9LAK3, wherein the mutant contains the following mutations / modifications compared to the wild-type: I53G and N -Terminal histidine tag MGHHHHHHH.

[0190] The α-2,3 / α-2,8-sialyltransferase derived from Campylobacter jejuni can also be called CST-II.

[0191] The term "enzyme with cytidine monophosphate kinase activity" may be used interchangeably with the terms "CMP kinase" or "CMK," and in the context of this invention, it refers to an enzyme that uses ATP as a preferred phosphoryl group donor to catalyze the phosphorylation of CMP (or dCMP).

[0192] In some embodiments, the enzyme having cytidine monophosphate kinase activity is derived from Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis The wild-type CMP kinase or its functional analogues derived from Mycobacterium tuberculosis correspond to the amino acid sequence of accession number WP_129368399 ( / / www.ncbi.nlm.nih.gov / genbank / ).

[0193] In some embodiments, the enzyme having CMP kinase activity is a recombinant CMP kinase derived from wild-type CMP kinase of Mycobacterium tuberculosis, wherein the recombinant CMP kinase comprises the following modifications compared to the wild-type: N -Terminal histidine tag MGHHHHHHH.

[0194] CMP kinase derived from Mycobacterium tuberculosis can also be called Mt CMK.

[0195] The term "enzyme with nucleoside diphosphate kinase activity" is used interchangeably with the terms "nucleoside diphosphate kinase" or "NDK," and in the context of this invention, refers to an enzyme that catalyzes the phosphorylation of nucleoside diphosphates.

[0196] In some preferred embodiments, the enzyme having nucleoside diphosphate kinase activity is a wild-type nucleoside diphosphate kinase derived from the Mycobacterium tuberculosis complex, or a functional analog thereof. The amino acid sequence of the wild-type nucleoside diphosphate kinase derived from the Mycobacterium tuberculosis complex corresponds to the amino acid sequence of accession number WP_003412592 (https: / / www.ncbi.nlm.nih.gov / genbank).

[0197] In some embodiments, the enzyme with NDK activity is a recombinant NDK derived from the wild-type NDK of the Mycobacterium tuberculosis complex, wherein the recombinant NDK contains the following modifications compared to the wild-type: N -Terminal histidine tag MGHHHHHHH.

[0198] Nucleoside diphosphate kinases derived from the Mycobacterium tuberculosis complex can also be called... Mt NDK.

[0199] The term "having" N The term "enzyme with α-acyl-neuraminidine cytidine transferase activity" can be related to the term "[...]". N"-Acylneuraminic acid cytidine transferase" or "CSS" is used interchangeably, and in the context of this invention, it refers to the catalytic transfer of CMP from CTP to N - An enzyme containing acetylneuraminic acid (Neu5Ac).

[0200] In some implementations, having N The enzyme with α-acylneuraminic acid cytyltransferase activity is derived from Neisseria meningitidis ( Neisseria meningitidis ) wild type N -Acylneuraminic acid cytidine transferase or its functional analogues. Wild-type Neisseria meningitidis. N The amino acid sequence of -acylneuraminic acid cytyltransferase corresponds to the amino acid sequence of accession number WP_061726245 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0201] In some embodiments, the enzyme with CSS activity is a recombinant CSS derived from the wild-type CSS of Neisseria meningitidis, wherein the recombinant CSS contains the following modifications compared to the wild-type: N -Terminal histidine tag MGHHHHHHH.

[0202] Originating from Neisseria meningitidis N -Acyl-neuraminidine cytidine transferase can also be called... Nm CSS.

[0203] The term "enzyme with inorganic diphosphatase activity" may be used interchangeably with the terms "inorganic diphosphatase" or "PPase," and in the context of this invention, it refers to an enzyme that catalyzes the hydrolysis of pyrophosphate (Ppi).

[0204] In some embodiments, the enzyme having inorganic diphosphatase activity is derived from *Escherichia coli* (E. coli). Escherichia coli Wild-type inorganic bisphosphatase or its functional analogues derived from *Escherichia coli* correspond to the amino acid sequence of accession number WP_073849715 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0205] In some embodiments, the enzyme with PPase activity is a recombinant PPase derived from wild-type PPase of Escherichia coli, wherein the recombinant PPase comprises the following modifications compared to the wild-type: N -Terminal histidine tag MGHHHHHHH.

[0206] Inorganic bisphosphatases derived from Escherichia coli can also be called... Ec PPase.

[0207] The term "enzyme with polyphosphate kinase activity" may be used interchangeably with the terms "polyphosphate kinase" or "PPK," and in the context of this invention, it refers to an enzyme that catalyzes the phosphorylation of ADP.

[0208] In some preferred embodiments, the enzyme having polyphosphate kinase activity is derived from *Thermus rubrum* (…). Meiothermus ruber Wild-type polyphosphate kinase or its functional analogues from strain DSM 1279. The amino acid sequence of the wild-type polyphosphate kinase from strain DSM 1279 corresponds to the amino acid sequence of accession number ADD29239 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0209] In some embodiments, the enzyme with PPK activity is derived from a strain of *Thermus rubrum*. DSM 1279 Recombinant PPK of wild-type PPK, wherein the recombinant PPK contains the following modifications compared to wild-type: N -Terminal histidine tag MGHHHHHHH.

[0210] Polyphosphoric acid kinase derived from *Thermomyces rubrum* can also be called... Mr PPK.

[0211] The mutant or recombinant variants used in the sialyltransferase cycle, as well as the wild-type enzyme, can be purchased from existing manufacturers or produced by methods known to the art. For example, mutants of wild-type α-2,3 / α-2,8-sialyltransferase derived from Campylobacter jejuni can be produced by Gilbert et al. Biological Chemistry The wild-type enzyme is produced by the method described in 2002, 277, 327-337. The wild-type enzyme can be produced by the method described in the following examples.

[0212] For the sialyltransferase cycle, the concentrations or amounts of various reactants used in the process depend on a number of factors, including reaction conditions (such as temperature and pH) and the selection and amount of the acceptor sugar to be sialylated. Because the sialylation process allows for the regeneration of activated nucleotides, activation of donor sugars, and removal of generated PPi in the presence of catalytic amounts of enzymes, the process is limited by the concentrations or amounts of stoichiometric substances. According to the method of the invention, the upper limit of usable reactant concentrations is determined by the solubility of these reactants. Preferably, the selection of the concentrations of activated nucleotides, phosphate (salt) donors, donor sugars, and enzymes should allow the glycosylation reaction to continue until the acceptor is depleted.

[0213] The second step, enzymatic sialylation, may also include other components that promote sialyl transferase activity. These components may include divalent cations (such as Mg2+). +2 or Mn +2The reaction medium may contain solubilizers (e.g., Triton or SDS) and organic solvents (e.g., methanol or ethanol), or cyclodextrins. Materials required for ATP regeneration, such as phosphate ions, are also present.

[0214] In a preferred embodiment, the reaction medium comprises cyclodextrin.

[0215] In some embodiments, the cyclodextrin is selected from β-cyclodextrin, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin. In some preferred embodiments, the cyclodextrin is β-cyclodextrin.

[0216] Cyclodextrin is typically used in amounts between about 0.1 equivalents and about 1 equivalent based on the glycosphingolipid receptor. In some preferred embodiments, cyclodextrin is used in amounts between about 0.1 equivalents and about 0.5 equivalents based on the glycosphingolipid receptor. Therefore, in some preferred embodiments, cyclodextrin is used in amounts of about 0.1, 0.2, 0.3, 0.4, or 0.5 equivalents based on the glycosphingolipid receptor.

[0217] The use of cyclodextrins offers several advantages, such as high yields and improved accessibility of the glycosyl moieties of glycosphingolipids without the need for detergents or organic solvents. However, detergents or organic solvents may also be used in the method of the present invention.

[0218] In the optimized reaction, the above components can be combined by mixing in an aqueous reaction medium (solution) with a pH of approximately 6 to approximately 8.5. This medium does not contain enzyme-binding cofactors such as Mg. +2 or Mn +2 The chelating agent. The choice of medium is based on its ability to maintain the desired pH level. Therefore, in some embodiments, the medium is buffered to a pH of about 6.5 to about 8.5. If a buffer is not used, the pH of the medium should be maintained at about 6.5 to 8.0, preferably about 7.3 to 8.0, by adding an alkali. A suitable alkali is NaOH. Therefore, in some preferred embodiments, the pH is buffered or maintained at about 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0219] The temperature range for the above process can range from slightly above the freezing point to the temperature at which enzyme denaturation is most sensitive. This temperature range is preferably from about 0°C to about 45°C, and more preferably from about 20°C to 37°C.

[0220] The resulting reaction mixture needs to be kept long enough for sialyltransferase to sialylate a high percentage of the receptors. Typically, the reaction is often allowed to proceed for about 8 to about 240 hours, preferably about 24 to 48 hours.

[0221] NAcetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), polyphosphate, all enzymes required for the sialyltransferase cycle, and any other components used in the cycle may be added to the reaction mixture as a solid or dissolved in a solvent, and in any amount and manner effective for the desired results of the process.

[0222] In the context of this invention, the amount or concentration of all enzymes is expressed in activity units, which are a measure of the initial catalytic rate. One activity unit catalyzes the formation of 1 μmol of product per minute at a given pH and temperature.

[0223] Enzymes can be utilized in solution or bound to carriers such as polymers.

[0224] Enzymes can be provided as purified proteins, cell-free extracts, or lysates.

[0225] In some implementations, the enzyme is provided as a purified protein with a purity of about 50% to about 95%.

[0226] In some embodiments, the enzyme is provided as a cell-free extract, wherein the cell-free extract comprises about 5 wt% to about 70 wt% of the enzyme. Preferably, the cell-free extract comprises about 20 wt% to about 70 wt% of the enzyme.

[0227] Enzymes are typically present in catalytic amounts. The catalytic amount of a particular enzyme varies depending on the concentration of its substrate and reaction conditions such as temperature, time, and pH. Methods for determining the catalytic amount of a given enzyme under preselected substrate concentrations and reaction conditions are well known to those skilled in the art.

[0228] The sialylated glycosphingolipids, such as those of formula (2) or formulas (11)-(18), are subjected to the second enzymatic sialylation step as described in the above embodiments, resulting in the formation of sialylated glycosphingolipids of formula (19): in X is a glycosyl moiety containing at least two sialic acid units. R 1 R 2 R 3 R 4 s and keys As defined for glycosphingolipids in equation (1).

[0229] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 R 3and R 4 It is hydrogen, and the bond is... It is a double bond.

[0230] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 OR 5 , where R 5 For hydrogen, R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0231] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0232] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 C 10 -C 17 1-Hydroxyalkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0233] In some embodiments, the sialylated glycosphingolipid of formula (19) is a sialylated glycosphingolipid selected from formulas (20), (21), (22), and (23): .

[0234] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a double bond.

[0235] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R2 OR 5 , where R 5 For hydrogen, R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0236] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0237] In some embodiments, for the sialylated glycosphingolipid of formula (19), R 1 C 10 -C 17 1-Hydroxyalkyl, R 2 and R 3 For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a double bond.

[0238] In some embodiments, the sialylated glycosphingolipid of formula (19) is a sialylated glycosphingolipid selected from formulas (24), (25), (26), and (27): .

[0239] In some embodiments, X of the sialylated glycosphingolipids of formulas (19) and (20)-(27) is a glycosyl moiety selected from the following glycosyl moieties, or a salt thereof: In some embodiments, the X of the sialylated glycosphingolipid of formula (19) and the sialylated glycosphingolipids of formulas (20)-(27) is Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ1-.

[0240] In some embodiments, the sialylated glycosphingolipid of formula (19) is the sialylated glycosphingolipid of formula (20), and wherein X of the sialylated glycosphingolipid of formula (20) is Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (19) is the sialylated glycosphingolipid of formula (20), and wherein the sialylated glycosphingolipid of formula (20) is...N -lyso-GD3.

[0241] In some embodiments, the sialylated glycosphingolipid of formula (19) is the sialylated glycosphingolipid of formula (24), and wherein X of the sialylated glycosphingolipid of formula (24) is Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (19) is the sialylated glycosphingolipid of formula (24), and wherein the sialylated glycosphingolipid of formula (24) is GD3.

[0242] The sialylated glycosphingolipids obtained after the second enzymatic sialylation step can be separated from the reaction mixture. This separation can be performed using standard methods known to those skilled in the art, such as extraction with organic solvents, chromatography, and / or ion-exchange chromatography.

[0243] In some embodiments, the present invention describes a method for generating sialylated glycosphingolipids, the method comprising the following steps: - Provides glycosphingolipid and sialic acid donors, enzymes with transsialylase activity, and enzymes with β-galactosidase activity. - In the presence of the enzyme with transsialyzedase activity, the glycosphingolipid is mixed with the sialic acid donor to produce the sialylated glycosphingolipid, and then: - Add an enzyme with β-galactosidase activity. - Perform nanofiltration on the mixture from the preceding steps. - Separate the sialylated glycosphingolipids produced in the aforementioned steps. - The resulting sialylated glycosphingolipids are reacted with a mixture containing sialic acid, cytidine monophosphate (CMP), nucleoside triphosphate, and at least five enzymes, wherein the at least five enzymes include at least one enzyme with sialyl transferase activity, at least one enzyme with... N An enzyme with α-acyl-neuraminidine cytidine transferase activity, at least one enzyme with inorganic diphosphatase activity, and at least two enzymes with kinase activity, and - Separate the sialylated glycosphingolipids produced in the aforementioned steps.

[0244] A preferred method for separation includes percolation filtration (DF) of the reaction mixture, wherein the DF is performed as described in the above embodiments.

[0245] In some embodiments, the separation step is followed by a spray drying or spray granulation step of sialylated glycosphingolipids, wherein the spray drying or spray granulation is performed as described in the embodiments above.

[0246] In some embodiments, the glycosphingolipids or sialylated glycosphingolipids according to the invention can be produced or utilized in the form of salts, preferably in the form of pharmaceutically acceptable salts.

[0247] In some embodiments, the salt 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, methanesulfonic acid, trifluoromethanesulfonic acid, and perchloric acid.

[0248] Example The following working examples describe non-limiting embodiments of the present invention and are given for illustrative purposes only.

[0249] General methods and materials LCMS analysis was performed using 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).

[0250] HPLC analyses were performed on a Dionex Ultimate 3000 HPLC system coupled with a Corona VeoCharged Aerosol Detector using an Accucore aQ (150 mm x 4.6 mm, 2.6 μm) column.

[0251] Mutant TcTS was expressed from E. coli strains, according to Paris et al. Glycobiology 2001, 11, 305-311 or Buschiazzo et al., Molecular Cell The method described in 2002, 10, 757-768, or the general method described in Example 11.

[0252] Mutant CST-II (wild-type Q9LAK3, mutation / modification: I53G and) N - The terminal histidine tag (MGHHHHHH) was expressed from E. coli strains, according to Gilbert et al., Biological Chemistry The method described in 2002, 277, 327-337, or by the general method described in Example 11.

[0253] Reorganization Mt CMK (wild-type: WP_129368399, modified: N-terminal histidine tag MGHHHHHHH), recombinant Mt NDK (wild-type: WP_003412592, modified: N-terminal histidine tag MGHHHHHH), recombinant MrPPK (wild-type: ADD29239, modified: N-terminal histidine tag MGHHHHHHH), recombinant Nm CSS (wild-type: WP_061726245, modified: N-terminal histidine tag MGHHHHHH) and recombinant Ec PPase (wild-type: WP_073849715, modified: N-terminal histidine tag MGHHHHHHH) was expressed from Escherichia coli strains following the general method described in Example 11.

[0254] Example 1. General procedure for sialylation of glycosphingolipids using 2,3-transsialidase The 2,3-transsialylase-catalyzed sialylation reaction was carried out in an aqueous solution at a pH of approximately 6.5 to 7.0. A typical reaction mixture contained a glycosphingolipid receptor (1 equivalent (eq.)), 3'-sialyl lactose (1.5–2.0 eq.), 2,3-transsialylase (TcTS, 0.4 g / L), and β-galactosidase (0.5 g / L). Nanofiltration was employed during the reaction. The reaction mixture was nanofiltered using a 300–500 Da membrane at a pressure of 15–20 bar and a temperature of approximately 30–40 °C for approximately 6–8 hours. The NF-retentive (NFR) was heated at approximately 60–95 °C for approximately 10–60 minutes and then percolated as described in Example 7.

[0255] Example 2. α-N-acetylneuraminoyl-(2-) 3)- O -β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 1')-D-erythrosphospirin ( N The generation of -lyso-GM3) N -lyso-GM3 was generated using lactosylsphingosine as a glycosphingolipid receptor following the general steps described in Example 1.

[0256] LC / MS: Rt = 4.38 min; for [C 41 H 74 N2O 20 Calculated ESI-MS: 914, Measured value: 915 [M+H] + 913 [MH] - .

[0257] Example 3. α- N -acetylceramide-(2 3)- O -β-D-galactopyranosyl-(1 3)- O -2-(acetamido)-2-deoxy-β-D-galactopyranosyl-(1 4)- O -[α-N-acetylceramide-(2-) 3)]- O -β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 Production of 1'-D-erythrosphospirin (N-lyso-GD1a) N-lyso-GD1a usage N -lyso-GM1a was generated as a glycosphingolipid receptor according to the general steps described in Example 1.

[0258] LC / MS: Rt = 7.8 min; for [C 66 H 114 N4O 38 Calculated ESI-MS value: 1571, measured value: 1572 [M+H] + 1570 [MH] - .

[0259] Example 4. α- N -acetylceramide-(2 3)- O -β-D-galactopyranosyl-(1 3)- O -2-(acetamido)-2-deoxy-β-D-galactopyranosyl-(1 4)- O -[α- N -acetylceramide-(2 3)]- O -β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 Production of 1'-N-stearoyl-D-erythrosphoside (GD1a) GD1a was generated using GM1a as a glycosphingolipid receptor following the general steps described in Example 1.

[0260] LC / MS: Rt = 12.5 min; for [C 84 H 148 N4O 39 Calculated ESI-MS value: 1837, measured value: 1836 [MH] - .

[0261] Example 5. General procedures for sialylation of glycosphingolipids using a sialyltransferase cycle 5.1. Sialyltransferase cycle with CMP-sialic acid generation / regeneration The sialyltransferase cycle is carried out in aqueous solution at a pH of approximately 6.5 to 7.5 and a temperature of approximately 37°C. A typical reaction mixture contains sialylated glycosphingolipid receptor (1 eq.), N-acetylneuraminic acid (Neu5Ac, 2.5 eq.), β-cyclodextrin (0.5 eq.), ATP (3.5 eq.), CMP (0.27 eq.), MgCl2 (20 mM), and the following enzymes: mutant CST-II (5 g / L), recombinant… Mt CMK (12 g / L), recombinant Mt NDK (6 g / L), recombinant Nm CSS (1 g / L), Recombinant Ec PPase (2.5 μL / mL). Sialization cycles were monitored by LCMS (methods and conditions are described in Example 10).

[0262] 5.2 Sialyltransferase cycle with CMP-sialic acid generation / regeneration and ATP regeneration The sialyltransferase cycle is carried out in aqueous solution at a pH of approximately 7.0 to 8.0 and a temperature of approximately 37°C. A typical reaction mixture contains sialylated glycosphingolipid receptor (1 eq.), N-acetylneuraminic acid (Neu5Ac, 1.5 eq.), β-cyclodextrin (0.5 eq.), ATP (approximately 0.1 eq. to approximately 0.5 eq.), polyphosphate (2.5 eq.), CMP (0.27 eq.), MgCl2 (20 mM), and the following enzymes: mutant CST-II (3 g / L), recombinant... Mt CMK (2.5 g / L), recombinant Mt NDK (2.5 g / L), recombinant Nm CSS (0.5 g / L), Recombinant Ec PPase (40 to 60 mg / mL), recombinant Mr PPK (40 to 60 mg / mL). Sialization cycle was monitored by LCMS (methods and conditions are described in Example 10).

[0263] Example 6. α-N-acetylneuraminoyl-(2-) 8)- O -α-N-acetylceramide-(2 3)- O -β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 1')-D-erythrosphospirin ( NThe generation of -lyso-GD3) N-lyso-GD3 was generated using N-lyso-GM3 as a sialylated glycosphingolipid receptor according to the general procedure described in Example 5.

[0264] LC / MS: Rt = 4.74 min; for [C 52 H 91 N3O 28 Calculated ESI-MS value: 1205, Measured value: 1206 [M+H] + 1204 [MH] - .

[0265] Example 7. Isolation of sialylated glycosphingolipids The sialylated glycosphingolipids produced as described in Examples 1-6 were separated from the reaction mixture by percolation (DF). DF was performed using a 250 kDa spiral wound membrane with a membrane area of ​​approximately 0.668 m². 2 The flow rate is approximately 10 l / h, the transmembrane pressure is approximately 8-10 bar, the temperature is approximately 20-25°C, and the volume relative to the feed liquid volume is approximately 2-10 DF. During percolation, the flow rate is maintained at approximately 15.3-18.1 l / m³. 2 High throughput. The DF-retentive medium (DFR) containing sialylated glycosphingolipids was subjected to high throughput at Mobile Minor under the following conditions. ® Spray drying is performed on a (GEA) spray dryer: Inlet flow rate: 45-50 g / min Atomizer speed: 20,000 rpm Inlet temperature: 160℃ Outlet temperature: 85℃ Following this procedure, a spray-dried powder is obtained, which contains approximately 70-90 wt .% of one or more sialylated glycosphingolipids.

[0266] Example 8. Particle size analysis and moisture content of spray-dried powder The average particle size and D(0.1), D(0.5), and D(0.9) values ​​were measured using laser diffraction particle size analysis with a Malvern Mastersizer 3000 (Malvern Instruments). The spray-dried powder obtained in Example 7 was dispersed in cyclohexane containing 0.1% soybean lecithin. The sample was sonicated to disperse aggregated particles before particle size measurement.

[0267] The moisture content of the spray-dried powder obtained in Example 2 was determined by thermogravimetric analysis (TG) and differential scanning calorimetry (DSC) or by Karl Fischer titration. TG and DSC measurements were performed on a Setaram Labsys Evo (Setaram). The spray-dried powder typically contains approximately 2-3... wt .% water.

[0268] Example 9. Includes N -lyso-GM3 spray-dried powder The product obtained according to the steps of Example 7 includes N The DFR of -lyso-GM3 was spray-dried under the conditions of Example 8 to obtain a spray-dried powder with the following characteristics: Example 10: LC / MS and HPLC analysis LC / MS analysis Take 50 μL of the sample from the reaction mixtures of Examples 5.1 and 5.2, mix with DMSO (950 μL), and centrifuge (16,000 rpm, 5 min). For the analysis of N-lyso-GD1a and GD1a, the eluent consisted of solvent D (2 mM ammonium formate, 0.2% v / v formic acid, 75% v / v MeOH, 25% v / v ACN) - solvent C (2 mM ammonium formate, 0.2% v / v formic acid aqueous solution); for the analysis of N-lysoGM3 and GD3, the eluent consisted of solvent D (2 mM ammonium formate, 0.2% v / v formic acid, 75% v / v MeOH, 25% v / v ACN) - solvent C (2 mM formic acid filtered aqueous solution), applying the following gradient: N-lyso GM3 70-95% (D in C). N -lyso-GD1a 60-85% (D in C), GD1a 60-85% (D in C), N-lyso-GD3 70-98% (D in C).

[0269] HPLC analysis Include N The glycosphingolipid content of the spray-dried powder of -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; Solvent B: 1 L MeOH + 1 L acetonitrile + 4 mL formic acid + 4 mmol ammonium formate.

[0270] The powder was eluted with a gradient of 50-100% B in A for 13 min; followed by isocratic elution with 100% B for 18 min; and finally isocratic elution with 50% B in A for 40 min. The glycosphingolipid content of the powder was quantified by peak area analysis using the external standard method.

[0271] Example 11. Enzyme Production The gene encoding the enzyme is typically ordered as a synthetic gene with codon-optimized expression for use in *E. coli*. The synthetic construct contains a forked end with a BsaI restriction site for golden-gate cloning into a pET28a-based expression vector (carrying the introduced BsaI restriction site and a fluorescent deletion cassette). The gene is expressed under the regulation of a lactose-inducible T7 promoter.

[0272] Overview of the present invention (SEQ ID NO). sequence list SEQ ID NO: 1: SEQ ID NO: 2: SEQ ID NO: 3:MGHHHHHH This disclosure should not be construed as limiting in any way to the described embodiments, and those skilled in the art will foresee the possibility of various modifications thereto.

[0273] The above implementation methods can be used in combination.

[0274] The appended claims further illustrate specific embodiments of this disclosure.

Claims

1. Method for producing a sialylated glycosphingolipid, the method comprising the steps of: - providing a glycosphingolipid, a sialic acid donor and an enzyme having transsialidase activity, - mixing the glycosphingolipid with the sialic acid donor in the presence of the enzyme having transsialidase activity, thereby producing the sialylated glycosphingolipid, wherein the glycosphingolipid is a compound of formula (1): wherein W is a Galβ1- or a glycosyl moiety carrying one or more terminal β-galactopyranosyl units, R 1 is hydrogen, aryl, or substituted or unsubstituted C 1-50 alkyl, preferably substituted or unsubstituted C 1-17 alkyl, more preferably substituted or unsubstituted C 10-17 alkyl, R 2 is hydrogen or -OR 5 wherein R 5 is selected from hydrogen, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 2-6 acyl, preferably R 5 is hydrogen, bond In R 2 is a double bond or a single bond, or in R 2 is -OR 5 is a single bond, R 3 hydrogen, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 acyl, preferably hydrogen, R 4 selected from hydrogen, substituted or unsubstituted aryl, heteroalkyl, substituted or unsubstituted C 2-32 acyl, and wherein the sialic acid donor is 3'-sialyllactose.

2. The method according to claim 1, further comprising the step of adding an enzyme having β-galactosidase activity.

3. The method according to claim 1 or 2, further comprising a step of nanofiltration.

4. The method according to any one of claims 1 to 3, further comprising a step of isolating the sialylated glycosphingolipid.

5. The method according to any one of claims 1 to 4, wherein R of the glycosphingolipid of formula (1) is hydrogen, and wherein R is C-C alkyl, preferably C-C alkyl. 5 4 1 13 17 13 5. The method according to any one of claims 1 to 4, wherein R of the glycosphingolipid of formula (1) is hydrogen, and wherein R is C-C alkyl, preferably C-C alkyl. 5 4 1 13 17 13 5. The method according to any one of claims 6. The method according to any one of claims 1 to 5, wherein W of the glycosphingolipid of formula (1) is a glycosyl moiety selected from Galβ1-, Galβ1-4Glc1-, and Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-.

7. The method according to any one of claims 1 to 6, wherein the glycosphingolipid of formula (1) is selected from the group consisting of sphingosylgalactoside, lactosylsphingosine, and N-lyso-GM1a.

8. The method according to any one of claims 1 to 7, wherein the glycosphingolipid is lactosylsphingosine.

9. The method according to any one of claims 1 to 8, wherein the enzyme having transsialidase activity is a transsialidase derived from Trypanosoma cruzi ( Trypanosoma cruzi ), Trypanosoma congolense ( Trypanosoma congolense ), or Trypanosoma brucei ( Trypanosome brucei ), or a functional analogue thereof, preferably a transsialidase derived from Trypanosoma cruzi ( Trypanosoma cruzi ).

10. The method of any one of claims 1 to 9, wherein the enzyme having trans-sialidase activity is a mutant of a trans-sialidase derived from Trypanosoma cruzi (TcTS). Trypanosoma cruzi 10. The method of any one of claims 1 to 9, wherein the enzyme having trans-sialidase activity is a mutant of a trans-sialidase derived from Trypanosoma cruzi (TcTS).​ 11. The method according to any one of claims 2 to 10, wherein the enzyme having beta-galactosidase activity is a beta-galactosidase derived from Aspergillus oryzae (A. oryzae) or a functional analogue thereof. Aspergillus oryzae 11. The method according to any one of claims 2 to 10, wherein the enzyme having beta-galactosidase activity is a beta-galactosidase derived from Aspergillus oryzae (A. oryzae) or a functional analogue thereof.​ 12. The method according to any one of claims 1 to 11, wherein the sialylated glycosphingolipid is a compound of formula (2): wherein Y is a glycosyl moiety carrying at least one sialic acid unit, R 1 , R 2 , R 3 , R 4 and a bond as defined for the sugar-sphingoid base of formula (1).

13. The method according to claim 12, wherein the R of the sialylated glycosphingolipid of formula (2) 4 It is hydrogen, and R is therein 1 C 13 -C 17 Alkyl group, preferably C 13 alkyl.

14. The method according to claim 12 or 13, wherein Y of the sialylated glycosphingolipid of formula (2) is a glycosyl moiety selected from Neu5Acα2-3Galβ1-, Neu5Acα2-3Galβ1-4Glc1-, and Neu5Acα2-3Gal1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-.

15. The method according to any one of claims 12 to 14, wherein the sialylated glycosphingolipid of formula (2) is selected from N - lyso-GM4, N - lyso-GM3 and N - lyso-GD1a.

16. The method according to any one of claims 12 to 15, wherein the sialylated glycosphingolipid of formula (2) is N - lyso-GM3.

17. The method according to any one of claims 1 to 16, wherein the method further comprises the step of reacting the sialylated glycosphingolipid produced according to any one of claims 1 to 16 with a mixture comprising sialic acid, cytidine monophosphate (CMP), nucleoside triphosphates and at least five enzymes, wherein the at least five enzymes comprise at least one enzyme having sialyltransferase activity, at least one enzyme having acylneuraminate cytidyltransferase activity, at least one enzyme having inorganic diphosphatase activity and at least two enzymes having kinase activity. N - acylneuraminate cytidyltransferase activity, at least one enzyme having inorganic diphosphatase activity and at least two enzymes having kinase activity.

18. The method according to claim 17, wherein the nucleoside triphosphate is adenosine 5'-triphosphate (ATP), and wherein the at least five enzymes comprise the following enzymes: - an enzyme having sialyltransferase activity, - an enzyme having cytidine monophosphate kinase activity, - an enzyme having nucleoside diphosphate kinase activity, - having N - an enzyme having acylneuraminate cytidylyltransferase activity, and - an enzyme having inorganic diphosphatase activity.

19. The method according to claim 17 or 18, wherein the step of reacting the sialylated glycosphingolipid produced according to any one of claims 1 to 16 with a mixture comprising sialic acid, cytidine monophosphate (CMP), a nucleoside triphosphate and at least five enzymes is carried out in the presence of a cyclodextrin, wherein the cyclodextrin is preferably selected from a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a derivative thereof, more preferably the cyclodextrin is β-cyclodextrin.

20. The method of any one of claims 17 to 19, wherein the enzyme having nucleoside diphosphokinase activity is an enzyme derived from Mycobacterium tuberculosis complex or a functional analog thereof and the enzyme having cytidine monophosphokinase activity is an enzyme derived from Mycobacterium tuberculosis or a functional analog thereof. Mycobacterium tuberculosis ​

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