Sialylation method

Abstract

The present invention relates to a novel and efficient method for sialylation of a glycoside comprising mixing said glycoside with sialic acid, cytidine monophosphate, nucleoside triphosphate and one or more cell-free extracts of a microorganism, the present invention relates to a microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein the one or more cell-free extracts comprise: at least one polypeptide having cytidine monophosphate kinase activity, at least one polypeptide having N-acyl neuraminic acid cytidyltransferase activity, and at least one polypeptide having sialyltransferase activity, thereby sialylating the glycoside.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel and effective method for sialylation of glycosides or analogs thereof. BACKGROUND

[0002] Glycosylation reactions are widespread in nature and are essential for the physiological and pathological functionality of cells. In fact, glycosylation reactions often confer additional specific biological functions to the glycosylated products. For example, certain carbohydrate moieties play an important functional role in the regulation of various biological processes such as intercellular recognition, communication and intercellular adhesion.

[0003] One important type of glycosylation reactions is the sialylation reaction, in which one or more sialic acid units are added to biological molecules such as oligosaccharides, lipids or proteins.

[0004] Sialic acid is a unique monosaccharide. It belongs to the family of nine-carbon sugars and is negatively charged at physiological pH. In addition, sialic acid can be modified at multiple positions with acetyl, sulfate and other groups.

[0005] Sialic acid is naturally present at the non-reducing end of the sugar chain of biological molecules such as oligosaccharides, glycoproteins, glycolipids, often responsible for the in vivo activity of these biological molecules.

[0006] In humans, sialic acid is present in the brain and is an essential part of the structure of gangliosides. In particular, gangliosides are involved in synapse formation and neurotransmission, as well as in neurological diseases, in particular Alzheimer's disease, Parkinson's disease and Huntington's disease (Chiricozzi E. et al., Biochimica et Biophysica Acta, 2020, 21, 868). In addition, certain gangliosides are found in the intestinal mucosa, capable of promoting intestinal health, as well as acting as anti-infective agents (E. J. Park et al., Glycobiology, 2005, 15, 935-942). Human milk also contains sialic acid, which is bound to the end of free oligosaccharides or glycolipids such as lactose or lactosylceramide. In particular, human milk oligosaccharides such as 3'-sialyllactose, or human milk gangliosides such as GM3 and GD3, confer antiviral, anti-inflammatory and immunomodulatory properties to human milk (Quitadamo et al., Nutrients, 2021, 8, article 589736). Int. J. Mol. Sci. 2020, 21, 868). In addition, certain gangliosides are found in the intestinal mucosa, capable of promoting intestinal health, as well as acting as anti-infective agents (E. J. Park et al., Glycobiology, 2005, 15, 935-942). Human milk also contains sialic acid, which is bound to the end of free oligosaccharides or glycolipids such as lactose or lactosylceramide. In particular, human milk oligosaccharides such as 3'-sialyllactose, or human milk gangliosides such as GM3 and GD3, confer antiviral, anti-inflammatory and immunomodulatory properties to human milk (Quitadamo et al., Nutrients, 2021, 8, article 589736). Glycobiology 2005, 15, 935-942). Human milk also contains sialic acid, which is bound to the end of free oligosaccharides or glycolipids such as lactose or lactosylceramide. In particular, human milk oligosaccharides such as 3'-sialyllactose, or human milk gangliosides such as GM3 and GD3, confer antiviral, anti-inflammatory and immunomodulatory properties to human milk (Quitadamo et al., Nutrients, 2021, 8, article 589736). Frontiers in Public Health 2005, 15, 935-942). Human milk also contains sialic acid, which is bound to the end of free oligosaccharides or glycolipids such as lactose or lactosylceramide. In particular, human milk oligosaccharides such as 3'-sialyllactose, or human milk gangliosides such as GM3 and GD3, confer antiviral, anti-inflammatory and immunomodulatory properties to human milk (Quitadamo et al., Nutrients, 2021, 8, article 589736).

[0007] Sialylated biological molecules and oligosaccharides have a great potential as therapeutic drugs and as food ingredients, however they are difficult to obtain for basic and clinical research. In fact, they are characterized by a high structural complexity, their preparation is extremely challenging.

[0008] A large number of attempts have been made to develop methods for sialylation of glycosides.

[0009] For example, sialylated sugars and glycosphingolipids can be obtained via chemical synthesis (JA Morales-25 Serna, Carbohydr. Res. 2007, Yukishige et al.). Tetrahedron (1990, 46, 89-102). The drawbacks of this method are that its stereochemistry and regiochemistry are difficult to control, it requires multiple protecting group operations, purification is difficult, and scale-up is challenging.

[0010] Alternatively, enzymatic synthesis can be used for the sialylation of carbohydrates and glycolipids.

[0011] Compared to purely chemical routes, enzymatic synthesis offers many advantages, such as high control over regiochemistry and stereochemistry, no need for protecting groups, and typically occurs under mild conditions.

[0012] A common approach for the enzymatic sialylation of glycosides is based on the use of sialyltransferases, in which sialic acid is transferred from CMP-sialic acid to the glycoside acceptor. CMP-sialic acid is a relatively expensive and unstable reagent, and methods for the in situ generation and / or regeneration of this glyconucleotide have been described (Yu et al., Org Biomol Chem. 2018, 4076-4080, WO9928491).

[0013] The drawbacks of these methods include the use of multiple purification enzymes and expensive reagents such as cytidine triphosphate and phosphoenolpyruvate, which makes large-scale scaling difficult.

[0014] Therefore, there is a need to develop novel methods that are technically feasible and cost-effective for the efficient and large-scale production of sialylated biomolecules and their analogues. Summary of the Invention

[0015] In a first aspect, the present invention relates to a method for sialylation of a glycoside of formula (1) or a salt thereof: in X represents the glycosyl moiety, which is preferably selected from Gal1- or has one or more terminal galactose units and / or one or more terminal galactose units. N -The glycosyl moiety of an acetylgalactosamine unit and / or one or more terminal sialic acid units; Y is selected from hydroxyl groups, fluorides, or portions of formula (2) or their salts: in R 1It 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, 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, The method includes: The glycoside of formula (1) is mixed with sialic acid, cytidine monophosphate, nucleoside triphosphate, and one or more cell-free extracts of microorganisms, wherein the microorganisms comprise one or more endogenous polypeptides with inorganic diphosphatase activity and one or more endogenous polypeptides with phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity, This allows the glycoside to be sialylated.

[0016] In a second aspect, the present invention relates to a sialytizing agent comprising one or more cell-free extracts of microorganisms, said microorganisms comprising one or more endogenous polypeptides having inorganic bisphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity. Attached Figure Description

[0017] Figure 1 : Schematic diagram of the sialyl transferase cycle, in which CMP-Neu5Ac is generated / regenerated.

[0018] Figure 2 : A schematic diagram of the sialyl transferase cycle, in which CMP-Neu5Ac is generated / regenerated and ATP is regenerated. Detailed Implementation

[0019] This invention describes a novel and efficient in vitro sialylation method for biomolecules and their analogues, catalyzed by sialyltransferases, wherein expensive nucleotide donors are generated in situ and regenerated during sialylation cycles. In particular, the method is characterized by the use of one or more cell-free extracts of microorganisms containing all the enzymes required for sialylation cycles, and wherein the endogenous enzyme activity of the microorganisms is utilized. Advantages of the method described herein include avoiding additional purification steps for enzyme isolation and reducing the number of specific enzymes used during sialylation cycles by utilizing the activity of naturally occurring enzymes in the cell-free extracts of microorganisms. Furthermore, the method uses inexpensive reagents. Therefore, the method is suitable for large-scale production of sialylated glycosides and sugars, such as gangliosides, sialylated glycosyl fluorides, and human lactose oligosaccharides, comprising glycosides of a mixture of formula (1) or salts thereof: in X represents the glycosyl moiety, which is preferably selected from Gal1- or has one or more terminal galactose units and / or one or more terminal galactose units. N -The glycosyl moiety of an acetylgalactosamine unit and / or one or more terminal sialic acid units; Y is selected from hydroxyl groups, fluorides, or portions of formula (2) or their salts: in R 1 It is hydrogen, aryl, or C 1-50 Alkyl group, preferably C 1-17 Alkyl, more preferably C 10-17 Alkyl groups, which may be saturated or contain one or more double and / or triple bonds, and / or may contain one or more functional groups, preferably selected from hydroxyl, amino, alkoxy, acyloxy, amide, thiol, thioether, or phosphorus-containing functional groups. R 2 For hydrogen or -OR 5 , where R 5Selected from hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 2-6 Acyl group, 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, The method includes: The glycoside of formula (1) is mixed with sialic acid, cytidine monophosphate, nucleoside triphosphate, and one or more cell-free extracts of microorganisms, wherein the microorganisms comprise one or more endogenous polypeptides with inorganic bisphosphatase activity and one or more endogenous polypeptides with phosphotransferase activity. and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity, This allows the glycoside to be sialylated.

[0020] The following describes non-limiting embodiments of different aspects of the present invention and illustrates them by way of non-limiting examples.

[0021] The terms, definitions, and implementation methods described throughout this specification relate to all aspects and implementations of the invention.

[0022] 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 "glycoside" does not refer to providing only one single glycoside, but rather to providing multiple glycosides of the same kind.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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), C2-6 -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.

[0028] 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.

[0029] With regard to the term polypeptide, the term "functional analog" refers to a protein whose amino acid sequence shares 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 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. 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, or a fusion protein that retains the same functional activity as a reference protein.

[0030] 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.

[0031] Table 1 Amino acid codes: Those skilled in the art will understand that in the chemical formula of a particular compound, such as formula (2) or (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.

[0032] 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 the compound shown in, for example, formula (3) or the part shown in, for example, formula (2).

[0033] In the context of this invention, the terms “about,” “approximately,” or “around” are used interchangeably to indicate a specific value (e.g., “pH about 7.0,” “pH about 7.0,” or “pH around 7.0”) or range (e.g., “from about 10% to about 99% conversion,” “from about 10% to about 99% conversion,” or “from about 10% to about 99% conversion”) to indicate a deviation from that specific value or range of 0.1% to 10%.

[0034] 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 the desired compound and other compounds. In this context, other compounds in the mixture are considered contaminants. The terms "separation" and "separation out" are used interchangeably.

[0035] 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-lacticoyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, and 9-azido-9-deoxy-Neu5Ac. A review of the sialic acid family is provided. 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).

[0036] As used herein, the term "cell-free extract" refers to a mixture of biomolecules (e.g., proteins, nucleic acids, etc.) and cellular debris (e.g., cell membranes, organelles, etc.), rather than living cells. Preferably, the cell-free extract lacks the genetic material and membranes inherent in living cells and contains components necessary for carrying out the desired biochemical processes. Typically, cell-free extracts are prepared by disrupting biological cells, for example, through chemical or mechanical cell lysis. Cell lysis can be performed using methods known to those skilled in the art, such as those described by Cole et al. Synthetic and Systems Biotechnology The method described in 2020, 5, 252-267.

[0037] In the context of the term microorganisms, the term "genetic engineering" refers to a microorganism whose genetic material is not part of the organism's natural genome (i.e., wild-type genome). Genetically engineered microorganisms are, for example, microorganisms whose own DNA sequence contains at least one artificially altered (i.e., through laboratory genomic manipulation) phenotype, with the aim of conferring a specific desired phenotype. DNA alterations can be, for example, the insertion or deletion of DNA segments in the genome, or the introduction of expression vectors carrying endogenous or heterologous genes into the cell. In this paper, DNA sequence alterations are particularly achieved through the expression of heterologous nucleic acid sequences (especially heterologous nucleic acid sequences encoding specific polypeptides). Genome editing can be achieved, for example, through common recombinant nucleic acid techniques, as described in, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). CRISPR technology can also be used for genetic modification.

[0038] The term "heterogeneous" is understood to mean that it is not naturally present within a particular host microorganism or is not inherent to that host microorganism. When used herein to describe a polypeptide or polypeptide sequence, the term heterogeneous includes, for example, polypeptides that are not naturally produced by a particular microorganism, synthetic polypeptides or other polypeptides that are not naturally present and / or their sequences, and any polypeptide sequence that has been artificially manipulated to achieve a non-natural level or activity in a particular host cell or microorganism.

[0039] The term "glycoside," as used herein, refers to a chemical compound in which a sugar moiety is linked to a non-sugar chemical moiety via a glycosidic bond. The sugar moiety may be referred to as a "glycone," and the non-sugar chemical moiety as an "aglycone." A "sugar residue" may consist of a single sugar unit (monosaccharide), two sugar units (disaccharide), or multiple sugar units (oligosaccharide).

[0040] In the context of this invention, a compound of formula (1) represents a "glycoside" in which the glycosyl moiety (sugar residue) X is linked to the aglycone Y via a glycosidic bond. This glycosidic bond can be an α-glycosidic bond or a β-glycosidic bond.

[0041] In some embodiments, X of the glycoside in formula (1) is a glycosyl moiety, wherein the glycosyl moiety is Gal1-.

[0042] In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units.

[0043] In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more ends. N - Acetylgalactosamine unit.

[0044] In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal sialic acid units, or salts thereof.

[0045] In some embodiments, X of the glycoside in formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units, one or more terminal... N - Acetylgalactosamine unit and one or more terminal sialic acid units.

[0046] In some embodiments, X of the glycoside in formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units and one or more terminal... N - Acetylgalactosamine unit.

[0047] In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units and one or more terminal sialic acid units.

[0048] In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety has one or more ends. N - Acetylgalactosamine unit and one or more terminal sialic acid units.

[0049] In some embodiments, the Y of the glycoside of formula (1) is selected from a hydroxyl group, a fluoride, or a portion of formula (2) or a salt thereof.

[0050] In some embodiments, the Y in the glycoside of formula (1) is a hydroxyl group. Therefore, in some embodiments, the glycoside of formula (1) is a sugar.

[0051] In some embodiments, the Y in the glycoside of formula (1) is a fluoride. Therefore, in some embodiments, the glycoside of formula (1) is a glycoside of formula (4): Where X is the glycosyl moiety as defined for glycosides in equation (1).

[0052] The glycosides of formula (4) can also be called glycosyl fluorides.

[0053] In some embodiments, the glycoside of formula (4) is an α-glycosyl fluoride.

[0054] In some preferred embodiments, the Y of the glycoside of formula (1) is a portion of formula (2) or a salt thereof. Therefore, in some preferred embodiments, the glycoside of formula (1) is a glycoside of formula (3) or a salt thereof: in X is defined as for glycosides in equation (1); 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; 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.

[0055] In some embodiments, for the glycosides of formula (3), R 1 For saturated unsubstituted C 10-17 Alkyl, R 2 For -OR 5 , where R 5 For hydrogen, R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0056] In some embodiments, for the glycosides of formula (3), R 1 For saturated unsubstituted C 10-17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0057] In some embodiments, for the glycosides of formula (3), R1 C 10-17 1-Hydroxyalkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0058] In some embodiments, the glycoside of formula (3) is selected from formulas (5), (6), (7) and (8): .

[0059] Where X is the glycosyl moiety as defined for glycosides in equation (1).

[0060] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety is Gal1-.

[0061] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units.

[0062] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more ends. N - Acetylgalactosamine unit.

[0063] In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal sialic acid units, or salts thereof.

[0064] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units, one or more terminal... N - Acetylgalactosamine unit and one or more terminal sialic acid units.

[0065] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units and one or more terminal... N - Acetylgalactosamine unit.

[0066] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more terminal galactose units and one or more terminal sialic acid units.

[0067] In some embodiments, X of the glycoside in formula (3) is a glycosyl moiety, wherein the glycosyl moiety has one or more ends. N - Acetylgalactosamine unit and one or more terminal sialic acid units.

[0068] In some embodiments, for the glycosides of formula (3), 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.

[0069] In some embodiments, for the glycosides of formula (3), R 1 For saturated unsubstituted C 10-17 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.

[0070] In some embodiments, for the glycosides of formula (3), 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.

[0071] In some embodiments, for the glycosides of formula (3), 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.

[0072] In some embodiments, the glycoside of formula (3) is a glycoside selected from formulas (9), (10), (11), and (12): .

[0073] Where X is the glycosyl moiety as defined for glycosides in equation (1).

[0074] The glycosides of formulas (3) and (5)-(12) can also be called glycosphingolipids.

[0075] 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.

[0076] The sphingolipid portion of the glycosphingolipids of this invention is typically derived from aliphatic amino alcohols, such as sphingosine bases or ceramides.

[0077] In the context of this invention, sphingosine bases refer to naturally occurring sphingosine bases, their analogues, or their derivatives.

[0078] Naturally occurring sphingosine bases are D-erythrosphingosine (S) and 6-hydroxy-D- erythro Sphingosine (H), D- ribose - Phytosphingosine (P) or DL- erythro Dihydrosphingosine (DS), where the number of carbon atoms in sphingosine can be indicated in parentheses after the letters S, H, P, and DS.

[0079] The letters S, H, P, and DS refer to those created by Motta et al. Biochim Biophys Acta. Developed by Rabionet, 1993, 1182:147-151 Biochim Biophys Acta. 2014, 1841:422-434 and Masukawa et al., Journal of Lipid Research , 2008, 49, 1466-1476 Extended abbreviation naming convention. According to INCI nomenclature, D- erythro Dihydrosphingosine can also be represented by the letter G.

[0080] 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 described by Motta et al. Biochim Biophys Acta. Developed by Rabionet, 1993, 1182:147-151 Biochim Biophys Acta. 2014, 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- erythro Sphingosine (S), 6-hydroxy-D- erythro Sphingosine (H), D-ribose-phytosphingosine (P) and D- erythro 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- erythro The INCI name for dihydrosphingosine.

[0081] The glycosyl moiety of the glycoside according to the invention 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 bonded 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.

[0082] 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.

[0083] 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.

[0084] In some embodiments, for glycosides of formulas (1), (3), (4), or (5)-(12), the glycosyl moiety X is selected from Gal1-, or has one or more terminal galactose units and / or one or more terminal galactose units. N The glycosyl moiety of -acetylgalactosamine unit and / or one or more terminal sialic acid units.

[0085] In some preferred embodiments, for glycosides of formula (1), (3) or (4) or (5)-(12), the glycosyl moiety X is a glycosyl moiety selected from Gal1- and Galβ1-4Glc1-.

[0086] In some preferred embodiments, for glycosides of formulas (1), (3) or (4) or (5)-(12), the glycosyl moiety X is a glycosyl moiety selected from the following glycosyl moieties or their salts: Neu5Acα2-3Galβ1-4Glc-, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-.

[0087] In some preferred embodiments, the glycoside of formula (1) is the glycoside of formula (3), and wherein the glycoside of formula (3) is the glycoside of formula (5).

[0088] In some preferred embodiments, the glycoside of formula (1) is the glycoside of formula (5).

[0089] In some embodiments, the X of the glycoside in formula (5) is selected from Gal1- or Galβ1-4Glc1-. Therefore, in some preferred embodiments, the glycoside in formula (3) is the glycoside in formula (5), wherein the glycoside in formula (5) is selected from sphingosine galactoside or lactosylsphingosine.

[0090] In some preferred embodiments, the X of the glycoside of formula (5) is selected from Neu5Acα2-3Galβ1-4Glc-, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, or a salt thereof. Therefore, in some preferred embodiments, the glycoside of formula (3) is a glycoside of formula (5), wherein the glycoside of formula (5) is selected from... N -lyso-GM3 and N -lyso-GM1a.

[0091] N -lyso-GM3 and N -lyso-GM1a represents lysosphingolipids. Lysosphingolipids are generally defined as the breakdown products of sphingolipids, lacking the amide-linked fatty acyl group at the 2-position of their sphingosine base backbone. Therefore, for each parent sphingolipid, a corresponding lysosphingolipid exists, possessing the same head group at the 1-position but lacking the amide-linked fatty acyl group at the 2-position (Hannun et al.). Science 1989, 243, 500-507).

[0092] In some embodiments, the sialylation of the glycosides according to the invention is carried out in the presence of cyclodextrin.

[0093] 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 1-6 Heteroalkyl, 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.

[0094] In some embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a derivative thereof.

[0095] 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.

[0096] This invention describes a method for sialylation of glycosides, wherein sialylation is typically performed as part of a sialyl transferase cycle, comprising a CMP-sialyl recycling system, wherein CMP-sialyl is generated / regenerated from sialyl and CMP.

[0097] CMP-sialic acid is a relatively expensive glyconucleotide. Therefore, in-situ generation and regeneration of sialic acid donors have economic advantages and enable large-scale production.

[0098] The sialyl transferase cycle described in this invention typically comprises sialic acid, cytidine monophosphate (CMP), nucleoside triphosphate, and one or more cell-free extracts using microorganisms, wherein the one or more cell-free extracts contain the enzymatic activity required for the sialyl transferase cycle. The enzymatic activities required for the sialyl transferase cycle include: - At least one phosphotransferase activity, - At least one inorganic bisphosphatase activity, - At least one cytidine monophosphate kinase activity, - at least oneN -Acylneuraminic acid cytidine transferase activity, and - At least one sialyl transferase activity.

[0099] 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).

[0100] In some preferred embodiments, the nucleoside triphosphate is adenosine-5'-triphosphate (ATP).

[0101] Therefore, in some preferred embodiments, the sialyl transferase cycle includes N - Acetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), and one or more cell-free extracts of microorganisms, wherein the one or more cell-free extracts comprise a polypeptide with cytidine monophosphate kinase activity (CMK) (for phosphorylation of CMP), a polypeptide with phosphotransferase activity (for phosphorylation of CDP), a polypeptide with N-acylneuraminic acid cytidine transferase activity (CSS) (for transferring CMP from CTP to Neu5Ac), a polypeptide with sialyl transferase activity (for transferring Neu5Ac from CMP-Neu5Ac to the receptor substrate), and a polypeptide with inorganic diphosphatase activity (PPase) (for degrading inorganic pyrophosphate (PPi) formed during the cycle). Furthermore, the polypeptide with phosphotransferase activity and the polypeptide with inorganic diphosphatase activity are endogenously expressed by the microorganism. The sialyl transferase cycle described in this preferred embodiment is as follows: Figure 1 As shown.

[0102] In some embodiments, the sialyltransferase cycle also includes the regeneration of ATP, wherein the regeneration of ATP is achieved by using a phosphate source and a polypeptide with kinase activity.

[0103] 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.

[0104] In some implementations, ATP regeneration is achieved by using polyphosphate as a phosphate source and a polypeptide with polyphosphatase kinase activity.

[0105] Therefore, in some embodiments, the sialyl transferase cycle includes: N- Acetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), polyphosphates, and one or more cell-free extracts of microorganisms, wherein one or more cell-free extracts contain a polypeptide with cytidine monophosphate kinase activity (CMK) (for CMP phosphorylation), a polypeptide with phosphotransferase activity (for CDP phosphorylation), and a polypeptide with... N The peptides include α-acyl-neuraminidine cytidine transferase (CSS) activity (for transferring CMP from CTP to Neu5Ac), sialyltransferase activity (for transferring Neu5Ac from CMP-Neu5Ac to the receptor substrate), polyphosphatase kinase (PPK) activity (for phosphorylation of ADP), and inorganic bisphosphatase (PPase) activity (for degrading inorganic pyrophosphate (PPi) formed during the cycle). The phosphatase activity peptides and inorganic bisphosphatase activity peptides (PPase) are expressed endogenously by the microorganism. The sialyltransferase cycle described in this embodiment is as follows: Figure 2 As shown.

[0106] In some preferred embodiments, one or more cell-free extracts have reduced or no β-galactosidase activity.

[0107] Typically, one or more cell-free extracts are derived from microorganisms, wherein the microorganisms contain at least one endogenous polypeptide with phosphotransferase activity and at least one endogenous polypeptide with bisphosphatase activity, and wherein the microorganisms are genetically engineered to express one or more polypeptides selected from: - At least one polypeptide with cytidine monophosphate kinase activity, - At least one polypeptide having N-acyl-neuraminidine cytidine transferase activity, - At least one polypeptide with sialyl transferase activity.

[0108] In some embodiments, one or more cell-free extracts are derived from microorganisms, wherein the microorganisms are genetically engineered to express and / or overexpress one or more polypeptides selected from the following: - At least one endogenous polypeptide with phosphotransferase activity, - At least one endogenous polypeptide with bisphosphatase activity, - At least one polypeptide with cytidine monophosphate kinase activity, - At least one polypeptide having N-acyl-neuraminidine cytidine transferase activity, - At least one polypeptide with sialyl transferase activity.

[0109] At least one polypeptide with cytidine monophosphate kinase activity, at least one polypeptide with N The polypeptide with α-acyl-neuraminidine cytidine transferase activity and at least one polypeptide with sialyl transferase activity can be endogenous or heterologous polypeptides.

[0110] In some embodiments, one or more cell-free extracts are derived from microorganisms, wherein the microorganisms comprise at least one endogenous polypeptide with phosphotransferase activity and at least one endogenous polypeptide with bisphosphatase activity, and wherein the microorganisms are genetically engineered to express one or more polypeptides selected from: - At least one heterologous polypeptide with cytidine monophosphate kinase activity, - At least one heterologous polypeptide with N-acyl-neuraminidine cytidine transferase activity, - At least one heterologous polypeptide with sialyl transferase activity.

[0111] In some embodiments, one or more cell-free extracts are derived from microorganisms, wherein the microorganisms are genetically engineered to express and / or overexpress one or more polypeptides selected from the following: - At least one endogenous polypeptide with phosphotransferase activity, - At least one endogenous polypeptide with bisphosphatase activity, - At least one heterologous polypeptide with cytidine monophosphate kinase activity, - At least one heterologous polypeptide having N-acylneuraminic acid cytidine transferase activity; - At least one heterologous polypeptide with sialyl transferase activity.

[0112] The microorganisms according to the invention preferably contain reduced or absent β-galactosidase activity. Reduction or knockout of β-galactosidase activity can be achieved, for example, by genetic manipulation of the gene encoding a polypeptide having β-galactosidase activity, such as by introducing a mutation that results in the expression of an inactive enzyme, gene knockout, or other methods.

[0113] The microorganism according to the present invention can be yeast or bacteria, preferably bacteria.

[0114] In some implementations, the microorganism is Escherichia coli (E. coli) E. coli ).

[0115] In some implementations, the microorganism is Escherichia coli strain BL21(DE3).

[0116] Those skilled in the art will understand that, in the context of this invention, the term "microorganism" is intended to encompass living cells, such as bacterial or yeast cells, and may include one or more variants of said living cells (also referred to herein as "strains" of said microorganisms). The term "strain" also includes microbial variants artificially (recombinantly) constructed by genetic engineering of said microorganisms.

[0117] In some implementations, one or more strains of microorganisms are constructed by genetically engineering Escherichia coli strain BL21(DE3).

[0118] In some implementations, the microorganism comprises a strain constructed by genetic engineering of Escherichia coli BL21(DE3).

[0119] In some implementations, the microorganisms comprise two strains of Escherichia coli BL21(DE3) that have been genetically engineered.

[0120] In some preferred embodiments, the microorganisms comprise three strains constructed through genetic engineering of Escherichia coli BL21(DE3).

[0121] In some implementations, the microorganisms comprise four strains constructed through genetic engineering of Escherichia coli BL21(DE3).

[0122] Escherichia coli BL21(DE3) cells are available from existing manufacturers such as ThermoFischer Scientific.

[0123] Typically, one or more strains of microorganisms are used, via methods known to those skilled in the art (e.g., Cole et al.). Synthetic and Systems Biotechnology (2020, 5, 252-267) produces one or more cell-free extracts.

[0124] Using one or more cell-free extracts offers several advantages, such as avoiding additional purification steps for enzyme isolation and reducing the amount of specific enzymes used during the sialylation cycle by utilizing the activity of enzymes naturally present in microorganisms, thus making the sialylation process cost-effective and suitable for the industrial production of sialylated glycosides.

[0125] In some embodiments, the method includes using a cell-free extract of a microorganism, wherein the cell-free extract comprises a polypeptide having cytidine monophosphate kinase activity, and having N The polypeptide with α-acylneuraminic acid cytidine transferase activity, and one or more enzymes with sialyl transferase activity, wherein the cell-free extract is derived from microorganisms that endogenously express a polypeptide with inorganic diphosphatase activity and at least one polypeptide with phosphotransferase activity.

[0126] In some embodiments, the method includes using two cell-free extracts of microorganisms, wherein the first cell-free extract comprises a polypeptide having cytidine monophosphate kinase activity and a... N The first is a polypeptide with acyl-neuraminidine cytidine transferase activity, and the second cell-free extract contains at least one polypeptide with sialyl transferase activity, wherein both cell-free extracts are derived from microorganisms that endogenously express a polypeptide with inorganic diphosphatase activity and at least one polypeptide with phosphotransferase activity.

[0127] In some embodiments, the method includes using three cell-free extracts of the microorganism, wherein the first cell-free extract contains a polypeptide having cytidine monophosphate kinase activity, the second cell-free extract contains... N The third cell-free extract contains a polypeptide with sialyl neuraminic acid cytidine transferase activity, and all three cell-free extracts are derived from microorganisms that endogenously express a polypeptide with inorganic diphosphatase activity and at least one polypeptide with phosphotransferase activity.

[0128] In some embodiments, the method includes using four cell-free extracts of microorganisms, wherein the first cell-free extract contains a polypeptide having cytidine monophosphate kinase activity, the second cell-free extract contains... N The first cell-free extract contains a polypeptide with acyl-neuraminidine cytidine transferase activity, the second cell-free extract contains a polypeptide with sialyl transferase activity, and the third cell-free extract contains a polypeptide with sialyl transferase activity, wherein the four cell-free extracts are derived from microorganisms that endogenously express polypeptides with inorganic diphosphatase activity and at least one polypeptide with phosphotransferase activity.

[0129] The term "polypeptide with sialyl transferase activity" may be used interchangeably with the term "sialyl transferase," and in the context of this invention, it refers to an enzyme belonging to class EC 2.4.99.

[0130] Sialotransferases applicable to the context of this invention are those capable of catalyzing the transfer of sialic acid residues to β-linked galactose residues of glycoside receptors. O -3 and / or α-2-3 linked sialic acid residues of glycoside receptors O -8 position sialyltransferase.

[0131] Wild-type sialyltransferases can be derived from microorganisms such as bacteria, yeast, ascomycetes, actinomycetes, filamentous fungi, basidiomycetes, or mammals.

[0132] Wild-type sialyltransferase can be derived from Trehalobacterium biberstein (Bibersteinia trehalosi ), Neisseria meningitidis ( Neisseria meningitidis ), Vibrio bacteria ( Vibrio sp. Pasteurella multocida ( ) Pasteurella multocida ) and / or Campylobacter jejuni ( Campilobacter jejuni ).

[0133] 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.

[0134] In some embodiments, the polypeptide with sialidyltransferase activity is derived from Campylobacter jejuni ( Campylobacter jejuni Wild-type α-2,3 / α-2,8-sialyltransferases from strain OX=197. The amino acid sequence of the wild-type α-2,3 / α-2,8-sialyltransferases from Campylobacter jejuni strain OX=197 corresponds to the amino acid sequence of accession number Q9LAK3 (https: / / www.ncbi.nlm.nih.gov / protein / ).

[0135] In some embodiments, the polypeptide having sialyltransferase activity is a mutant of wild-type α-2,3 / α-2,8-sialyltransferase Q9LAK3, wherein the mutant preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1, wherein the mutant contains the following mutation / modification compared to the wild type: Ile53Ser, with a 32-amino acid deletion at the C-terminus.

[0136] In some embodiments, the polypeptide with sialyltransferase activity is a mutant of wild-type α-2,3 / α-2,8-sialyltransferase Q9LAK3, wherein the mutant preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2, and wherein, compared with the wild type, the mutant contains the following mutation / modification: Ile53Ser, N -Terminal histidine tag, 32 amino acids deleted at the C-terminus.

[0137] In some embodiments, the polypeptide with sialidyltransferase activity is a mutant of the wild-type α-2,3 / α-2,8-sialidyltransferase derived from Campylobacter jejuni strain OX=197 (Q9LAK3), wherein the mutant preferably comprises or consists of the amino acid sequence of SEQ ID NO: 3, and wherein, compared with the wild type, the mutant contains the following mutation / modification: Ile53Gly, N -Terminal histidine tag, C-terminal deletion of 32 amino acids.

[0138] α-2,3 / α-2,8-sialoyltransferases or their functional analogs derived from Campylobacter jejuni can also be called CST-II.

[0139] In some embodiments, the polypeptide having sialyltransferase activity is an α-2,3-sialyltransferase derived from the *Trebrospinal trehalose* strain DSM 23101 or a functional analog thereof.

[0140] In some embodiments, the polypeptide possessing sialidyltransferase activity is a wild-type α-2,3-sialotyltransferase derived from *Trebrospinal trehalose* strain DSM 23101. The amino acid sequence of the wild-type *Trebrospinal trehalose* α-2,3-sialotyltransferase corresponds to the amino acid sequence of accession number WP_0252672561 (https: / / www.ncbi.nlm.nih.gov / protein).

[0141] In some embodiments, the polypeptide with sialyltransferase activity is a mutant derived from wild-type α-2,3-sialyltransferase WP_025267256, wherein the mutant contains the following mutations / modifications compared to the wild type: N -Terminal histidine tag MGHHHHHH.

[0142] Derived from trehalose-bibstein bacteria ( Bibersteinia trehalose α-2,3 sialyltransferase or its functional analogues can also be called... Bt SiaT.

[0143] The term “polypeptide with cytidine monophosphate kinase activity” may be used interchangeably with the term “CMP kinase” or “CMK”, and in the context of this invention, refers to an enzyme of class EC 2.7.4.25, which typically uses ATP as the preferred phosphate donor to catalyze the phosphorylation of CMP (or dCMP).

[0144] Wild-type CMK can be derived from Mycobacterium tuberculosis ( Mycobacterium tuberculosis ), Escherichia coli, Yersinia pseudotuberculosis ( Yersinia pseudotuberculosis ) or Bacillus subtilis ( Bacillus subtilis ).

[0145] Wild-type CMP kinases can be derived from any known CMP kinase sequence or from any unidentified CMP kinase sequence. Unidentified CMP kinases can be identified using sequence databases and sequence alignment algorithms, such as the publicly available GenBank database and the BLAST alignment algorithm.

[0146] In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a CMP kinase derived from Mycobacterium tuberculosis or a functional analogue thereof.

[0147] In some embodiments, the polypeptide having CMK kinase activity is a wild-type CMK kinase derived from Mycobacterium tuberculosis. The amino acid sequence of the wild-type CMP kinase derived from Mycobacterium tuberculosis corresponds to the amino acid sequence of accession number WP_129368399 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0148] In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a mutant derived from wild-type CMP kinase WP_129368399, wherein the mutant contains the following mutation / modification compared to the wild type: N-terminal histidine tag MGHHHHHHH.

[0149] CMP kinase or its functional analogues derived from Mycobacterium tuberculosis can also be called... Mt CMK.

[0150] In some preferred embodiments, the enzyme having cytidine monophosphate kinase activity is a CMP kinase derived from Bacillus subtilis or a functional analogue thereof.

[0151] In some embodiments, the enzyme having cytidine monophosphate kinase activity is a CMP kinase derived from Bacillus subtilis strain 168 or a functional analogue thereof.

[0152] In some embodiments, the enzyme having cytidine monophosphate kinase activity is a wild-type CMP kinase derived from Bacillus subtilis strain 168. The amino acid sequence of the wild-type CMP kinase derived from Bacillus subtilis strain 168 corresponds to the amino acid sequence of accession number AAC83961 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0153] In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a mutant derived from wild-type CMP kinase AAC83961, wherein the mutant contains the following mutation / modification compared to the wild type: N-terminal histidine tag MGHHHHHHH.

[0154] CMP kinase derived from Bacillus subtilis or its functional analogues can also be called... Bs CMK.

[0155] The term "having" N The term "peptide with α-acyl-neuraminidine cytyltransferase activity" can be related to the term "peptide with α-acyl-neuraminidine cytyltransferase activity". N"-Acylneuraminic acid cytidine transferase" or "CSS" is used interchangeably, and in the context of this invention, refers to an enzyme of class EC 2.7.7.43 that catalyzes the transfer of CMP from CTP to... N - Acetylneuraminic acid (Neu5Ac).

[0156] Wild-type CSS can originate from any known CSS sequence or any CSS sequence that has not yet been identified. Unidentified CSS can be identified using sequence databases and sequence alignment algorithms, such as the publicly available GenBank database and the BLAST alignment algorithm.

[0157] In some implementations, having N The polypeptide with α-acylneuraminic acid cytyltransferase activity is CSS derived from Neisseria meningitidis or its functional analogues.

[0158] In some implementations, having N The polypeptide with α-acyl-neuraminidine cytidine transferase activity is derived from the wild-type CSS of Neisseria meningitidis. The amino acid sequence of the wild-type CSS of Neisseria meningitidis corresponds to the amino acid sequence of accession number WP_061726245 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0159] In some implementations, having N The polypeptide with α-acylneuraminic acid cytyltransferase activity is a mutant derived from wild-type CSS WP_061726245, which contains or consists of the amino acid sequence of SEQ ID NO: 4, wherein, compared with the wild type, the mutant contains the following mutation / modification: N-terminal histidine tag MGHHHHHHH.

[0160] Originating from Neisseria meningitidis N -Acyl-neuraminidyltransferase or its functional analogues can also be called... Nm CSS.

[0161] The term "polypeptide with inorganic bisphosphatase activity" may be used interchangeably with the terms "inorganic bisphosphatase" or "PPase," and in the context of this invention, it refers to an enzyme of class EC 3.6.1.1 that catalyzes the hydrolysis of pyrophosphate (PPi).

[0162] In some preferred embodiments, the polypeptide having inorganic bisphosphatase activity is a wild-type PPase derived from *Escherichia coli*. The amino acid sequence of the wild-type inorganic bisphosphatase derived from *E. coli* corresponds to the amino acid sequence of accession number WP_073849715 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0163] Inorganic bisphosphatases derived from Escherichia coli can also be called... Ec PPase.

[0164] "Polypeptides with phosphotransferase activity" are selected from polypeptides with nucleoside diphosphate kinase activity and / or polypeptides with myokinase activity.

[0165] In some embodiments, a "peptide with phosphotransferase activity" is a peptide with nucleoside diphosphate kinase activity.

[0166] The term "polypeptide with nucleoside diphosphate kinase activity" may be used interchangeably with the term "nucleoside diphosphate kinase" or "NDK," and in the context of this invention, it refers to an enzyme of class EC 2.7.4.6 that catalyzes the phosphorylation of nucleoside diphosphates.

[0167] In some preferred embodiments, the polypeptide having nucleoside diphosphate kinase activity is a wild-type NDK derived from Escherichia coli strain BL21(DE3). The amino acid sequence of the wild-type NDK derived from Escherichia coli strain BL21(DE3) corresponds to the amino acid sequence of accession number ACT44230 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0168] Nucleoside diphosphatase derived from Escherichia coli strain BL21(DE3) can also be simply referred to as Ec NDK.

[0169] In some embodiments, the polypeptide with phosphotransferase activity is a polypeptide with myokinase activity.

[0170] The term "polypeptide with myokinase activity" may be used interchangeably with the terms "myokinase," "adenosine kinase," or "ADK," and in the context of this invention, refers to an enzyme of class EC 2.7.4.3 that catalyzes the interconversion of various adenosine phosphates (e.g., ATP, ADP, and AMP).

[0171] In some embodiments, the polypeptide with myokinase activity is a wild-type myokinase derived from Escherichia coli strain BL21(DE3). The amino acid sequence of the wild-type ADK derived from Escherichia coli strain BL21(DE3) corresponds to the amino acid sequence of accession number ACT42324 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0172] Myokinase derived from Escherichia coli strain BL21(DE3) can also be abbreviated as... Ec ADK.

[0173] The term "enzyme with amylase activity" is used interchangeably with the term "amylase," and in the context of this invention, it refers to an enzyme belonging to class EC 3.2.1 that typically catalyzes the hydrolysis of S- and / or O-glycosyl compounds. Wild-type forms of amylase can be derived from microorganisms such as bacteria, yeasts, ascomycetes, actinomycetes, filamentous fungi, basidiomycetes, etc.

[0174] Wild-type amylases can be derived from Bacillus thermophilus and oleophilus (Bacillus) Geobacillus thermoleovorans ), Yellow fever anaerobic spores ( Anoxybacillus flavithermus ) or fireball bacteria ( Pyrococcus furiosus ).

[0175] Wild-type amylases can be derived from microorganisms with a vector that has been linked to or inserted with a gene encoding wild-type amylase.

[0176] Wild-type amylases can originate from any known amylase sequence or from any unidentified amylase sequence. Unidentified amylases can be identified using sequence databases and sequence alignment algorithms, such as the publicly available GenBank database and the BLAST alignment algorithm.

[0177] In some embodiments, the enzyme with amylase activity is a wild-type amylase derived from *Bacillus thermophilus* or a functional analogue thereof. The amino acid sequence of the wild-type amylase derived from *Bacillus thermophilus* corresponds to the amino acid sequence of accession number AFM43699 (https: / / www.ncbi.nlm.nih.gov / protein / ).

[0178] In the context of this invention, amylases derived from Bacillus thermophilus and oleophilus may also be referred to as maltodextrin-producing amylases or... Gt CDase.

[0179] In some embodiments, the enzyme with amylase activity is a wild-type amylase derived from *Bacillus flavus* or a functional analogue thereof. The amino acid sequence of the wild-type amylase derived from *Bacillus flavus* corresponds to the amino acid sequence of accession number AMB26774 (https: / / www.ncbi.nlm.nih.gov / protein / ).

[0180] In the context of this invention, amylases derived from *Bacillus flavus* can also be referred to as cyclic maltodextrinase or... Af CDase.

[0181] In some embodiments, the enzyme with amylase activity is a wild-type amylase derived from *Vallococcus* or a functional analogue thereof. The amino acid sequence of the wild-type amylase derived from *Vallococcus* corresponds to the amino acid sequence of accession number WP_011013079 (https: / / www.ncbi.nlm.nih.gov / protein / ).

[0182] In the context of this invention, amylases derived from *Pyrococcus* may also be referred to as α-amylases or... Pf CDase.

[0183] 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.

[0184] In some embodiments, the enzyme with polyphosphate kinase activity is derived from *Thermus rubrum* (a type of fungus). Meiothermus ruber PPK of strain DSM 1279, or its functional analogues.

[0185] In some embodiments, the enzyme with polyphosphate kinase activity is a wild-type PPK derived from the *Subthermomyces rubrum* strain DSM 1279. The amino acid sequence of the wild-type PPK derived from *Subthermomyces rubrum* strain DSM 1279 corresponds to the amino acid sequence of accession number ADD29239 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0186] In some embodiments, the polypeptide with polyphosphoric acid kinase activity is a mutant derived from wild-type polyphosphoric acid kinase ADD29239, wherein the mutant contains the following mutation / modification compared to the wild type: N-terminal histidine tag MGHHHHHHH.

[0187] Polyphosphoryl kinases derived from *Thermomyces rubrum* or their functional analogues can also be called... Mr PPK.

[0188] One or more cell-free extracts according to the invention comprise polypeptides or enzymes having the activity required to perform the sialyl transferase cycle. Non-limiting examples of polypeptides or enzymes having the activity required to perform the sialyl transferase cycle are summarized in Table 2, wherein more than one polypeptide or enzyme may be appropriate for a particular enzyme activity.

[0189] Table 2: Overview of suitable enzymes for the sialic acidification cycle In some embodiments, the method includes using three cell-free extracts of Escherichia coli strain BL21(DE3), wherein the first cell-free extract contains wild-type... MtCMK (GenBank accession number: WP_129368399), the second cell-free extract contains SEQ ID NO: 4. Nm CSS, the third cell-free extract, comprises CSTII selected from SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the *E. coli* strain BL21(DE3) endogenously expresses the following enzymes: Ec PPase (GenBank login number: WP_073849715) Ec NDK (GenBank login number: ACT44230) and EcADK (GenBank login number: ACT42324).

[0190] In some embodiments, the method includes using three cell-free extracts of Escherichia coli strain BL21(DE3), wherein the first cell-free extract contains wild-type... Bs CMK (GenBank accession number: AAC83961), the second cell-free extract contains SEQ ID NO: 4. Nm CSS, the third cell-free extract, comprises CSTII selected from SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the *E. coli* strain BL21(DE3) endogenously expresses the following enzymes: Ec PPase (GenBank login number: WP_073849715) Ec NDK (GenBank login number: ACT44230) and Ec ADK (GenBank login number: ACT42324).

[0191] In some embodiments, the method includes using three cell-free extracts of Escherichia coli strain BL21(DE3), wherein the first cell-free extract contains wild-type... Mt CMK (GenBank accession number: WP_129368399), the second cell-free extract contains SEQ ID NO: 4. Nm CSS, the third cell-free extract containing wild type Bt SiaT (GenBank accession number: WP_025267256), and the Escherichia coli strain BL21(DE3) endogenously expresses the following enzymes: Ec PPase (GenBank login number: WP_073849715) EcNDK (GenBank login number: ACT44230) and Ec ADK (GenBank login number: ACT42324).

[0192] In some embodiments, the method includes using three cell-free extracts of Escherichia coli strain BL21(DE3), wherein the first cell-free extract contains wild-type... Bs CMK (GenBank accession number: AAC83961), the second cell-free extract contains SEQ ID NO: 4. Nm CSS, the third cell-free extract containing wild type Bt SiaT (GenBank accession number: WP_025267256), and the Escherichia coli strain BL21(DE3) endogenously expresses the following enzymes: Ec PPase (GenBank login number: WP_073849715) Ec NDK (GenBank login number: ACT44230) and Ec ADK (GenBank login number: ACT42324).

[0193] In some embodiments, the method includes using four cell-free extracts of Escherichia coli strain BL21(DE3), wherein the first cell-free extract contains wild-type... Mt CMK (GenBank accession number: WP_129368399), the second cell-free extract contains SEQ ID NO: 4. Nm The third cell-free extract contains CSTII selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; the fourth cell-free extract contains wild-type CSTII. Bt SiaT (GenBank accession number: WP_025267256), and the Escherichia coli strain BL21(DE3) endogenously expresses the following enzymes: Ec PPase (GenBank login number: WP_073849715) Ec NDK (GenBank login number: ACT44230), and Ec ADK (GenBank login number: ACT42324).

[0194] In some embodiments, the method includes using four cell-free extracts of Escherichia coli strain BL21(DE3), wherein the first cell-free extract contains wild-type... BsCMK (GenBank accession number: AAC83961), the second cell-free extract contains SEQ ID NO: 3. Nm CSS, the third cell-free extract contains CSTII of SEQ ID NO: 1 or SEQ ID NO: 2, and the fourth cell-free extract contains wild-type CSTII. Bt SiaT (GenBank accession number: WP_025267256), and the Escherichia coli strain BL21(DE3) endogenously expresses the following enzymes: Ec PPase (GenBank login number: WP_073849715) Ec NDK (GenBank login number: ACT44230) and Ec ADK (Login: ACT42324).

[0195] According to the present invention, the selection of sialyltransferase contained in one or more cell-free extracts depends on the selection of glycosyl receptors.

[0196] Typically, for glycosyl receptors with a terminal galactosyl unit, α-2,3-sialyltransferase or a combination of α-2,3-sialyltransferase and α-2,3 / α-2,8-sialyltransferase is selected, while for those glycosyl receptors with a glycosyl moiety containing a terminal sialic acid unit, α-2,3 / α-2,8-sialyltransferase is usually selected.

[0197] In some embodiments, the glycosyl receptor is sphingosine galactoside or lactosysphingosine, and α-2,3-sialyl transferase is selected, preferably wild-type. Bt SiaT (Login ID: WP_025267256).

[0198] In some embodiments, the glycosyl receptor is sphingosine galactoside or lactosysphingosine, and both α-2,3-sialyltransferase and α-2,3 / α-2,8-sialyltransferase are selected, preferably wild-type. Bt SiaT (accession number: WP_025267256), and a mutant CSTII selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0199] In some implementations, the glycosyl receptor is N -lyso-GM3 or N-lyso-GM1a, and select α-2,3 / α-2,8-sialylate transferase, preferably a mutant CSTII selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0200] 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 glycoside to be sialylated. Because the sialylation process allows for the regeneration of activated nucleotides, activated donor sugars, and the removal of generated PPi in the presence of catalytic amounts of enzyme, the process is limited by the concentrations or amounts of stoichiometric substances. The upper limit of reactant concentrations usable according to the method of the invention is determined by the solubility of these reactants. Preferably, the concentrations of activated nucleotides, phosphate donors, donor sugars, and enzymes are selected such that the glycosylation reaction continues until the acceptor is depleted.

[0201] The sialyl transferase cycle according to the method of the present invention may also include other components that promote sialyl transferase activity. These components may include divalent cations (e.g., Mg). 2+ or Mn 2+ The reaction medium may contain solubilizers (such as Triton or SDS) and organic solvents, such as methanol or ethanol, or cyclodextrin. Materials required for ATP regeneration, phosphate ions, etc.

[0202] In a preferred embodiment, sialylation is performed in the presence of cyclodextrin.

[0203] 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.

[0204] 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.

[0205] The use of cyclodextrins offers 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.

[0206] Preferably, when sialylation is performed in the presence of cyclodextrin, the method further includes using a polypeptide with amylase activity. The polypeptide with amylase activity is added to the sialylation mixture when a certain conversion rate of the glycoside is achieved, preferably when the glycoside conversion rate reaches at least about 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. The glycoside conversion rate can be determined by standard methods known to those skilled in the art. Typically, the glycoside conversion rate is determined by HPLC and can be expressed as mol.% or... wt. % is given.

[0207] In some embodiments, the present invention describes a method for sialylation of glycosides of formula (1) or their salts: in X represents the glycosyl moiety, which is preferably selected from Gal1-, or has one or more terminal galactose units and / or one or more terminal... N -The glycosyl moiety of an acetylgalactosamine unit and / or one or more terminal sialic acid units; Y is selected from hydroxyl groups, fluorides, or portions of formula (2) or their salts: in 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, 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, The method includes the following steps: - The glycoside of formula (1) is mixed with sialic acid, cytidine monophosphate, nucleoside triphosphate, cyclodextrin, and one or more cell-free extracts of microorganisms, wherein the microorganisms comprise one or more endogenous polypeptides with inorganic diphosphatase activity and one or more endogenous polypeptides with phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity, This allows for sialylation of the glycoside, and the process continues: - Add a polypeptide with amylase activity.

[0208] This polypeptide with amylase activity can be advantageously used to degrade cyclodextrin. Degradation of cyclodextrin facilitates the separation of sialylated glycosphingolipid products in high yield and purity.

[0209] Peptides with amylase activity can be provided as purified peptides, cell-free extracts, or lysates.

[0210] In some embodiments, the polypeptide with amylase activity is provided as a purified protein with a purity of about 50% to about 95%.

[0211] In some embodiments, the polypeptide with amylase activity is provided as a cell-free extract, wherein the cell-free extract contains about 5 wt% to about 70 wt% of the enzyme. Preferably, the cell-free extract contains about 20 wt% to about 70 wt% of the enzyme.

[0212] 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 chelating agents that bind to enzyme cofactors such as Mg. +2 or Mn +2 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, or 8.0.

[0213] 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.

[0214] The resulting reaction mixture is kept long enough for sialylation of a high percentage of receptors by sialyltransferase. Typically, the reaction is often allowed to proceed for about 8 to about 240 hours, preferably about 24 to 48 hours.

[0215] N Acetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), polyphosphates, cell-free extracts, 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.

[0216] In the context of this invention, the enzyme activity of cell-free extracts 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. The enzyme activity of cell-free extracts can be measured according to the methods described in the following examples.

[0217] According to the method of the present invention, sialylation of the glycoside of formula (1) results in the formation of the sialylated glycoside of formula (13): in J is a glycosyl moiety containing one or more sialic acid units. Y is as defined for the glycosides in formula (1).

[0218] In some embodiments, the Y in the sialylated glycoside of formula (13) is a hydroxyl group. Therefore, in some embodiments, the sialylated glycoside of formula (13) is a sialylated sugar.

[0219] In some embodiments, the Y in the sialylated glycoside of formula (13) is a fluoride. Therefore, in some embodiments, the sialylated glycoside of formula (13) is the sialylated glycoside of formula (14): Where J is the glycosyl moiety as defined for sialylated glycosides of formula (13).

[0220] In some embodiments, the sialylated glycoside of formula (14) is an α-glycoside.

[0221] In some preferred embodiments, the Y of the sialylated glycoside of formula (13) is a portion of formula (2) or a salt thereof. Therefore, in some preferred embodiments, the glycoside of formula (13) is a glycoside of formula (15) or a salt thereof: in J is the glycosyl moiety as defined for sialylated glycosides of formula (13). 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, 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.

[0222] In some embodiments, for the sialylated glycoside of formula (15), R 1 For saturated unsubstituted C 10-17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0223] In some embodiments, for the sialylated glycoside of formula (15), R 1 For saturated unsubstituted C 10-17 Alkyl, R 2 For -OR 5 , where R 5 For hydrogen, R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0224] In some embodiments, for the glycoside of formula (15), R 1 For saturated unsubstituted C 10-17 Alkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a single key.

[0225] In some embodiments, for the glycoside of formula (15), R 1 C 10-17 1-Hydroxyalkyl, R 2 R 3 and R 4 It is hydrogen, and the bond is... It is a double bond.

[0226] In some embodiments, the sialylated glycoside of formula (13) is the sialylated glycoside of formula (15), wherein the sialylated glycoside of formula (15) is a glycoside selected from the glycosides of formulas (16), (17), (18) and (19): .

[0227] J is the glycosyl moiety as defined for sialylated glycosides of formula (13).

[0228] In some embodiments, for the sialylated glycoside of formula (15), 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.

[0229] In some embodiments, for the glycoside of formula (15), R 1 For saturated unsubstituted C 10-17 Alkyl, R 2 For -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.

[0230] In some embodiments, for the glycoside of formula (15), R 1 For saturated unsubstituted C 10 -C 17 Alkyl, R 2 and R 3For hydrogen, R 4 C with or without substitution 16-32 Acyl group, and bond It is a single key.

[0231] In some embodiments, for the glycoside of formula (15), 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.

[0232] In some embodiments, the glycoside of formula (15) is a glycosphingolipid selected from formulas (20), (21), (22), or (23): .

[0233] Where J is the glycosyl moiety as defined for sialylated glycosides in equation (13).

[0234] Sialidized glycosides of formulas (15)-(23) can also be called sialylated glycosphingolipids.

[0235] In some embodiments, the J of the sialylated glycosides of formulas (13)-(23) is selected from the following glycosyl moieties or salts thereof: Neu5Acα2-3Gal1-, Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1- or Neu5Acα2-8Neu5Acα2-3Galβ1-3GalNAcβ1-4Gal β1-4Glcβ1-.

[0236] In some embodiments, the J of the sialylated glycosides of formulas (13)-(23) is Neu5Acα2-3Galβ1-4Glcβ1.

[0237] In some embodiments, the sialylated glycoside of formula (13) is the sialylated glycoside of formula (16), and wherein the J of the sialylated glycoside of formula (16) is Neu5Acα2-3Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycoside of formula (13) is the sialylated glycoside of formula (16), and wherein the sialylated glycoside of formula (16) is... N -lyso-GM3.

[0238] In some embodiments, the J of the sialylated glycosphingolipids of formulas (13)-(23) is Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ1-.

[0239] In some embodiments, the sialylated glycoside of formula (13) is the sialylated glycosphingolipid of formula (16), and wherein the J of the sialylated glycosphingolipid of formula (16) is Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycoside of formula (13) is the sialylated glycoside of formula (16), and wherein the sialylated glycoside of formula (16) is... N -lyso-GD3.

[0240] In some embodiments, the sialylated glycoside of formula (13) is the sialylated glycoside of formula (16), and wherein the J of the sialylated glycosphingolipid of formula (16) is Neu5Acα2-8Neu5Acα2-3Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-. Therefore, in some embodiments, the sialylated glycosphingolipid of formula (13) is the sialylated glycosphingolipid of formula (16), and wherein the sialylated glycosphingolipid of formula (16) is... N -lyso-GD1a.

[0241] In some embodiments, according to the method of the invention, sialylation of the glycoside of formula (1) results in the formation of a sialylated glycoside, wherein the sialylated glycoside is a mixture of more than one sialylated glycoside with different degrees of sialylation.

[0242] In some embodiments, sialylated glycosides are N -lyso-GM3 and N A mixture of -lyso-GD3.

[0243] In some embodiments, sialylated glycosides are N -lyso-GM3 and N A mixture of -lyso-GD3, wherein the mixture contains N -lyso-GD3 and N The weight ratio of -lyso-GM3 is approximately 1:3.

[0244] In some embodiments, sialylated glycosides are N -lyso-GM3 and N A mixture of -lyso-GD3, wherein the mixture contains N -lyso-GD3 and N The weight ratio of -lyso-GM3 is approximately 1:1.

[0245] In some embodiments, sialylated glycosides are N -lyso-GM3 and N A mixture of -lyso-GD3, wherein the mixture contains N -lyso-GD3 and N The weight ratio of -lyso-GM3 is approximately 4:1.

[0246] In some embodiments, the method further includes separating sialylated glycosides.

[0247] In some embodiments, the present invention describes a method for sialylation of a glycoside of formula (1) or a salt thereof, the method comprising the steps of: - The glycoside of formula (1) is mixed with sialic acid, cytidine monophosphate, nucleoside triphosphate, cyclodextrin, and one or more cell-free extracts of microorganisms, wherein the microorganisms comprise one or more endogenous polypeptides with inorganic diphosphatase activity and one or more endogenous polypeptides with phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity, This allows the glycoside to be sialylated. - Separate the sialylated glycosides produced in the aforementioned steps.

[0248] In some embodiments, the present invention describes a method for sialylation of a glycoside of formula (1) or a salt thereof, the method comprising the steps of: - The glycoside of formula (1) is mixed with sialic acid, cytidine monophosphate, nucleoside triphosphate, cyclodextrin, and one or more cell-free extracts of microorganisms, wherein the microorganisms comprise one or more endogenous polypeptides with inorganic diphosphatase activity and one or more endogenous polypeptides with phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity, This allows for sialylation of the glycoside, and the process continues: - Add a peptide with amylase activity. - Separate the sialylated glycosides produced in the aforementioned steps.

[0249] Sialidized glycosides can be separated from the reaction mixture. Separation can be achieved by standard methods known to those skilled in the art, such as organic solvent extraction, chromatography, and / or ion-exchange chromatography. Methods for separating sialylated glycosphingolipids from enzymatic reaction mixtures are described, for example, in Bai et al.'s Current Protocols. 2021 , 1, e91. doi: 10.1002 / cpz1.91.

[0250] 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 about 100-150 kDa, 150-200 kDa, 200-250 kDa, or 250-300 kDa.

[0251] It is noteworthy that even though the MWCO of approximately 100-300 kDa is much higher than the molecular weight of sialylated glycosphingolipids, sialylated glycosphingolipids still accumulate in the DF throttling fluid (DFR).

[0252] It has been described that ganglioside GM1 can form 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 (DBGammak, Biochem J 1963, 88, 373). This property may prevent gangliosides (such as GM1) from permeating ultrafiltration membranes with a MWCO higher than that of GM1.

[0253] Surprisingly, the inventors have discovered that gangliosides lacking fatty acid chains... N -hemolytic form ( N Neither lysine 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.

[0254] 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.

[0255] A concentration step can be optionally performed after the DF step.

[0256] In some embodiments, the method further includes a step of concentrating DFR, wherein the step of concentrating DFR is performed.

[0257] 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.

[0258] DFRs rich in sialylated glycosphingolipids are subsequently spray-dried or spray-granulated.

[0259] In some preferred embodiments, the DFR containing the glycosphingolipid of formula (1) is spray-dried.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] Spray drying of the DFR retentate will result in a spray-dried powder containing sialylated glycosphingolipids.

[0264] 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.

[0265] The span of the particles is typically less than about 3, preferably less than about 2.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] Spray-dried powders with such low specific volume (i.e., less than 4 ml / g) are generally preferred because they have improved flow properties.

[0270] The glycosphingolipid content of the spray-dried powder obtained according to the method of the present invention will typically be at least about 65%. wt. %, usually at least about 70% wt. %, preferably at least about 75% wt. %, more preferably at least about 85% wt. %.

[0271] In some embodiments, the spray-dried powder contains at least about 50 wt. %of N -lyso-GD3 and N A mixture of -lyso-GM3, or at least about 60 wt. of N -lyso-GD3 and N A mixture of -lyso-GM3, or at least about 70 wt. %of N -lyso-GD3 and N A mixture of -lyso-GM3, or at least about 80 wt. %of N -lyso-GD3 and N A mixture of -lyso-GM3, and wherein the mixture contains N -lyso-GD3 and N The weight ratio of -lyso-GM3 is approximately 1:10 to approximately 10:1.

[0272] In some embodiments, the mixture contains N -lyso-GD3 and NThe weight ratio of -lyso-GM3 is approximately 1:3.

[0273] In some embodiments, the mixture contains N -lyso-GD3 and N The weight ratio of -lyso-GM3 is approximately 1:1.

[0274] In some embodiments, the mixture contains N -lyso-GD3 and N The weight ratio of -lyso-GM3 is approximately 4:1.

[0275] In some embodiments, the spray-dried powder contains about 40-55 wt. of N -lyso-GD3 and approximately 10-15 wt. of N -lyso-GM3, and the spray-dried powder therein also contains approximately 3-6 erythro %of N -lyso-GT3, approximately 4-6 wt. % lactose-D- erythro Sphingosine and approximately 0.1-1.0 wt. % of glucosyl D- wt. Sphingosine.

[0276] In some embodiments, the spray-dried powder contains about 15-20 wt. %of N -lyso-GD3 and approximately 50-60 wt. %of N -lyso-GM3, and the spray-dried powder therein also contains approximately 0.1-0.5 erythro %of N -lyso-GT3, approximately 4-7 wt. % lactose-D- erythro Sphingosine and approximately 0.1-1.0 wt. % of glucosyl D- wt. Sphingosine.

[0277] In some embodiments, the spray-dried powder contains about 35-40 wt. %of N -lyso-GD3 and approximately 25-40 wt. %of N -lyso-GM3, and the spray-dried powder therein also contains approximately 5-6 Gt % lactosyl D-erythrosine and approximately 0.5-1.0 Af % of glucosyl D-erythrosphoprotein.

[0278] In some embodiments, the glycosides and sialylated glycosides according to the invention can be utilized or generated in the form of salts, preferably in the form of pharmaceutically acceptable salts.

[0279] In some embodiments, the salt comprises the following cation: Na + K + Mg 2+ Ca 2+ NH4 + Et3NH + .

[0280] In some embodiments, the salt contains the following anion: Cl - ,Br - CH3CO2 - CO3 2- SO4 2- HPO4 - .

[0281] In some embodiments, the present invention describes a sialytizing agent comprising one or more cell-free extracts of microorganisms, said microorganisms comprising one or more endogenous polypeptides having inorganic bisphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity.

[0282] In some embodiments, the sialytic agent also comprises sialic acid, cytidine monophosphate, and nucleoside triphosphate.

[0283] Example The following working examples describe non-limiting embodiments of the present invention, which are given only for illustrative purposes.

[0284] General methods and materials LCMS analysis was performed using a Shimadzu ECO 2020 LC system coupled with a Shimadzu MS-2020 system. A sample (50 µL) was taken from the reaction mixture, mixed with DMSO (950 µL), centrifuged (16,000 rpm, 5 min), and analyzed using the Shimadzu ECO 2020 LC system coupled with a Shimadzu LCMS-2020 system. HPLC analysis was performed using a Merck Ascentis Express RP-Amide column (15 cm × 4.6 mm, 2.7 µm).

[0285] amylase Pf CDase E. coli CDase and Mt CDase was expressed from Escherichia coli strains according to the methods described in Metha et al., PLOS ONE 2013, 8, e73612, Aliakbari et al., Starch 2019, 71, 1800133, and Yand et al., Applied and Environmental Microbiology 2004, 70, 5988.

[0286] The following cell-free extracts were obtained from Escherichia coli (E. coli) Bs Production of expression strains: i. From wild-type cytidine monophosphate kinase (CMP) genetically engineered for expression of Mycobacterium tuberculosis. Bt CMK (Login: WP_129368399) Escherichia coli BL21(DE3) Cell-free extract of LacZ; ii. Wild-type cytidine monophosphate kinase derived from genetically engineered Bacillus subtilis expression ( Nm CMK, registration number: AAC83961) of Escherichia coli BL21(DE3) Cell-free extract of LacZ; iii. Derived from a mutant strain genetically engineered to express SEQ ID NO: 4 N Escherichia coli BL21(DE3) with α-acylneuraminic acid cytyltransferase Cell-free extract of LacZ; iv. Wild-type sialyltransferase from genetically engineered strain DSM 23101 for expressing trehalose-Bibresia bishops ( Ec SiaT, login: WP025267256) Escherichia coli BL21(DE3) Cell-free extract of LacZ; v. From *Escherichia coli* BL21(DE3) genetically engineered to express α-2,3 / α-2,8-sialyltransferase of SEQ ID NO: 1. Cell-free extract of LacZ.

[0287] vi. From *Escherichia coli* BL21(DE3) genetically engineered to express α-2,3 / α-2,8-sialyltransferase SEQ ID NO: 2. Cell-free extract of LacZ.

[0288] vii. From Escherichia coli BL21(DE3) genetically engineered to express α-2,3 / α-2,8-sialyltransferase SEQ ID NO: 3. Cell-free extract of LacZ.

[0289] Cell-free extracts (i)-(vii) were produced and characterized according to the steps described in Examples 1.1-1.3 and Examples 2.1-2.3.

[0290] Example 1: Cloning The gene encoding the enzyme is typically ordered as a codon-optimized synthetic gene for optimized expression in *E. coli* host strains. The synthetic construct contains a forked end with a BsaI restriction site for golden gate cloning into a pET28a-based expression vector (with the introduced BsaI restriction site and a fluorescent deletion cassette). The resulting plasmid is used to transform *E. coli* BL21(DE3). LacZ.

[0291] Example 2: Expression The preculture of the expression strain was prepared in 10 mL of LB medium supplemented with the appropriate antibiotic and incubated overnight at 37°C with shaking. The preculture was then diluted 1:100 to TB medium supplemented with the appropriate antibiotic, and the expression strain was cultured. The culture was incubated at 37°C until OD... 600 Reach 0.7-1.0. Cool the culture to the desired expression temperature, induce with 0.5 mM IPTG, and incubate for the desired expression time.

[0292] Example 3: Preparation of cell-free extract Cells were collected by centrifugation and resuspended in water. Cell lysis was achieved by sonication. The resulting lysed cell suspension was centrifuged to separate the cell-free extract containing soluble enzymes and cell debris. The supernatant containing the cell-free extract was freeze-dried to dryness.

[0293] Example 4: Activity measurement of α-2,3-sialyltransferase (α-2,3-SiaT) from cell-free extract To quantify the α-2,3-sialyltransferase activity from cell-free extracts, and to study the synthesis of lactosysphingosine from lactosylsphingosine... N The determination was performed using DMSO-GM3. The reaction was sampled at a given reaction time, the sample was quenched by adding DMSO, and the synthesized product was quantified by LC / MS. N The amount of α-2,3-SiaT3 was used to intermittently determine the reaction progress. One α-2,3-SiaT unit (U) corresponds to the reaction at 25 °C and pH 7.5, containing 20 mM Mg. 2+ In the solution, 1 μmol of lactosylsphingosine is converted to lactosylsphingosine per minute. N -lyso-GM3.

[0294] Example 5: Activity measurement of α-2,8-sialyltransferase (α-2,8-SiaT) from cell-free extracts To quantify the α-2,8-sialyltransferase activity from cell-free extracts, the activity of α-2,8-sialyltransferase from cell-free extracts was analyzed. N -lyso-GM3 Synthesis N The determination was performed using lyso-GD3. The reaction was sampled at a given reaction time, the sample was quenched by adding DMSO, and the synthesized product was quantified by HPLC. N The amount of α-lyso-GD3 was used to intermittently determine the reaction progress. One α-2,8-SiaT unit (U) corresponds to the reaction at 37°C and pH=8.0, containing 20 mM Mg. 2+ In the buffer solution, 1 μmol per minute N -lyso-GM3 is converted to N -lyso-GD3.

[0295] Example 6: Measurement of cytidine monophosphate kinase (CMK) activity in cell-free extracts To quantify the CMK activity in cell-free extracts, a pyruvate kinase-lactate dehydrogenase coupled enzyme method was used, and spectrophotometric readings were performed on NADH oxidation at 340 nm. This was described by Blodin et al., Anal. Biochem. 1994, 220, 219. One CMK unit (U) corresponds to the concentration of 20 mM Mg at 30 °C and pH 7.5. 2+ In a buffer solution of 0.5 M KCl, 1 µmol of CMP is converted to CDP per minute.

[0296] Example 7: Cell-free extract N Measurement of acetylneuraminic acid cytyltransferase (CSS) activity To quantify the cell-free extract Ec The activity of CSS (GenBank accession number: WP_061726245) was measured by determining the phosphate released by the hydrolysis of pyrophosphate (PPi) formed during the synthesis of CMP-sialic acid from sialic acid and cytidine triphosphate (CTP). Overexpression of endogenous inorganic bisphosphatase (CSS) was also performed. Ec Cell-free extracts of *E. coli* (PPase, GenBank accession number: WP_073849715.1) were added to the reaction mixture, and the reaction time was recorded. At selected time points, samples were taken and mixed with the working reagents (4 parts 10% ascorbic acid solution and 1 part 15 mM zinc acetate and 10 mM ammonium molybdate solution). After color development at 30°C for 10 minutes, the absorbance at 630 nm was measured. One CSS unit (U) corresponds to the hydrolysis of 1 µmol PPi per minute at 37°C in 50 mM TRIS buffer (pH=7.2), which in turn represents the conversion of 1 µmol CTP to CMP-sialic acid per minute under these conditions.

[0297] Example 8: Measurement of endogenous inorganic bisphosphatase (PPase) activity To determine the overexpression or endogenous expression in cell-free extracts Nm PPase (GenBank accession number: WP_073849715.1) activity was determined by recording the reaction time progression to measure the phosphate released from the hydrolysis of sodium pyrophosphate. Samples were taken at selected time points and mixed with the working reagent (containing four portions of 10% ascorbic acid solution and one portion of 15 mM zinc acetate and 10 mM ammonium molybdate solution). After color development at 30°C for 10 minutes, absorbance was measured at 630 nm. The reaction was carried out in TRIS buffer at pH 7.2. One inorganic bisphosphatase unit (U) corresponds to the hydrolysis of 1 µmol PPi per minute at 37°C in 50 mM TRIS buffer (pH 7.2).

[0298] Example 9: Measurement of endogenous nucleoside diphosphate kinase (NDK) activity To determine the endogenous content in cell-free extracts Ec NDK (GenBank accession number: YP_490746) activity was measured by determining the phosphate released by the hydrolysis of pyrophosphate (PPi) formed during the synthesis of CMP-sialic acid from sialic acid, cytidine diphosphate (CDP), and adenosine 5'-triphosphate (ATP). The expression... Gt CSS (GenBank login ID: WP_061726245) and AfCell-free extracts of *E. coli* containing PPase (GenBank accession number: WP_073849715) were added to the reaction mixture. The reaction time was recorded. At selected time points, samples were taken and mixed with the working reagent (containing four parts of 10% ascorbic acid solution and one part of 15 mM zinc acetate and 10 mM ammonium molybdate solution). After color development at 30°C for 10 minutes, the absorbance at 630 nm was measured. The reaction was carried out in TRIS buffer at pH 7.2. One NDK unit (U) corresponds to the hydrolysis of 1 µmol of PPi per minute at 37°C in 50 mM TRIS buffer (pH 7.2), which in turn corresponds to the conversion of 1 µmol of CDP to CTP per minute under these conditions.

[0299] Example 10: General Procedure for Sialization of Glycoside Receptors The sialyl transferase cycle is carried out in aqueous solution at a pH of about 7.0 to about 7.5, and at a temperature range of about 25°C to about 37°C. A typical reaction mixture contains a glycoside acceptor (1 equivalent (eq.)). N - Acetylneuraminic acid (Neu5Ac, 1.2–2.5 eq.), β-cyclodextrin (0.5 eq.), ATP (2.0–3.5 eq.), CMP (0.1–0.3 eq.), MgCl2 (0.5 M), and cell-free extracts containing the desired enzymes. Sialization cycling was monitored by LCMS (methods and conditions see Example 22). Conversion rates were typically 10–99%.

[0300] When the desired conversion rate is reached, add to the reaction mixture Pf CDase, or Gt CDase or Af CDase (1000 to 5000 U / L) and heated to 65°C.

[0301] Example 11: α- N -acetylceramide-(2 3)-O-β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 Production of 1'-D-erythrosphospirin (N-lyso-GM3) N -lyso-GM3 is produced using lactosylsphingosine as a glycoside receptor, following the general procedure described in Example 10, wherein the following three cell-free extracts are used: cell-free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), and cell-free extract (iv) (2960 U / L).

[0302] LC / MS: Rt = 4.38 min; for [C 41 H 74 N2O 20 Calculated ESI-MS: 914, Measured value: 915 [M+H] + 913 [MH] - .

[0303] Example 12: Generation of α-N-acetylneuraminoyl-(2-)- from lactosylsphingosine 8)- O -α- N -acetylceramide-(2 3)- O -β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 1')-D-erythrosphospirin ( N -lyso-GD3) N -lyso-GD3 is produced using lactosylsphingosine as a glycoside receptor, following the general steps described in Example 10, which utilize the following four cell-free extracts: cell-free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), cell-free extract (iv) (2960 U / L), and cell-free extract (v), or (vi) or (vii) (163-1000 U / L).

[0304] 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] - .

[0305] Example 13: From N -lyso-GM3 produces α-N-acetylneuraminoyl-(2-) 8)- O -α- N -acetylceramide-(2 3)- O -β-D-galactopyranosyl-(1 4)-β-D-glucopyranosyl-(1 1')-D-erythrosphospirin ( N -lyso-GD3) N -lyso-GD3 usage N -lyso-GM3, as a glycoside receptor, was produced according to the general steps described in Example 10, using the following three cell-free extracts: cell-free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), and cell-free extract (v), or (vi) or (vii) (163-500 U / L).

[0306] 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] - .

[0307] Example 14: N -lyso-GD3 and N Production of a mixture of -lyso-GM3 Different proportions N A mixture of -lyso-GD3 and N-lyso-GM3 is used N -lyso-GM3, as a glycoside receptor, was produced according to the general steps described in Example 10, wherein the following three cell-free extracts were used: cell-free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), and cell-free extract (v), or (vi), or (vii) (163-500 U / L). The sialylation cycle was monitored by LCMS, and when the desired... N When the -lyso-GM3 conversion rate (corresponding to a specific ratio) is reached, the reaction is quenched by enzyme inactivation (preferably via heat treatment).

[0308] Following the steps above, three mixtures are obtained, namely: - A 4:1 mixture of N-lyso-GD3 and N-lyso-GM3 - A 1:3 mixture of N-lyso-GD3 and N-lyso-GM3 - A 1:1 mixture of N-lyso-GD3 and N-lyso-GM3.

[0309] Example 15: α- N -acetylceramide-(2 3)-O-β-D-galactopyranosyl-(1 4) Formation of α-D-pyranose glucopyranosyl fluoride (3'SL-fluoride) 3'SL-fluoride is produced using α-D-lactosyl fluoride as a glycoside acceptor, following the general procedure described in Example 10, without the addition of amylase ( Pf CDase, or Gt CDase, or Af CDase) and a final heating step. The following three cell-free extracts were used: cell-free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), and cell-free extract (iv) (2960 U / L).

[0310] Example 16: α- N -acetylceramide-(2 8)- O -α- N -acetylceramide-(2 3)- O -β-D-galactopyranosyl-(1 4) Formation of β-D-glucopyranosyl fluoride The title compound was produced from α-D-lactosyl fluoride according to the general steps described in Example 10, without the addition of amylase ( Pf CDase, or wt. CDase, or wt. CDase) and the final heating step. The following cell-free extracts are used: cell-free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), cell-free extract (iv) (2960 U / L) and cell-free extract (v), or (vi) or (vii) (163-500 U / L).

[0311] Example 17: Isolation of sialylated glycosphingolipids The sialylated glycosphingolipids produced as described in Examples 10-14 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 During percolation (DF), maintain a flow rate of approximately 10 l / h, a transmembrane pressure of approximately 8–10 bar, a temperature of approximately 20–25 °C, and a pH of 7.0–7.5. Continue filtration until approximately 2–10 DF volumes have passed relative to the feed volume. During percolation, maintain a flow rate of approximately 15.3–18.1 l / m³. 2High 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 ​ % of one or more sialylated glycosphingolipids.

[0312] Example 18. 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 17 was dispersed in cyclohexane containing 0.1% soybean lecithin. The sample was sonicated to disperse aggregated particles before particle size measurement.

[0313] The moisture content of the spray-dried powder obtained in Example 17 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... ​ % of water.

[0314] Example 19. Contains N -lyso-GD3 and N -lyso-GM3 4:1 mixture spray-dried powder The product obtained according to the steps of Example 17 includes N -lyso-GD3 and N The DFR of a 4:1 mixture of -lyso-GM3 was spray-dried under the conditions of Example 17 to obtain a spray-dried powder with the following characteristics: Example 20. Includes N -lyso-GD3 and N -lyso-GM3 1:3 mixture spray-dried powder The product obtained according to the steps of Example 17 includes N -lyso-GD3 and NThe DFR of a 1:3 mixture of -lyso-GM3 was spray-dried under the conditions of Example 17 to obtain a spray-dried powder with the following characteristics: Example 21. Includes N -lyso-GD3 and N spray-dried powder of a 1:1 mixture of -lyso-GM3 The product obtained according to the steps of Example 17 includes N -lyso-GD3 and N The DFR of a 1:1 mixture of -lyso-GM3 was spray-dried under the conditions of Example 17 to obtain a spray-dried powder with the following characteristics: Example 22: LC / MS Analysis A sample (50 µL) was taken from the reaction mixtures of Examples 5.1 and 5.2, mixed with DMSO (950 µL), centrifuged (16,000 rpm, 5 min), and the supernatant was analyzed. 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 (filtered aqueous solution of 2 mM formic acid), using the following gradient: N -lysoGM3 70-95% (D in C). N -lyso-GD3 70-98% (D in C).

[0315] sequence list Overview of the present invention SEQ ID NO This disclosure should not be construed as limiting the described embodiments in any way, and those skilled in the art will foresee many possibilities for its modification.

[0316] The above embodiments are combined. The appended claims further illustrate specific embodiments of this disclosure.

Claims

1. A method for sialylation of glycosides or their salts of formula (1): in X represents a glycosyl moiety, wherein the glycosyl moiety is preferably selected from Gal1-, or has one or more terminal galactose units and / or one or more terminal... N -The glycosyl moiety of an acetylgalactosamine unit and / or one or more terminal sialic acid units; Y is selected from hydroxyl groups, fluorides, or portions of formula (2) or their salts: in 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, key In R 2 When it is hydrogen, it is a double bond or a single bond, or in R 2 For -OR 5 Time 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, The method includes: The glycoside of formula (1) is mixed with sialic acid, cytidine monophosphate, nucleoside triphosphate, and one or more cell-free extracts of microorganisms, wherein the microorganisms comprise one or more endogenous polypeptides with inorganic diphosphatase activity and one or more endogenous polypeptides with phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity, This allows the glycoside to be sialylated.

2. The method of claim 1, wherein the microorganism is genetically engineered to express one or more polypeptides selected from: - At least one polypeptide with cytidine monophosphate kinase activity, - At least one polypeptide having N-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity.

3. The method according to claim 1 or 2, wherein the microorganism is *Escherichia coli* (E. coli). Escherichiacoli ).

4. The method according to any one of claims 1 to 3, wherein the microorganism comprises one or two endogenous polypeptides having phosphotransferase activity and one endogenous polypeptide having inorganic diphosphatase activity.

5. The method according to claim 3 or 4, wherein the one or two endogenous polypeptides having phosphotransferase activity are selected from endogenous Escherichia coli nucleoside diphosphate kinase and / or endogenous Escherichia coli myokinase.

6. The method according to any one of claims 3 to 5, wherein the endogenous polypeptide having inorganic diphosphatase activity is an endogenous Escherichia coli inorganic diphosphatase.

7. The method according to any one of claims 3 to 6, wherein the one or more cell-free extracts comprise a polypeptide having cytidine monophosphate kinase activity, and wherein the polypeptide has an amino acid sequence derived from Mycobacterium tuberculosis (…). Mycobacterium tuberculosis ) or Bacillus subtilis ( Bacillus subtilis ( ) heterologous cytidine monophosphate kinase, or its functional analogues.

8. The method according to any one of claims 3 to 7, wherein the one or more cell-free extracts comprise a heterologous polypeptide having N -Acyl-neuraminidine cytidine transferase activity, and wherein the polypeptide is an amino acid sequence derived from Neisseria meningitidis ( Neisseria meningitidis heterogeneity N -Acylneuraminic acid cytidine transferase, or its functional analogues.

9. The method according to any one of claims 3 to 8, wherein the one or more cell-free extracts comprise a polypeptide having sialyl transferase activity, wherein the polypeptide has an amino acid sequence derived from Campylobacter jejuni (… Campylobacter jejuni Heterosial sialyl transferase of strain OX=197, or its functional analogue.

10. The method according to any one of claims 3 to 8, wherein the one or more cell-free extracts comprise a polypeptide having sialyl transferase activity, wherein the polypeptide has an amino acid sequence derived from *Trehalobacterium bibersteine* (TBI). Bibersteinia trehalosi Heterosial sialylate of strain DSM 23101, or its functional analogue.

11. The method according to any one of claims 3 to 8, wherein the one or more cell-free extracts comprise two polypeptides having sialyl transferase activity, and wherein the two polypeptides are heterologous sialyl transferases according to claims 9 and 10.

12. The method according to any one of claims 1 to 11, wherein the nucleoside triphosphate is adenosine 5'-triphosphate (ATP).

13. The method according to any one of claims 1 to 12, wherein the method further comprises using cyclodextrin.

14. The method of claim 13, wherein the cyclodextrin is β-cyclodextrin.

15. The method of claim 13 or 14, wherein the method further comprises using a polypeptide having amylase activity.

16. The method of claim 15, wherein the polypeptide having amylase activity is derived from the amino acid sequence of Bacillus thermophilus and oleophilus (Bacillus). Geobacillus thermoleovorans ), Yellow fever anaerobic spores ( Anoxybacillus flavithermus ) or fireball bacteria ( Pyrococcus furiosus (Amylase, or its functional analogues) 17. The method according to any one of claims 1 to 16, wherein the glycoside of formula (1) is a glycoside of formula (3): Where X is as defined for the glycosides of equation (1), the bond R 1 R 2 R 3 and R 4 As defined for the part of equation (2).

18. The method according to claim 17, wherein for the glycoside of formula (3), R 2 R 3 and R 4 It is hydrogen, and R 1 It is C 13-17 Alkyl group, preferably C 13 alkyl.

19. The method according to any one of claims 1 to 18, wherein X is a glycosyl moiety selected from Gal1β-, Galβ1-4Glc1β-, Neu5Acα2-3Galβ1-4Glc1β- or Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-.

20. The method according to any one of claims 17 to 19, wherein the glycoside of formula (3) is N -lyso-GM3.

21. The method according to any one of claims 1 to 20, wherein the method further comprises separating sialylated glycosides.

22. A sialytizing agent comprising one or more cell-free extracts of microorganisms, said microorganisms comprising one or more endogenous polypeptides having inorganic bisphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and The one or more cell-free extracts comprise: - At least one polypeptide with cytidine monophosphate kinase activity, - at least one with N Peptides with α-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity.

23. The sialylation agent of claim 22, wherein the microorganism is genetically engineered to express one or more polypeptides selected from: - At least one polypeptide with cytidine monophosphate kinase activity, and - At least one polypeptide having N-acyl-neuraminidine cytidine transferase activity, and - At least one polypeptide with sialyl transferase activity.

24. The saliva acidifying agent according to claim 21 or 22, wherein the microorganism is *Escherichia coli* (…). Escherichia coli ).

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