Sialylated milk-N-tetrasaccharide as well as synthesis method and application thereof

Abstract

The invention discloses sialylated milk-N-tetrasaccharide as well as a synthesis method and application of the sialylated milk-N-tetrasaccharide. The sialylated milk-N-tetrasaccharide is disialylated milk-N-tetrasaccharide and / or trisialylated milk-N-tetrasaccharide.

Description

Technical Field

[0001] The invention relates to a high-efficiency preparation method of sialylated lacto-N-tetraose and provides novel sialylated lacto-N-tetraose. Background Art

[0002] Carbohydrates in breast milk exist in various forms, including monosaccharides, oligosaccharides, glycoproteins, glycopeptides and glycolipids. Human milk oligosaccharides (HMOs) are a class of highly complex non-binding polysaccharides in breast milk, the third largest component after lactose and lipids, accounting for 1% of the total breast milk. Human milk oligosaccharides (HMOs) are abundant and unique in human milk. Currently, more than 200 different HMOs have been found in breast milk, of which about 150 structures have been characterized. In addition, the content of HMOs in human milk is 10 to 100 times higher than that in other mammals11, which is incomparable to cow's milk (<1g / L)

[12] . Some components, such as lacto-N-fucopentaose (LNFP), are present in high levels in human milk but not in cow's milk. The content of lacto-N-tetraose (LNT) and sialylated lacto-N-tetraose (LST) in human milk is relatively high, but extremely low in cow's milk. The difference in composition and content between human milk and cow's milk is also one of the reasons why breastfeeding is more advantageous than artificial feeding today.

[0003] HMOs have prebiotic effects and can maintain the balance of intestinal ecology; they are antibacterial and anti-adhesive, and HMOs can serve as soluble decoy receptors for pathogens or virulence agents (such as toxins); they mediate immune cell responses and regulate intestinal microorganisms and neonatal immunity. Taking one of the oligosaccharides, the tetrasaccharide Galpβ1-3GlcNAcpβ1-3Galpβ1-4Glc (lacto-N-tetraose, LNT) as an example, LNT acts as a bacterial receptor for pneumococci and is found to be used to identify the receptor specificity of glycosyltransferases, the substrate specificity of glycosidases, and the structure of antigenic determinants. In the human body, sialylated polysaccharide structures mediate or regulate many biological processes, including subcellular and cellular transport, cell-to-cell adhesion, signal transduction, and microbial attachment. At the same time, sialic acid is an essential nutrient for brain development, and sialylated HMOs are also the main source of sialic acid in the brain. Based on the important biological functions and physiological activities of the above-mentioned oligosaccharides, it is necessary to provide a large number of such compounds with uniform structures.

[0004] However, to date, there is no route to obtain a large amount of LNT and its sialylated derivatives, and this problem is not easy to overcome by using a variety of separation, biotechnology and synthesis methods. Since LNT and its sialylated derivatives are unique components in human milk, they do not exist in cow's milk or exist in trace amounts, and there are a large number of similar oligosaccharides in human milk, it is quite difficult to separate them from human milk even in milligram quantities. Therefore, obtaining such compounds by artificial synthesis becomes the best choice. However, since sialylated HMOs have complex structures, their chemical or (chemical) enzymatic synthesis is challenging and involves many difficulties, such as stereochemical control, formation of specific bonds, availability of raw materials, etc. Therefore, commercially available sialylated HMOs are very expensive due to the small number of their natural sources, especially derivatives of di / tri-sialic acid HMOs.

[0005] Up to now, the methods for obtaining oligosaccharides mainly include chemical synthesis, enzymatic synthesis and chemoenzymatic synthesis. The chemical method can be used to generate specific glycosidic bonds at any position, which makes up for the shortcomings of limited enzyme types and can flexibly synthesize natural and non-natural oligosaccharides, but the application of chemical methods inevitably increases the reaction steps and purification work. Enzymatic synthesis, enzyme-catalyzed glycosylation reaction, can make full use of the high stereoselectivity and regioselectivity of enzymes, avoid the cumbersome protection and deprotection operations in the chemical synthesis process, reduce the purification work and also improve the reaction yield. However, the building blocks synthesized by enzymes require further protection group operations for use in chemical glycosylation reactions. The chemoenzymatic synthesis strategy cleverly combines the flexibility of chemical methods with the stereo and regioselectivity of enzyme methods. However, although the combined application of chemistry and enzyme methods has played their respective advantages, they also have their own limitations. And the above methods basically have problems with unsatisfactory yields, yields, and conversion rates.

[0006] Based on this, there is still a need for a preparation method that can solve the preparation problem for the disialylated derivatives and / or trisialylated derivatives (abbreviated as: disialylated / trisialylated derivatives) of lacto-N-tetraose. Summary of the invention

[0007] 1. Problem to be solved

[0008] One of the purposes of the present invention is to provide a method for preparing a sialylated derivative of lacto-N-tetraose, which method helps to increase the yield of the sialylated derivative of lacto-N-tetraose;

[0009] A preferred object of the present invention is to provide a cost-effective method for preparing sialylated derivatives of lacto-N-tetraose.

[0010] In addition, the present invention also provides a disialylated derivative, a trisialylated derivative of lacto-N-tetraose having a novel structure, and a mixture of the disialylated derivative and the trisialylated derivative.

[0011] 2. Technical solution

[0012] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0013] According to the purpose of the present invention, the present invention provides a method for synthesizing sialylated lacto-N-tetraose, the method comprising the following steps:

[0014] The following steps are involved:

[0015] a) Provide a mixture containing the following substances

[0016] Substance (I) comprises at least one compound (LSTa) represented by formula 1:

[0017]

[0018] Substance (II) comprising at least one α2,6-sialyltransferase:

[0019] Substance (III), cytidine monophosphate-N-acetylneuraminic acid (hereinafter referred to as CMP-Neu5Ac); and / or, the generation of CMP-Neu5Ac;

[0020] Substance (IV), buffer containing metal ions;

[0021] Wherein, the molar ratio of the compounds LSTa and CMP-Neu5Ac does not exceed 10.

[0022] b) placing the mixture provided in a) in a reactor to react and produce di / trisialyl lacto-N-tetraose.

[0023] Optionally, the method for synthesizing di / trisialyl lacto-N-tetraose further comprises:

[0024] c) separating and purifying the product obtained in step d).

[0025] According to any embodiment of the first aspect of the present invention, the method for synthesizing sialylated lacto-N-tetraose comprises the following steps: a) providing a mixture containing the following substances:

[0026] LSTa;

[0027] α2,6-sialyltransferase;

[0028] CMP-Neu5Ac, and / or, generation of CMP-Neu5Ac;

[0029] Buffers containing metal ions;

[0030] b) placing the mixture provided in a) in a reactor for reaction.

[0031] Optionally, the method for synthesizing di / trisialyl lacto-N-tetraose further comprises:

[0032] c) separating and purifying the product obtained in step d).

[0033] According to any embodiment of the first aspect of the present invention, the generation of CMP-Neu5Ac comprises generating CMP-Neu5Ac from N-acetylneuraminic acid (Neu5Ac) in the presence of a CMP-Neu5Ac generating enzyme set.

[0034] According to any embodiment of the first aspect of the present invention, the generation of CMP-Neu5Ac further comprises cytidine triphosphate (CTP).

[0035] According to any embodiment of the first aspect of the present invention, the generation of CMP-Neu5Ac includes: generating CMP-Neu5Ac with N-acetylneuraminic acid (hereinafter referred to as: Neu5Ac) and CTP as substrates in the presence of a CMP-Neu5Ac generating enzyme group.

[0036] According to any embodiment of the first aspect of the present invention, the CMP-Neu5Ac generating enzyme group includes CMP-Neu5Ac synthetase (CSS).

[0037] According to any embodiment of the first aspect of the present invention, the CMP-Neu5Ac generating enzyme group comprises CMP-Neu5Ac synthetase (CSS) and pyrophosphorylase (PPA).

[0038] Based on the above, the preparation system of di / trisialyl lacto-N-tetraose has the following different situations:

[0039] Case 1: The following components are mixed in a single container to form a reaction system: the compound LSTa represented by Formula 1, α2,6 sialyltransferase, CMP-Neu5Ac, and a buffer containing metal ions;

[0040] Case 2: The following components are mixed in a single container to form a reaction system: the compound LSTa represented by Formula 1, α2,6 sialyltransferase, CSS, CTP, Neu5Ac, and a buffer solution containing metal ions;

[0041] Case 3: The following components are mixed in a single container to form a reaction system: the compound LSTa represented by Formula 1, α2,6 sialyltransferase, CSS, PPA, CTP, Neu5Ac, and a buffer solution containing metal ions.

[0042] Furthermore, in case 1 (one-pot single enzyme):

[0043] The content of the compound LSTa is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the best is 200mM;

[0044] The content of α2,6 sialyltransferase is 0.1-25 μM, preferably 1-25 μM, 5-25 μM, 5-20 μM, 10-20 μM, 12-17 μM, and 15 μM;

[0045] The content of CMP-Neu5Ac is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the best is 200mM;

[0046] The content of metal ions is 5-200mM, preferably 10-200mM, 15-200mM, 20-180mM, 30-150mM, 30-100mM, 30-80mM, 40-80mM, 50-70mM, 60mM;

[0047] The content of the buffer is 50-500mM, preferably 50-400mM, 50-350mM, 100-350mM, 100-300mM, 150-350mM, 150-250mM, and 200mM.

[0048] In case 2 (two enzymes in one pot):

[0049] The content of the compound LSTa is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the best is 200mM;

[0050] The content of α2,6 sialyltransferase is 0.1-25 μM, preferably 1-25 μM, 5-25 μM, 5-20 μM, 10-20 μM, 12-17 μM, and 15 μM;

[0051] The content of Neu5Ac is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the most preferred content is 200mM;

[0052] The content of CSS is 0.1-50 μM, preferably 1-50 μM, 5-50 μM, 10-50 μM, 10-40 μM, 15-40 μM, 20-40 μM, and 35 μM;

[0053] The content of CTP is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the most preferred content is 200mM;

[0054] The content of metal ions is 5-200mM, preferably 10-200mM, 15-200mM, 20-180mM, 30-150mM, 30-100mM, 30-80mM, 40-80mM, 50-70mM, 60mM;

[0055] The content of the buffer is 50-500mM, preferably 50-400mM, 50-350mM, 100-350mM, 100-300mM, 150-350mM, 150-250mM, and 200mM.

[0056] It should be further explained here that, based on the amount of Neu5Ac, the amounts of CSS and CTP need to be sufficient to completely convert Neu5Ac into CMP-Neu5Ac.

[0057] Case 3 (three enzymes in one pot):

[0058] The content of the compound LSTa is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the best is 200mM;

[0059] The content of α2,6 sialyltransferase is 0.1-25 μM, preferably 1-25 μM, 5-25 μM, 5-20 μM, 10-20 μM, 12-17 μM, and 15 μM;

[0060] The content of CSS is 0.1-50 μM, preferably 1-50 μM, 5-50 μM, 10-50 μM, 10-40 μM, 15-40 μM, 20-40 μM, and 35 μM;

[0061] The content of PPA is 0.1-40 μM, preferably 1-40 μM, 5-40 μM, 10-40 μM, 10-35 μM, 15-35 μM, 20-35 μM, 20-30 μM, 25 μM;

[0062] The content of Neu5Ac is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the most preferred content is 200mM;

[0063] The content of CTP is 0.1-300mM, preferably 1-300mM, 10-300mM, 50-300mM, 75-300mM, 100-300mM, 125-300mM, 150-300mM, 150-250mM, 175-250mM, 175-225mM, and the most preferred content is 200mM;

[0064] The content of metal ions is 5-200mM, preferably 10-200mM, 15-200mM, 20-180mM, 30-150mM, 30-100mM, 30-80mM, 40-80mM, 50-70mM, 60mM;

[0065] The content of the buffer is 50-500mM, preferably 50-400mM, 50-350mM, 100-350mM, 100-300mM, 150-350mM, 150-250mM, and 200mM.

[0066] It should be further explained here that, based on the amount of Neu5Ac, the amounts of CSS, CTP, and PPA need to be sufficient to completely convert Neu5Ac into CMP-Neu5Ac.

[0067] According to any embodiment of the first aspect of the present invention, the α2,6 sialyltransferase has an activity capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule; the sialic acid residue is preferably an N-acetylneuraminic acid (Neu5Ac) residue, and the sialyltransferase activity is capable of transferring the N-acetylneuraminic acid moiety from CMP-Neu5Ac to the acceptor molecule, wherein the acceptor molecule is a sugar molecule to provide sialylated sugar.

[0068] According to any embodiment of the first aspect of the present invention, the α2,6-sialyltransferase (EC2.4.99.1) includes but is not limited to Photobacterium sp. JT-ISH-224 (Accession No.: BAF92026), Photobacterium damselae (Accession No.: BAA25316), Photobacterium leiognathi JT-SHIZ-145 (Accession No.: BAF91416), Photobacterium leiognathi JT-SHIZ-119 (Accession No.: BAI49484), Helicobacter acinonychis (Accession No.: NC_008229), Helicobacter cetorum (Accession No.: WP_014661583), Streptococcus suis (Accession No.: EEF64774), Streptococcus suis (Accession No.: AGL48117), Photobacterium phosphoreum (accession number: BAF63530)_, Homo sapiens (accession number: Q9BVH7, Q969X2), Alistipestimonensis (accession number: WP_026020701), Haemophilusparasuis (accession number: KEZ22922), Acinetobacter pittii (accession number: EOQ74854), Porphyromonas sp. (accession number: KGO00880), Pasteurella multocida (accession number: DQ087233), Actinobacillus ureae (accession number: WP_005624339), Actinobacillus equuli (accession number: WP_039196193), Acinetobacter calcoaceticus (accession number: WP_017391462), Acinetobacter baumannii (accession number: WP_032039596), Acinetobacter oleivorans (accession number: WP_042898369), Acinetobacter gyllenbergii (accession number: WP_032865812), Shigella boydii (accession number: ACD37071), Kingella kingae (accession number: WP_026036248), Alysiella crassa (accession number: WP_034294366), Alistipes sp.(Accession No.: EFR56292), Fusobacterium mortiferum (Accession No.: EEO36694), Porphyromonascatoniae (Accession No.: WP_005467220), Vibrio sp. (Accession No.: BAF91160), Pasteurella dagmatis (Accession No.: AFY98851), Haemophilus ducreyi (Accession No.: AAP95068), Neisseria meningitidis (Accession No.: U60660), Pasteurella multocida subsp.multocida (Accession No.: AAK03258), any one or more thereof, but not limited to these;.

[0069] According to any embodiment of the first aspect of the present invention, the CSS (EC 2.7.7.43) includes but is not limited to any one or more of Clostridium thermocellum (Accession No.: ZP_00314171), Streptococcus agalactiae (Accession No.: NP_688167), Escherichia coli (Accession No.: P13266), Neisseria meningitidis (Accession No.: Q57385), Haemophilus ducrey (Accession No.: NP_873215), Haemophilus influenzae (Accession No.: Q57140), C. jejuni (Accession No.: AAR82878), P. multocida (Accession No.: NP_245124);

[0070] According to any embodiment of the first aspect of the present invention, the PPA (EC 3.6.1.1) includes but is not limited to Anabaena sp. strain PCC 7120 (accession number: P80562), Aquifex aeolicus (accession number: O67501), Arabidopsis thaliana Col-0 (accession number: Q67YC0), Bacteroides thetaiotaomicron (accession number: Q8A5V9), Clostridium perfringens (accession number: Q8XIQ9), Desulfitobacterium hafniense DSM 10664 (accession number: B8FP42), Ethanoligenens harbinense (accession number: E6U5H4), Haloferax volcanii (accession number: D4GT97), Methanothrix thermoacetophila DSM 6194 (accession number: A0B5R0), Mycobacterium tuberculosis (accession number: P9WI55), Pseudanabaena sp. strain PCC 6903 (accession number: P58733), Pyrococcus furiosusATCC 43587 (accession number: Q8U438), Pyrococcus horikoshii DSM 12428 (accession number: O59570), Saccharomyces cerevisiae ATCC 204508 (accession number: P00817), Staphylococcus aureus (accession number: P65752), Streptococcus agalactiae (accession number: Q3K0B5), Streptococcus gordonii (accession number: P95765), Sulfolobus acidocaldarius DSM 639 (accession number: P50308), Sulfuris phaera tokodaii (accession number: F9VPB8), Synechocystis sp. strain PCC 6803 (accession number: P80507), Thermococcus thioreducens (accession number: H2L2L6), Thermoplasma acidophilum (accession number: P37981), Thermus thermophilus (accession number: P38576);

[0071] According to any embodiment of the first aspect of the present invention, the metal ions include but are not limited to Co 2+ , Mn 2+ Mg 2+ 、Ni 2+ , Ca 2+ 、Zn 2+ 、Cd 2+ . Any one or more of the following;

[0072] Furthermore, the buffer solution includes but is not limited to any one or more of Tris-HCl, triethanolamine-HCl, sodium barbital-HCl, disodium hydrogen phosphate-citric acid, phosphate, Britton-Robinson (BR), glycine-sodium hydroxide and other buffer solutions;

[0073] According to any embodiment of the first aspect of the present invention, the buffer has a pH value of 7.0-9.5;

[0074] According to any embodiment of the first aspect of the present invention, the generation of CMP-Neu5Ac in a) and the process of producing disialyl lacto-N-tetraose and trisialyl lacto-N-tetraose in b) are carried out in the same reactor.

[0075] According to any embodiment of the first aspect of the present invention, the temperature in the reactor is maintained at 28-37°C.

[0076] The second aspect of the present invention provides sialylated lacto-N-tetraose; the sialylated lacto-N-tetraose is characterized by general formula 2, and the compound represented by general formula 2 is as follows:

[0077]

[0078] In the formula, R1, R2, R3, and R4 are each independently Neu5Ac or H.

[0079] According to any embodiment of the second aspect of the present invention, the sialylated lacto-N-tetraose is selected from any one of the following formulae 2-1 to 2-5, wherein: the compound represented by 2-1:

[0080]

[0081] In the formula, R1 is H, R2 is H, R3 is Neu5Ac, and R4 is H;

[0082] The compound represented by 2-2:

[0083]

[0084] In the formula, R1 is H, R2 is Neu5Ac, R3 is H, and R4 is H;

[0085] The compound represented by 2-3:

[0086]

[0087] In the formula, R1 is Neu5Ac, R2 is H, R3 is Neu5Ac, and R4 is H;

[0088] The compound represented by 2-4:

[0089]

[0090] In the formula, R1 is Neu5Ac, R2 is Neu5Ac, R3 is H, and R4 is H;

[0091] The compound represented by 2-5:

[0092]

[0093] In the formula, R1 is H, R2 is Neu5Ac, R3 is H, and R4 is Neu5Ac.

[0094] According to any embodiment of the second aspect of the present invention, the sialylated lacto-N-tetraose is prepared based on the method provided by any embodiment of the first aspect of the present invention.

[0095] The third aspect of the present invention provides a human milk oligosaccharide mixture; the mixture contains any one or two or more of sialylated lacto-N-tetraose characterized by general formula 2; wherein the compound represented by general formula 2 is as follows:

[0096]

[0097] In the formula, R1, R2, R3, and R4 are each independently Neu5Ac or H.

[0098] According to any embodiment of the third aspect of the present invention, the mixture contains any one or two or more of the compounds represented by the following formulae 2-1 to 2-5; the compound represented by 2-1:

[0099]

[0100] In the formula, R1 is H, R2 is H, R3 is Neu5Ac, and R4 is H;

[0101] The compound represented by 2-2:

[0102]

[0103] In the formula, R1 is H, R2 is Neu5Ac, R3 is H, and R4 is H;

[0104] The compound represented by 2-3:

[0105]

[0106] In the formula, R1 is Neu5Ac, R2 is H, R3 is Neu5Ac, and R4 is H;

[0107] The compound represented by 2-4:

[0108]

[0109] In the formula, R1 is Neu5Ac, R2 is Neu5Ac, R3 is H, and R4 is H;

[0110] The compound represented by 2-5:

[0111]

[0112] In the formula, R1 is H, R2 is Neu5Ac, R3 is H, and R4 is Neu5Ac.

[0113] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1 and a compound represented by Formula 2-2.

[0114] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1 and a compound represented by Formula 2-3.

[0115] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1 and a compound represented by Formula 2-4.

[0116] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1 and a compound represented by Formula 2-5.

[0117] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2 and a compound represented by Formula 2-3.

[0118] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2 and a compound represented by Formula 2-4.

[0119] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2 and a compound represented by Formula 2-5.

[0120] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-3 and a compound represented by Formula 2-4.

[0121] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-3 and a compound represented by Formula 2-5.

[0122] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-4 and a compound represented by Formula 2-5.

[0123] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-2, and a compound represented by Formula 2-3.

[0124] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-2, and a compound represented by Formula 2-4.

[0125] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-2, and a compound represented by Formula 2-5.

[0126] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-3, and a compound represented by Formula 2-4.

[0127] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-3, and a compound represented by Formula 2-5.

[0128] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-4, and a compound represented by Formula 2-5.

[0129] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2, a compound represented by Formula 2-3, and a compound represented by Formula 2-4.

[0130] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2, a compound represented by Formula 2-3, and a compound represented by Formula 2-5.

[0131] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2, a compound represented by Formula 2-4, and a compound represented by Formula 2-5.

[0132] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-3, a compound represented by Formula 2-4, and a compound represented by Formula 2-5.

[0133] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-2, a compound represented by Formula 2-3, and a compound represented by Formula 2-4.

[0134] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-2, a compound represented by Formula 2-3, and a compound represented by Formula 2-5.

[0135] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-2, a compound represented by Formula 2-4, and a compound represented by Formula 2-5.

[0136] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-1, a compound represented by Formula 2-3, a compound represented by Formula 2-4, and a compound represented by Formula 2-5.

[0137] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by Formula 2-2, a compound represented by Formula 2-3, a compound represented by Formula 2-4, and a compound represented by Formula 2-5.

[0138] According to any embodiment of the third aspect of the present invention, the human milk oligosaccharide mixture includes a compound represented by formula 2-1, a compound represented by formula 2-2, a compound represented by formula 2-3, a compound represented by formula 2-4, and a compound represented by formula 2-5.

[0139] According to any embodiment of the second aspect of the present invention, the method for preparing sialyl lacto-N-tetraose comprises the steps of: a) providing a mixture containing the following substances:

[0140] LSTa;

[0141] α2,6-sialyltransferase;

[0142] CMP-Neu5Ac and / or a system for producing CMP-Neu5Ac;

[0143] Buffers containing metal ions;

[0144] b) placing the mixture provided in a) in a reactor for reaction.

[0145] The sialyl lacto-N-tetraose prepared by the preparation method of sialyl lacto-N-tetraose provided by any embodiment of the first aspect of the present invention, or the sialyl lacto-N-tetraose provided by the second aspect of the present invention; the third aspect of the present invention provides the use of sialyl lacto-N-tetraose, wherein the sialyl lacto-N-tetraose is used to prepare a nutritional composition or a nutritional preparation or a pharmaceutical composition or a pharmaceutical preparation.

[0146] Sialyl lacto-N-tetraose prepared by the preparation method of sialyl lacto-N-tetraose provided by any embodiment of the first aspect of the present invention, or sialyl lacto-N-tetraose provided by the second aspect of the present invention, or sialyl lacto-N-tetraose provided by the fourth aspect of the present invention; the fifth aspect of the present invention provides a composition comprising sialyl lacto-N-tetraose; the composition is any one or more of a nutritional composition, a nutritional preparation, a pharmaceutical composition, a pharmaceutical preparation, and a dietary supplement. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Figure 1 The figure is the synthesis result of disialyl lacto-N-tetraose by TLC (thin layer chromatography), the arrow at the top in the figure is the substrate compound (LSTa) represented by Formula 1, and the arrow at the bottom is disialyl lacto-N-tetraose (disialyl LNT);

[0148] Figure 2 for Figure 1 Mass spectrometry analysis of disialyl lacto- N -tetraose indicated by the arrow;

[0149] Figure 3 The figure is a TLC diagram of the synthesis result of di / trisialyl lacto-N-tetraose, in which the arrow at the top is disialyl lacto-N-tetraose (disialyl LNT), and the arrow at the bottom is trisialyl lacto-N-tetraose (trisialyl LNT);

[0150] Figure 4 for Figure 3 Mass spectrometry analysis of trisialyl lacto- N -tetraose indicated by the arrow;

[0151] Figure 5 HPLC analysis of the reaction product with a molar ratio of LSTa to sialic acid of 1:0.5 (ratio is 2) (the main product is disialyl LNT, with a small amount of trisialyl LNT and a large amount of remaining LSTa);

[0152] Figure 6 HPLC analysis of the reaction of LSTa and sialic acid at a molar ratio of 1:1 (ratio is 1) (the main product is trisialoyl LNT, followed by disialoyl LNT, and the remaining amount of LSTa is very small);

[0153] Figure 7 HPLC analysis of the reaction when the molar ratio of LSTa to sialic acid is 1:2 (ratio is 0.5) (almost all trisialic acid LNT is generated);

[0154] Figure 8 Disialyl lacto-N-tetraose 1 H NMR analysis;

[0155] Fig. 9 Disialyl lacto-N-tetraose 13 C NMR analysis;

[0156] Fig.10 trisialyl lacto-N-tetraose 1 H NMR analysis;

[0157] Fig.11 trisialyl lacto-N-tetrasaccharide 13 C NMR analysis.

[0158] Explanation of symbols

[0159] The abbreviations of the symbols in this article are shown in Table 1:

[0160] Table 1 Symbols

[0161] Abbreviation English name Chinese name Neu5Ac N-Acetylneuraminic acid N-Acetylneuraminic acid CMP-Neu5Ac Cytidine 5'-monophosphate-N-acetylneuramine acid Cytidine monophosphate-N-acetylneuraminic acid CTP Cytidine-5'-triphosphate Cytidine triphosphate CMP Cytidine 5'-monophosphate Cytidine 5'-monophosphate CSS CMP-Sialic acid synthetase CMP-Neu5Ac synthetase PPA Pyrophosphorylase Pyrophosphorylase LNT Lactose-N-tetraose DETAILED DESCRIPTION

[0162] The present disclosure may be more easily understood by reference to the following description in conjunction with the accompanying drawings and examples, all of which constitute a part of the present disclosure. It should be understood that the present disclosure is not limited to the specific products, methods, conditions or parameters described and / or shown herein. Further, the terms used herein are only used for the purpose of describing specific embodiments by way of example and are not intended to be limiting unless otherwise stated.

[0163] It should be noted that the terms used here are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. It should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments and are not intended to limit the scope of protection of the present invention. In the following specific embodiments, if the experimental methods of specific conditions are not indicated, they are generally in accordance with the conventional methods and conditions of molecular biology within the art, and such techniques and conditions are fully explained in the literature.

[0164] It should also be understood that, for the sake of clarity, certain features of the present disclosure may be described herein in the context of separate embodiments, but may also be provided in combination with each other in a single embodiment. That is, unless clearly incompatible or specifically not included, each separate embodiment is considered to be combinable with any other embodiment, and the combination is considered to represent another different embodiment. Conversely, for the sake of simplicity, the various features of the present disclosure described in the context of a single embodiment may also be provided individually or in any sub-combination. Finally, although a particular embodiment may be described as part of a series of steps or part of a more general structure, each step or substructure itself may also be considered to be an independent embodiment.

[0165] Unless otherwise indicated, it should be understood that each individual element in a list and each combination of individual elements in the list will be interpreted as a different embodiment. For example, a list of embodiments represented as "A, B, or C" should be interpreted to include embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0166] In the present disclosure, the singular forms of the articles "a," "an," and "the" also include the corresponding plural references, and a reference to a specific value includes at least that specific value unless the context clearly indicates otherwise. Thus, for example, a reference to "a substance" is a reference to at least one of that substance and equivalents thereof.

[0167] The terms including ordinal numbers such as "first" and "second" can be used to explain various components or fluids, but these components and fluids are not limited by these terms. Therefore, without departing from the teachings of the present disclosure, these terms are only used to distinguish the component / fluid from another component / fluid.

[0168] When items are described by using the conjunction terms "... and / or...," etc., the description should be understood to include any one of the associated listed items and all combinations of one or more thereof.

[0169] In general, the use of the term "about" indicates an approximate value that may vary depending on the desired properties obtained by the disclosed subject matter, and will be interpreted in a context-dependent manner based on function. Therefore, one of ordinary skill in the art will be able to interpret a certain degree of difference on a case-by-case basis. In some cases, the number of significant figures used when expressing a particular value can be a representative technique for determining the difference allowed by the term "about". In other cases, a gradient in a range of values ​​can be used to determine the range of differences allowed by the term "about". Further, all ranges in this disclosure are inclusive and combinable, and reference to the values ​​stated in the range includes each value within the range.

[0170] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs; the terms used herein and / or include any and all combinations of one or more of the associated listed items.

[0171] As described herein, the "enzymes" used in the present invention, including "CMP-Neu5Ac synthetase, pyrophosphorylase, α2,6-sialyltransferase, etc.", can be purchased through channels such as: Merck SKU US2076 α-2,6-sialyltransferase is derived from Photorhabditis mermani, EC No. 2.4.99.1 (BRENDA, IUBMB); Merck SKU I5907 pyrophosphorylase is derived from Escherichia coli, EC No. 3.6.1.1 (BRENDA, IUBMB); Merck SKU C1999 CMP-Neu5Ac synthetase is derived from Neisseria meningitidis, EC No. 2.7.7.43 (BRENDA, IUBMB); or, in fact, it can also be obtained by extraction from microbial cells such as bacteria, yeast or fungi in nature; or it can also be obtained by in vitro cloning and expression, see, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0172] The present invention is further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The essential characteristics and remarkable effects of the present invention can be reflected from the following examples, and the described embodiments are part of the embodiments of the present invention, rather than all the embodiments, and therefore, they do not limit the present invention in any way, and those skilled in the art make some non-essential improvements and adjustments based on the content of the present invention, which all belong to the protection scope of the present invention.

[0173] Description of the synthetic route of oligosaccharides

[0174] Based on the above, the present invention provides a route for catalytic synthesis of di / trisialic acid LNT using LSTa as a substrate:

[0175] Route 1:

[0176]

[0177] Route 2:

[0178]

[0179] Route 3:

[0180]

[0181] The molar ratio of the compound LSTa to CMP-Neu5Ac (or the raw material Neu5Ac for forming CMP-Neu5Ac) in the above routes 1 to 3 must be ensured to be 0.1 to 10; on this basis,

[0182] In order to ensure the synthesis of disialyl lacto-N-tetraose (yield not less than 10%) in the product, the molar ratio of the compound LSTa to CMP-Neu5Ac is preferably 2 to 10; for example, if the amount of LSTa is 1M, the amount of CMP-Neu5Ac is preferably 0.1 to 0.5M;

[0183] In order to ensure that the product contains both disialyl lacto-N-tetraose (yield not less than 10%) and trisialyl lacto-N-tetraose (yield not less than 30%, preferably not less than 50%), the molar ratio of the compound LSTa to CMP-Neu5Ac is preferably greater than or equal to 1 and less than 2, and the optimal molar ratio of the compound LSTa to CMP-Neu5Ac is 1.3-1.35; for example, if the amount of LSTa is 1M, the amount of CMP-Neu5Ac is preferably 0.5-1M, and the optimal amount of CMP-Neu5Ac is 0.75M;

[0184] In order to ensure the synthesis of trisialyl lacto-N-tetraose in the product as much as possible (yield not less than 50%, preferably not less than 60%), the molar ratio of the preferred compound LSTa to CMP-Neu5Ac is less than or equal to 1, and the optimal molar ratio of the compound LSTa to CMP-Neu5Ac is less than or equal to 0.5; for example, if the amount of LSTa is 1M, the amount of CMP-Neu5Ac is greater than or equal to 1M, and preferably the amount of CMP-Neu5Ac is greater than or equal to 2M;

[0185] In order to demonstrate the technical solutions and advantages of the present invention, the technical solutions in the embodiments of the present invention will be fully described below.

[0186] In the following embodiments: CMP-Neu5Ac synthetase, pyrophosphorylase, α2,6-sialyltransferase, etc. were purchased through the following channels: specifically: Merck SKU US2076 α-2,6-sialyltransferase is derived from Photorhabditis elegans, EC number 2.4.99.1 (BRENDA, IUBMB); Merck SKU I5907 pyrophosphorylase is derived from Escherichia coli, EC number 3.6.1.1 (BRENDA, IUBMB); Merck SKU C1999 CMP-Neu5Ac synthetase is derived from Neisseria meningitidis, EC number 2.7.7.43 (BRENDA, IUBMB).

[0187] Example 1

[0188] Experiment on the synthesis of disialyl lacto-N-tetraose catalyzed by α2,6-sialyltransferase: The reaction system contains 200mMTris buffer (pH=7.5), magnesium chloride (30mM), 1.5mM CMP-Neu5Ac, and 3mM LSTa, and α2,6 sialyltransferase (15μM) is added to the system. The reaction was carried out in a shaker at 37℃ for 12 hours. During the process, samples were taken for product detection by TLC (ethyl acetate: methanol: water: acetic acid = 4:6:1:0.2 volume ratio, sulfuric acid-ethanol staining method detection) and HPLC analysis (HPLC conditions: A: pure water, B: 100mM ammonium formate (pH3.2); C: acetonitrile. 0min: 15% A, 75% B, 10% C; 45min, 28% A, 62% B, 10% C), such as Figure 1 and Figure 5 As shown, the yield of disialyl lacto-N-tetraose was 34.2%.

[0189] Example 2

[0190] The experiment of "one-pot dual enzyme" catalyzing the synthesis of disialyl lacto-N-tetraose: the reaction system contains 100mM Tris buffer (pH 7.5), magnesium chloride (30mM), 1.5mM CTP, 1.5mM Neu5Ac, 3mM LSTa, CSS (35μM) and α2,6 sialyltransferase (15μM) are added to the system. The reaction is carried out in a shaker at 37℃ for 12 hours. The yield of disialyl lacto-N-tetraose is 30%.

[0191] Example 3

[0192] The experiment of synthesizing disialyl lacto-N-tetraose by "one pot three enzymes": the reaction system contains 100mM Tris buffer (pH 7.5), magnesium chloride (30mM), 1.5mM CTP, 1.5mM Neu5Ac, 3mM LSTa, CSS (35μM), α2,6 sialyltransferase (15μM), PPA (25μM) are added to the system. The reaction is carried out in a shaker at 37℃ for 12 hours. The yield of disialyl lacto-N-tetraose is 30%.

[0193] Example 4

[0194] Experiment on the synthesis of di / trisialyl lacto-N-tetraose catalyzed by α2,6-sialyltransferase: The reaction system contains 100mMTris buffer (pH 7.5), magnesium chloride (30mM), 3.0mM CMP-Neu5Ac, and 3mM LSTa, and α2,6 sialyltransferase (15μM) is added to the system. The reaction was carried out in a shaker at 37°C for 12 hours. During the process, samples were taken for product detection by TLC (ethyl acetate: methanol: water: acetic acid = 4:6:1:0.2 volume ratio, sulfuric acid-ethanol staining method detection) and HPLC analysis (HPLC conditions: A: pure water, B: 100mM ammonium formate (pH3.2); C: acetonitrile. 0min: 15% A, 75% B, 10% C; 45min, 28% A, 62% B, 10% C)). Figure 3 and Figure 6 The yields of disialyl lacto-N-tetraose and trisialyl lacto-N-tetraose were 34.8% and 60.9%, respectively.

[0195] Example 5

[0196] The experiment of "one-pot dual enzyme" catalyzing the synthesis of di- / tri-sialyl lacto-N-tetraose: the reaction system contains 100mM Tris buffer (pH 7.5), magnesium chloride (30mM), 3.0mM CTP, 3.0mM Neu5Ac, 3mM LSTa, CSS (35μM), α2,6 sialyltransferase (15μM) are added to the system. The reaction is carried out in a shaker at 37℃ for 12 hours. The yields of di- / tri-sialyl lacto-N-tetraose and tri-sialyl lacto-N-tetraose are 30% and 60%, respectively.

[0197] Example 6

[0198] The experiment of "one pot three enzymes" catalyzing the synthesis of di- / tri-sialyl lacto-N-tetraose: the reaction system contains 100mM Tris buffer (pH 7.5), magnesium chloride (30mM), 3.0mM CTP, 3.0mM Neu5Ac, 3mM LSTa, CSS (35μM), α2,6 sialyltransferase (15μM), PPA (25μM) are added to the system. The reaction was carried out in a shaker at 37℃ for 12 hours. The yields of di- / tri-sialyl lacto-N-tetraose and tri-sialyl lacto-N-tetraose were 30% and 60%, respectively.

[0199] Example 7

[0200] Experiment on the synthesis of trisialyl lacto-N-tetraose catalyzed by α2,6-sialyltransferase: The reaction system contains 100mMTris buffer (pH7.5), magnesium chloride (30mM), 6.0mM CMP-Neu5Ac, and 3mM LSTa, and α2,6 sialyltransferase (15μM) is added to the system. The reaction was carried out in a shaker at 37℃ for 12 hours. Samples were taken during the process and analyzed by HPLC (HPLC conditions: A: pure water, B: 100mM ammonium formate (pH3.2); C: acetonitrile. 0min: 15% A, 75% B, 10% C; 45min, 28% A, 62% B, 10% C)). Figure 7 The yield of trisialyl lacto-N-tetraose was >90%.

[0201] Example 8

[0202] The experiment of "one-pot dual enzyme" catalyzing the synthesis of trisialyl lacto-N-tetraose: the reaction system contains 100mM Tris buffer (pH 7.5), magnesium chloride (30mM), 6.0mM CTP, 6.0mM Neu5Ac, 3mM LSTa, CSS (35μM) and α2,6 sialyltransferase (15μM) are added to the system. The reaction is carried out in a shaker at 37℃ for 12 hours. The yield of trisialyl lacto-N-tetraose is >90%.

[0203] Example 9

[0204] The experiment of synthesizing trisialyl lacto-N-tetraose by "one pot three enzymes": the reaction system contains 100mM Tris buffer (pH 7.5), magnesium chloride (30mM), 6.0mM CTP, 6.0mM Neu5Ac, 3mM LSTa, CSS (35μM), α2,6 sialyltransferase (15μM), PPA (25μM) are added to the system. The reaction is carried out in a shaker at 37℃ for 12 hours. The yield of trisialyl lacto-N-tetraose is >90%.

[0205] Example 10

[0206] The present invention combines the two methods to obtain a relatively pure disialyl lacto-N-tetraose product.

[0207] The first step of separation and purification of the reaction solution obtained in Example 1 is as follows:

[0208] The reaction solution was concentrated to dryness using a rotary evaporator, and then the sample was redissolved with a 5mM NH4HCO3 solution. The separation column was eluted with different concentrations (5mM, 10mM, 20mM, 30mM, 50mM) of NH4HCO3 solution, and the eluents were collected respectively. The eluent was analyzed by TLC, the developing solvent was ethyl acetate: methanol: water: acetic acid = 4: 6: 1: 0.2, the color developer was concentrated sulfuric acid: ethanol = 1: 9, and it was placed in a 115℃ oven for color development. The target product was collected and concentrated according to the results. LSTa was eluted when the concentration of NH4HCO3 solution was 20mM, and disialyl lacto-N-tetraose was eluted when the concentration of NH4HCO3 solution was 50mM.

[0209] The concentrated target product was again separated and purified in the second step using a Bio-Gel P-2Fine (fine polyacrylamide) (Bio-Rad 1504114) gel filtration chromatography column (100 cm×2.5 cm), using water as the eluent. The target product was collected and concentrated according to the TLC results.

[0210] The obtained pure disialyl lacto-N-tetraose was freeze-dried to obtain a white powder. Its molecular weight was verified by mass spectrometry. Disialyl lacto-N-tetraose includes two mixtures of the structural formula 2-1 and the formula 2-2, and the expected m / z [M-2H] 2- 644.07, and the mass spectrometry result was 643.61, which was consistent with the expectation. Figure 2 shown.

[0211] The obtained pure disialyl lacto-N-tetrasaccharide 1 H NMR analysis Figure 8 As shown, 13 C NMR analysis Fig. 9 As shown, the analysis results are: 1 H NMR (600MHz, D2O) δ5.06,4.56,4.50,4.35,4.27,4.01,3.92,3.80-3.31,3.15,2.53-2.61,1.91,1.87,1.63,1.57. 13C NMR(600MHz,D2O)δ174.90,174.83,173.42,166.14,103.82,103.21,102.53,100.07,99.48, 95.63,83.95,83.76,83.32,79.57,75.29,74.66,74.31,73.82,73.71,73.30,72.80,72.51, 72.44,71.78,71.68,71.21,70.33,69.55,69.27,68.95,68.54,68.38,68.25,68.06,63.46,63.18,62.65,62.48,60.72,59.40,54.32,51.79,51.64,40.07,39.63,22.30,22.10,22.06.

[0212]

[0213] Formula 2-1, where R1 is H, R2 is H, R3 is Neu5Ac, R4 is H, and the molecular formula is C 48 H 79 N3O 37 ;

[0214]

[0215] Formula 2-2, where R1 is H, R2 is Neu5Ac, R3 is H, R4 is H, and the molecular formula is C 48 H 79 N3O 37 .

[0216] Embodiment 11

[0217] The present invention combines two purification methods to obtain a relatively pure trisialyl lacto-N-tetraose product.

[0218] The reaction solution obtained in Example 7 was subjected to the first step of separation and purification, and the specific steps are as follows:

[0219] The reaction solution was concentrated to dryness using a rotary evaporator, and then the sample was redissolved with a 5mM NH4HCO3 solution. The separation column was eluted with NH4HCO3 solutions of different concentrations (5mM, 10mM, 20mM, 30mM, 50mM, 60mM), and the eluents were collected respectively. The eluent was analyzed by TLC, with the developing solvent being ethyl acetate: methanol: water: acetic acid = 4: 6: 1: 0.2, and the color developing agent being concentrated sulfuric acid: ethanol = 1: 9. The product was placed in a 115°C oven for color development, and the target product was collected and concentrated according to the results. The trisialyl lacto-N-tetraose product was eluted when the concentration of the NH4HCO3 solution was 60mM.

[0220] The concentrated target product was again separated and purified in the second step using a Bio-Gel P-2Fine (fine polyacrylamide) (Bio-Rad 1504114) gel filtration chromatography column (100 cm×2.5 cm), using water as the eluent. The target product was collected and concentrated according to the TLC results.

[0221] The obtained pure trisialyl lacto-N-tetraose was freeze-dried to obtain a white powder. Its molecular weight was verified by mass spectrometry. Trisialyl lacto-N-tetraose includes three mixtures of the structural formulas 2-3, 2-4 and 2-5, with an expected m / z [M-2H] 2- 789.5, and the mass spectrometry result was 789.13, which was consistent with the expectation. Figure 4 shown.

[0222] The obtained pure trisialyl lacto-N-tetrasaccharide 1 H NMR analysis Fig.10 As shown, 13 C NMR analysis Fig.11 The analysis results are as follows: 1 H NMR (600MHz, D2O) δ5.14,4.66,4.59,4.36,4.24,4.17,4.10,4.02,3.94-3.44,3.23,2.68-2.60,1.97,1.85,1.94,1.71,1.57. 13 C NMR(600MHz,D2O)δ174.97,174.91,174.87,174.43,124.67,103.38,103.22,102.55,95.64,91.80,83.82,82.18, 82.10,79.75,79.64,76.59,75.20,75.07,74.66,74.62,73.72,73.27,72.79,72.52,71.83,71.76,71.05,70.69, 70.40,69.96,69.73,69.41,69.09,68.51,68.37,68.35,68.16,68.05,67.76,67.27,66.75,63.44,63.25,62.64,62.47,61.01,60.54,60.26,60.10,54.59,53.56,52.25,51.80,51.67,40.11,39.77,22.32,22.11,22.06,21.89.

[0223]

[0224] Formula 2-3, where R1 is Neu5Ac, R2 is H, R3 is Neu5Ac, R4 is H, and the molecular formula is C 59 H 96 N4O 45 ;

[0225]

[0226] Formula 2-4, where R1 is Neu5Ac, R2 is Neu5Ac, R3 is H, R4 is H, and the molecular formula is C 59 H 96 N4O 45 .

[0227]

[0228] Formula 2-5, where R1 is H, R2 is Neu5Ac, R3 is H, R4 is Neu5Ac, and the molecular formula is C 59 H 96 N4O 45 .

Claims

1. A method for preparing sialylated lacto-N-tetraose, wherein the sialylated lacto-N-tetraose is disialylated lacto-N-tetraose and / or trisialylated lacto-N-tetraose, characterized in that: The following steps are involved: a) Provide a mixture containing the following substances Substance (I) includes a compound LSTa represented by formula 1: Substance (II), including α2,6-sialyltransferase: Substance (III) comprising cytidine monophosphate-N-acetylneuraminic acid; and / or, the production of cytidine monophosphate-N-acetylneuraminic acid; Substance (IV), buffer containing metal ions; b) placing the mixture provided in a) in a reactor for reaction to obtain the sialylated lacto-N-tetraose; Wherein, the molar ratio of the compound LSTa to cytidine monophosphate-N-acetylneuraminic acid is no more than 10.

2. The method for preparing sialylated lacto-N-tetraose according to claim 1, characterized in that: The generation of cytidine monophosphate-N-acetylneuraminic acid includes: in the presence of a cytidine monophosphate-N-acetylneuraminic acid generating enzyme group, using N-acetylneuraminic acid and cytidine triphosphate as substrates to generate cytidine monophosphate-N-acetylneuraminic acid.

3. The method for preparing sialylated lacto-N-tetraose according to claim 2, characterized in that: The cytidine monophosphate-N-acetylneuraminic acid generating enzyme group includes cytidine monophosphate-N-acetylneuraminic acid synthetase.

4. The method for preparing sialylated lacto-N-tetraose according to claim 2, characterized in that: The cytidine monophosphate-N-acetylneuraminic acid generating enzyme group comprises cytidine monophosphate-N-acetylneuraminic acid synthetase and pyrophosphorylase.

5. The method for preparing sialylated lacto-N-tetraose according to any one of claims 2 to 4, characterized in that: The concentration of N-acetylneuraminic acid is 0.1-300 mM; the concentration of cytidine triphosphate is 0.1-300 mM; the concentration of cytidine monophosphate-N-acetylneuraminic acid synthetase is 0.1-50 μM; the concentration of pyrophosphorylase is 0.1-40 μM; Preferably, the concentration of N-acetylneuraminic acid is 10-300 mM; the concentration of cytidine triphosphate is 10-300 mM; the concentration of cytidine monophosphate-N-acetylneuraminic acid synthetase is 10-40 μM; the concentration of pyrophosphorylase is 10-40 μM; Further preferably, the concentration of N-acetylneuraminic acid is 175-250 mM; the concentration of cytidine triphosphate is 175-250 mM; the concentration of cytidine monophosphate-N-acetylneuraminic acid synthetase is 20-40 μM; The concentration of the pyrophosphorylase is 20-40 μM.

6. The method for preparing sialylated lacto-N-tetraose according to any one of claims 1 to 5, characterized in that: In the mixture provided in a), the amount of the compound LSTa is 0.1-300 mM, the amount of α2,6-sialyltransferase is 0.1-25 μM, the amount of cytidine monophosphate-N-acetylneuraminic acid or the amount of cytidine monophosphate-N-acetylneuraminic acid generated is 0.1-300 mM, and the amount of metal ions is 5-200 mM; Preferably, in the mixture provided in a), the amount of the compound LSTa is 10-300 mM, the amount of α2,6-sialyltransferase is 10-20 μM, the amount of cytidine monophosphate-N-acetylneuraminic acid or the amount of cytidine monophosphate-N-acetylneuraminic acid generated is 10-300 mM, and the amount of metal ions is 40-80 mM; Further preferably, in the mixture provided in a), the amount of compound LSTa is 175-250 mM, the amount of α2,6-sialyltransferase is 10-20 μM, the amount of cytidine monophosphate-N-acetylneuraminic acid or the amount of cytidine monophosphate-N-acetylneuraminic acid generated is 175-250 mM, and the amount of metal ions is 40-80 mM.

7. The method for preparing sialylated lactotetraose according to any one of claims 1 to 6, characterized in that: The production of cytidine monophosphate-N-acetylneuraminic acid in a) and the production of sialylated lacto-N-tetraose in b) are carried out in the same reactor; The temperature in the reactor was maintained at 25-45°C.

8. Sialyl lacto-N-tetraose, characterized in that The sialylated lacto-N-tetraose is characterized by the general formula 2: In formula 2, R1, R2, R3, and R4 are each independently Neu5Ac or H.

9. Sialyl lacto-N-tetraose, characterized in that The sialylated lacto-N-tetraose is selected from any one of the following formulas 2-1 to 2-5, wherein: In formula 2-1, R1 is H, R2 is H, R3 is Neu5Ac, and R4 is H; In formula 2-2, R1 is H, R2 is Neu5Ac, R3 is H, and R4 is H; In formula 2-3, R1 is Neu5Ac, R2 is H, R3 is Neu5Ac, and R4 is H; In formula 2-4, R1 is Neu5Ac, R2 is Neu5Ac, R3 is H, and R4 is H; In formula 2-5, R1 is H, R2 is Neu5Ac, R3 is H, and R4 is Neu5Ac.

10. The sialylated lacto-N-tetraose according to any one of claims 8 to 9, characterized in that The sialylated lacto-N-tetraose is prepared according to any one of claims 1-7.

11. A human milk oligosaccharide mixture, characterized in that The mixture contains any one or two or more sialylated lacto-N-tetraoses characterized by general formula 2.

12. A human milk oligosaccharide mixture, characterized in that The mixture contains any one or two or more of the compounds represented by Formula 2-1 to Formula 2-5.

13. The human milk oligosaccharide mixture according to claim 11 or 12, characterized in that: Prepared according to any one of claims 1 to 7.

14. Use of sialylacto-N-tetraose prepared according to the method of any one of claims 1 to 7, sialylacto-N-tetraose according to any one of claims 8 to 10, and human milk oligosaccharide mixture according to any one of claims 11 to 13, characterized in that: Used to manufacture nutritional compositions or nutritional preparations or pharmaceutical compositions or pharmaceutical preparations or cosmetics or health products or dietary supplements.

15. A composition, characterized in that The composition comprises sialyl lacto-N-tetraose prepared by the method of any one of claims 1 to 7, or sialyl lacto-N-tetraose as described in any one of claims 8 to 10, or the human milk oligosaccharide mixture as described in any one of claims 11 to 13; The composition is any one or more of a nutritional composition, a nutritional preparation, a pharmaceutical composition, a pharmaceutical preparation, a dietary supplement, a cosmetic, and a health product.

Images