A method for the synthesis of sulfated human milk oligosaccharide at position 6 of n-acetylglucosamine

Abstract

This invention provides a method for synthesizing N-acetylglucosamine sulfonated human milk oligosaccharides at position 6, belonging to the field of glycosyl compound preparation technology. In this invention, lactose is used as the starting substrate, and compound (1) is synthesized with UDP-GlcNAc under the action of NmLgtA. Compound (1) is then reacted with PAPS under the action of CHST2 to synthesize compound (2). Compound (2) is then reacted with different glycosyl donors under the action of the corresponding transferase of each donor through multiple enzymatic reactions to synthesize three N-acetylglucosamine sulfonated human milk oligosaccharides at position 6: DF-pLNH sulfate I, DF-pLNH sulfate II, and TF-pLNH sulfate. This method provides a simple and rapid in vitro synthesis of high-purity human milk oligosaccharides, enriching the human milk oligosaccharide library.

Description

Technical Field

[0001] This invention belongs to the field of glycosyl compound preparation technology, specifically relating to a method for synthesizing N-acetylglucosamine sulfonated at position 6 of human lactose oligosaccharide. Background Technology

[0002] Human milk oligosaccharides (HMOs) are naturally occurring, bioactive oligosaccharides found in human milk in free form. They are structurally diverse oligosaccharides composed of five basic monosaccharides linked in varying proportions: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (Sia). All these monosaccharides are linked to lactose through different glycosidic bonds, resulting in the diverse structures of HMOs. HMOs can be classified into neutral and acidic types. Neutral HMOs primarily consist of oligosaccharides containing fucose groups, while acidic HMOs contain sialic acid and its sulfate form. Human milk oligosaccharides (HMOs) are a complex mixture of numerous oligosaccharides. Currently, over 200 types of HMOs have been identified in human milk, while over 50 types are found in cow's milk. The content of HMOs differs significantly between human and cow's milk: human colostrum contains 22-23 g / L, mature milk contains 12-13 g / L, while cow's milk contains only trace amounts of HMOs.

[0003] Human breast milk contains three main organic molecules (HMOs): HMOs are the third largest solid component after lactose and fat. They play a variety of important physiological roles, including maintaining gut microbiota balance, inhibiting intestinal pathogen infection, regulating immune responses, and promoting infant brain development. Numerous studies have shown that HMOs are anti-adhesion antibiotics, acting as decoy receptors to prevent pathogens from adhering to the infant's mucosal surface, thus reducing the risk of viral, bacterial, and protozoan parasite infections. Furthermore, HMOs can regulate epithelial and immune cell responses, reduce mucus cell infiltration and activation, lower the risk of necrotizing enterocolitis, and provide sialic acid to infants. Therefore, HMOs are essential nutrients for brain development and cognitive growth.

[0004] Traditional methods for synthesizing human milk oligosaccharides (HMOs) mainly include chemical methods, enzymatic methods, whole-cell biocatalysis (microbial fermentation), and chemical-enzyme combined methods. Chemical synthesis methods offer high flexibility, theoretically capable of synthesizing HMOs of any structure. However, existing chemical synthesis methods are costly and difficult to control configuration, especially when synthesizing complex HMOs. These methods require numerous reaction steps, a large number of expensive chemical reagents, and complex subsequent purification processes, resulting in extremely low yields and high costs, making them unsuitable for large-scale industrial production. Whole-cell biocatalysis utilizes the powerful reproductive and metabolic capabilities of microorganisms, using inexpensive substrates, and can achieve ton-scale production at a much lower cost than other methods. However, this method has a long development cycle, requires complex metabolic engineering and strain modification, and optimizing fermentation conditions is time-consuming and labor-intensive. Currently, the products that can be synthesized in large quantities are at most pentasaccharides, and there are also many byproducts. Enzymes possess extremely high regio and stereoselectivity, enabling precise synthesis of target structures with fewer byproducts. Purification is relatively simple, and they are typically carried out under mild aqueous phases, neutral pH, and room temperature conditions, making them energy-efficient and environmentally friendly. Currently, there is no clear synthetic method for the three N-acetylglucosamine sulfonated human milk oligosaccharides DF-pLNH sulfate I, DF-pLNH sulfate II, and TF-pLNH sulfate, which mainly rely on separation from breast milk, thus limiting the subsequent structural characterization and physiological function research of this series of compounds. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for synthesizing N-acetylglucosamine sulfonated at the 6-position of human milk oligosaccharides. Through a multi-step enzymatic reaction, three structures of N-acetylglucosamine sulfonated at the 6-position of human milk oligosaccharides—DF-pLNH sulfate I, DF-pLNH sulfate II, and TF-pLNH sulfate—were successfully synthesized in vitro. This synthetic method is simple, rapid, and exhibits strong stereospecificity.

[0006] Specifically, the present invention adopts the following technical solution to achieve the above objectives:

[0007] A method for synthesizing N-acetylglucosamine sulfonated human lactic acid oligosaccharide DF-pLNH sulfate I, comprising the following steps:

[0008] S1, lactose and UDP-GlcNAc react under the action of NmLgtA to form compound (1).

[0009] S2, compound (1) reacts with PAPS under the action of CHST2 to generate compound (2);

[0010] S3-1, Compound (2) reacts with UDP-Gal under the action of Hpβ14GalT to generate compound (3).

[0011] S4-1, Compound (3) reacts with UDP-GlcNAc under the action of Hpβ3GlcNAcT to generate compound (4).

[0012] S5-1, Compound (4) reacts with GDP-Fuc under the action of Hp34FucT to generate compound (5);

[0013] S6-1, Compound (5) reacts with UDP-Gal under the action of Cvβ13GalT to generate compound (6).

[0014] S7-1, Compound (6) reacts with GDP-Fuc under the action of α12FucT to generate DF-pLNH sulfate I;

[0015] The structural formulas of compounds (1) to (6) and DF-pLNH sulfate I are as follows: Figure 1 As shown.

[0016] A method for synthesizing N-acetylglucosamine sulfonated human lactic acid oligosaccharide DF-pLNH sulfate II includes the following steps:

[0017] S1, lactose and UDP-GlcNAc react under the action of NmLgtA to form compound (1).

[0018] S2, compound (1) reacts with PAPS under the action of CHST2 to generate compound (2);

[0019] S3-2, Compound (2) reacts with CMP-Neu5Ac under the action of Pd2,6ST to generate compound (7);

[0020] S4-2, Compound (7) reacts with UDP-Gal under the action of Hpβ14GalT to generate compound (8).

[0021] S5-2, Compound (8) reacts with UDP-GlcNAc under the action of Hpβ3GlcNAcT to generate compound (9);

[0022] S6-2, Compound (9) reacts with UDP-Gal under the action of Cvβ13GalT to generate compound (10).

[0023] S7-2', compound (10) and GDP-Fuc react under the action of Hp34FucT to generate compound (11).

[0024] S8' and compound (11) were hydrolyzed by α-2,6-sialidase to give DF-pLNH sulfate II;

[0025] The structural formulas of compounds (1), (2), (7)~(11) and DF-pLNH sulfate II are as follows: Figure 2 As shown.

[0026] A method for synthesizing N-acetylglucosamine sulfonated human lactic acid oligosaccharide TF-pLNH sulfate, comprising the following steps:

[0027] S1, lactose and UDP-GlcNAc react under the action of NmLgtA to form compound (1).

[0028] S2, compound (1) reacts with PAPS under the action of CHST2 to generate compound (2);

[0029] S3-2, Compound (2) reacts with CMP-Neu5Ac under the action of Pd2,6ST to generate compound (7);

[0030] S4-2, Compound (7) reacts with UDP-Gal under the action of Hpβ14GalT to generate compound (8).

[0031] S5-2, Compound (8) reacts with UDP-GlcNAc under the action of Hpβ3GlcNAcT to generate compound (9);

[0032] S6-2, Compound (9) reacts with UDP-Gal under the action of Cvβ13GalT to generate compound (10).

[0033] S7-2'', Compound (10) reacts with GDP-Fuc under the action of α12FucT to generate compound (12), and Compound (12) reacts with GDP-Fuc under the action of Hp34FucT to generate compound (13).

[0034] S8” and compound (13) were hydrolyzed by α-2,6-sialidase to give TF-pLNH sulfate.

[0035] The structural formulas of compounds (1), (2), (7)~(10), (12), (13) and TF-pLNH sulfate are as follows: Figure 3 As shown.

[0036] In a preferred embodiment, the molar ratio of lactose to UDP-GlcNAc in step S1 is 1:1.5.

[0037] In a preferred embodiment, step S1 includes the following steps: adding lactose, UDP-GlcNAc, and Mg-containing... 2+ The solution and NmLgtA were mixed thoroughly, and the pH of the reaction system was adjusted to 7.0-8.0. The reaction system was then reacted at 30℃-37℃ until TLC detection showed that the reaction product no longer increased. The reaction was then terminated by boiling water bath. Lactose and Mg were present in the reaction system. 2+ The molar ratio of NmLgtA to UDP-GlcNAc is 1:1:1.5, and the final concentration of NmLgtA is 1 mg / mL.

[0038] In a further preferred embodiment, the developing solvent for TLC detection in step S1 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0039] In a further preferred embodiment, the buffer solution used to adjust the pH in step S1 is either Tris-HCl buffer or HEPES buffer.

[0040] In a preferred embodiment, in step S2, the molar ratio of compound (1) to PAPS is 1:3.

[0041] In a preferred embodiment, step S2 includes the following steps: mixing compound (1), PAPS, and Mg-containing compounds. 2+ The solution and CHST2 were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until TLC detection showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compounds (1), PAPS and Mg 2+ The molar ratio is 1:3:2, and the final concentration of CHST2 is 2 mg / mL.

[0042] In a further preferred embodiment, the developing solvent for TLC detection in step S2 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0043] In a further preferred embodiment, the buffer solution used to adjust the pH in step S2 is either Tris-HCl buffer or HEPES buffer.

[0044] In the preferred embodiment, in step S3-1, the molar ratio of compound (2) to UDP-Gal is 1:1.5.

[0045] In the preferred embodiment, step S3-1 includes the following steps: mixing compound (2), UDP-Gal, and Mg-containing compounds. 2+The solution and Hpβ14GalT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (2), UDP-Gal and Mg 2+ The molar ratio was 1:1.5:2, and the final concentration of Hpβ14GalT was 1 mg / mL.

[0046] In a further preferred embodiment, the developing solvent for TLC detection in step S3-1 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0047] In a further preferred embodiment, the buffer solution used to adjust the pH in step S3-1 is either Tris-HCl buffer or HEPES buffer.

[0048] In the preferred embodiment, the molar ratio of compound (3) to UDP-GlcNAc in step S4-1 is 1:1.5.

[0049] In the preferred embodiment, step S4-1 includes the following steps: mixing compound (3), UDP-GlcNAc, and Mn-containing compounds. 2+ The solution and Hpβ3GlcNAcT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (3), UDP-GlcNAc and Mn 2+ The molar ratio was 1:1.5:2, and the final concentration of Hpβ3GlcNAcT was 1 mg / mL;

[0050] In a further preferred embodiment, the developing solvent for TLC detection in step S4-1 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0051] In a further preferred embodiment, the buffer solution used to adjust the pH in step S4-1 is either Tris-HCl buffer or HEPES buffer.

[0052] In the preferred embodiment, in step S5-1, the molar ratio of compound (4) to GDP-Fuc is 1:1.5.

[0053] In the preferred embodiment, step S5-1 includes the following steps: mixing compound (4), GDP-Fuc, and Mg-containing compounds. 2+The solution and Hp34FucT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction products no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (4), GDP-Fuc and Mg 2+ The molar ratio was 1:1.5:2, and the final concentration of Hp34FucT was 1 mg / mL.

[0054] In a further preferred embodiment, the developing solvent for TLC detection in step S5-1 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0055] In a further preferred embodiment, the buffer solution used to adjust the pH in step S5-1 is either Tris-HCl buffer or HEPES buffer.

[0056] In the preferred embodiment, the molar ratio of compound (5) to UDP-Gal in step S6-1 is 1:1.5.

[0057] In the preferred embodiment, step S6-1 includes the following steps: mixing compound (5), UDP-Gal, and Mn-containing compounds. 2+ The solution and Cvβ13GalT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (5), UDP-Gal and Mn 2+ The molar ratio was 1:1.5:2, and the final concentration of Cvβ13GalT was 1 mg / mL.

[0058] In a further preferred embodiment, the developing solvent for TLC detection in step S6-1 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0059] In a further preferred embodiment, the buffer solution used to adjust the pH in step S6-1 is either Tris-HCl buffer or HEPES buffer.

[0060] In the preferred embodiment, the molar ratio of compound (6) to GDP-Fuc in step S7-1 is 1:1.5.

[0061] In the preferred embodiment, step S7-1 includes the following steps: mixing compound (6), GDP-Fuc, and Mn-containing compounds. 2+The solution and α12FucT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30~37℃ until the TLC detection results showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (6), GDP-Fuc and Mn 2+ The molar ratio is 1:1.5:2, and the final concentration of α12FucT is 1 mg / mL.

[0062] In a further preferred embodiment, the developing solvent for TLC detection in step S7-1 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0063] In a further preferred embodiment, the buffer solution used to adjust the pH in step S7-1 is either Tris-HCl buffer or HEPES buffer.

[0064] In the preferred embodiment, the molar ratio of compound (2) to CMP-Neu5Ac in step S3-2 is 1:1.5.

[0065] In the preferred embodiment, step S3-2 includes the following steps: mixing compound (2), CMP-Neu5Ac, and Mg-containing compounds. 2+ The solution and Pd2,6ST were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (2), CMP-Neu5Ac and Mg 2+ The molar ratio was 1:1.5:2, and the final concentration of Pd2,6ST was 1 mg / mL.

[0066] In a further preferred embodiment, the developing solvent for TLC detection in step S3-2 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0067] In a further preferred embodiment, the buffer solution used to adjust the pH in step S3-2 is Tris-HCl buffer or HEPES buffer.

[0068] In the preferred embodiment, the molar ratio of compound (7) to UDP-Gal in step S4-2 is 1:1.5.

[0069] In the preferred embodiment, step S4-2 includes the following steps: mixing compound (7), UDP-Gal, and Mg-containing compounds. 2+The solution and Hpβ14GalT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (7), UDP-Gal and Mg 2+ The molar ratio was 1:1.5:2, and the final concentration of Hpβ14GalT was 1 mg / mL.

[0070] In a further preferred embodiment, the developing solvent for TLC detection in step S4-2 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0071] In a further preferred embodiment, the buffer used to adjust the pH in step S4-2 is Tris-HCl buffer or HEPES buffer.

[0072] In the preferred embodiment, the molar ratio of compound (8) to UDP-GlcNAc in step S5-2 is 1:1.5.

[0073] In the preferred embodiment, step S5-2 includes the following steps: mixing compound (8), UDP-GlcNAc, and Mg-containing compounds. 2+ The solution and Hpβ3GlcNAcT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until TLC detection showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (8), UDP-GlcNAc and Mg 2+ The molar ratio was 1:1.5:2, and the final concentration of Hpβ3GlcNAcT was 1 mg / mL.

[0074] In a further preferred embodiment, the developing solvent for TLC detection in step S5-2 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0075] In a further preferred embodiment, the buffer solution used to adjust the pH in step S5-2 is Tris-HCl buffer or HEPES buffer.

[0076] In the preferred embodiment, the molar ratio of compound (9) to UDP-Gal in step S6-2 is 1:1.5.

[0077] In the preferred embodiment, step S6-2 includes the following steps: mixing compound (9), UDP-Gal, and Mn-containing compounds. 2+The solution and Cvβ13GalT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until TLC detection showed that the reaction product no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (9), UDP-Gal and Mn 2+ The molar ratio was 1:1.5:2, and the final concentration of Cvβ13GalT was 1 mg / mL.

[0078] In a further preferred embodiment, the developing solvent for TLC detection in step S6-2 is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0079] In a further preferred embodiment, the buffer solution used to adjust the pH in step S6-2 is Tris-HCl buffer or HEPES buffer.

[0080] In the preferred embodiment, the molar ratio of compound (10) to GDP-Fuc in step S7-2' is 1:3.

[0081] In the preferred embodiment, step S7-2' includes the following steps: mixing compound (10), GDP-Fuc, and Mg-containing compounds. 2+ The solution and Hp34FucT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction products no longer increased, and the reaction was terminated by boiling water bath; the reaction system contained compound (10), GDP-Fuc and Mg 2+ The molar ratio was 1:3:2, and the final concentration of Hp34FucT was 1 mg / mL.

[0082] In a further preferred embodiment, the developing solvent for TLC detection in step S7-2' is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0083] In a further preferred embodiment, the buffer used to adjust the pH in step S7-2' is Tris-HCl buffer or HEPES buffer.

[0084] In the preferred embodiment, the final concentration of compound (11) in step S8' is 5 mmol / L, and the final concentration of α-2,6-sialic acid glycosidase is 1 mg / mL.

[0085] In the preferred embodiment, step S8' includes the following steps: mixing compound (11) with α-2,6-sialic acid glycosidase, adjusting the pH of the mixture to 7.0~8.0 to obtain a reaction system, wherein the final concentration of compound (11) in the reaction system is 5 mmol / L and the final concentration of α-2,6-sialic acid glycosidase is 1 mg / mL; placing the reaction system at 30℃~37℃ until the product no longer increases as detected by TLC, and then terminating the reaction in a boiling water bath; centrifuging the reaction solution, and purifying the supernatant sequentially by a Bio-Gel P2 chromatography column and an amide column to obtain DF-pLNH sulfate II.

[0086] In a further preferred embodiment, the developing solvent for TLC detection in step S8' is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0087] In a further preferred embodiment, the buffer used to adjust the pH in step S8' is Tris-HCl buffer or HEPES buffer.

[0088] In the preferred embodiment, the molar ratio of compound (10) to GDP-Fuc in step S7-2" is 1:1.5; or / and the molar ratio of compound (12) to GDP-Fuc is 1:3.

[0089] In the preferred embodiment, step S7-2" includes the following steps: mixing compound (10), GDP-Fuc, and Mn-containing compounds. 2+ The solution and α12FucT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased. The reaction was terminated by boiling water bath. The reaction solution was centrifuged and the supernatant was compound (12). In the reaction system for synthesizing compound (12), compound (10), GDP-Fuc and Mn were present. 2+ The molar ratio was 1:1.5:2, and the final concentration of α12FucT was 1 mg / mL; compound (12), GDP-Fuc, and Mg-containing compounds were added. 2+ The solution and Hp34FucT were mixed evenly, and the pH of the reaction system was adjusted to 7.0~8.0. The reaction system was reacted at 30℃~37℃ until the TLC detection results showed that the reaction product no longer increased. The reaction was terminated by boiling water bath. The reaction solution was centrifuged, and the supernatant obtained was compound (13). Compound (12), GDP-Fuc and Mg were present in the reaction system. 2+ The molar ratio was 1:3:2, and the final concentration of Hp34FucT was 1 mg / mL.

[0090] In a further preferred embodiment, when synthesizing compound (12) in step S7-2", the developing solvent for TLC detection is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1; or / and when synthesizing compound (13), the developing solvent for TLC detection is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0091] In a further preferred embodiment, when synthesizing compound (12) in step S7-2", the buffer used to adjust the pH is Tris-HCl buffer or HEPES buffer; or / and when synthesizing compound (13), the buffer used to adjust the pH is Tris-HCl buffer or HEPES buffer.

[0092] In the preferred embodiment, the final concentration of compound (13) in step S8" is 5 mmol / L, and the final concentration of α-2,6-sialic acid glycosidase is 1 mg / mL.

[0093] In the preferred embodiment, step S8" includes the following steps: mixing compound (13) with α-2,6-sialic acid glycosidase, adjusting the pH of the mixture to 7.0~8.0 to obtain a reaction system, wherein the final concentration of compound (13) in the reaction system is 5 mmol / L and the final concentration of α-2,6-sialic acid glycosidase is 1 mg / mL; placing the reaction system at 30℃~37℃ until the product no longer increases as detected by TLC, and then terminating the reaction in a boiling water bath; centrifuging the reaction solution, and purifying the supernatant sequentially by a Bio-Gel P2 chromatography column and an amide column to obtain TF-pLNH sulfate.

[0094] In a further preferred embodiment, the developing solvent for TLC detection in step S8" is a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1.

[0095] In a further preferred embodiment, the buffer used to adjust the pH in step S8" is Tris-HCl buffer or HEPES buffer.

[0096] In this invention, lactose is used as the starting substrate, and UDP-GlcNAc is used as the glycosyl donor. Neisseria meningitidis Under the action of β-1,3-N-acetylglucosamine transferase (NmLgtA), compound (1) is generated. Compound (1) reacts with PAPS, a sulfonyl donor, in a reaction derived from... Homo sapiensCompound (2) was generated by GlcNAc-6-O-sulfotransferase (CHST2), and purified by DEAE ion exchange chromatography. Compound (2) was then reacted with different glycosyl donors and, through a multi-step enzymatic reaction with the corresponding transferase of each glycosyl donor, synthesized three N-acetylglucosamine 6-sulfonated human lactose oligosaccharides: DF-pLNH sulfate I, DF-pLNH sulfate II, and TF-pLNH sulfate.

[0097] Compared with existing technologies, the present invention has the following advantages: Using lactose as a starting substrate, the present invention successfully synthesized three structurally different N-acetylglucosamine sulfonated human milk oligosaccharides at position 6 through a multi-step enzymatic reaction, enriching the human milk oligosaccharide library and playing an important role in further studying the physiological functions of N-acetylglucosamine sulfonated human milk oligosaccharides at position 6. This method is simple, rapid, and exhibits strong stereospecificity. Attached Figure Description

[0098] Figure 1 This is a synthetic route diagram of DF-pLNH sulfate I in Example 1 of the present invention;

[0099] Figure 2 This is a synthetic route diagram of DF-pLNH sulfate II in Example 2 of the present invention;

[0100] Figure 3 This is a synthetic route diagram of TF-pLNH sulfate in Example 3 of the present invention;

[0101] Figure 4 The results of TLC detection of the reactions in steps S2 to S7 in Example 1 of the present invention; 1 to 6 in the figure are, in order, the reaction solution of compound (2), the reaction solution of compound (3), the reaction solution of compound (4), the reaction solution of compound (5), the reaction solution of compound (6) and the reaction solution of DF-pLNH sulfate I;

[0102] Figure 5 This is a HPLC-ELSD detection result of DF-pLNH sulfate I synthesized in Example 1 of the present invention;

[0103] Figure 6 This is a graph showing the MS (negative ion mode) detection results of DF-pLNH sulfate I synthesized in Example 1 of the present invention;

[0104] Figure 7 This is a chromatogram of the 1H-NMR detection results of DF-pLNH sulfate I synthesized in Example 1 of the present invention;

[0105] Figure 8 This is a HPLC-ELSD detection result of DF-pLNH sulfate II synthesized in Example 2 of the present invention;

[0106] Figure 9 This is a graph showing the MS (negative ion mode) detection results of DF-pLNH sulfate II synthesized in Example 2 of the present invention;

[0107] Figure 10 This is a chromatogram of the 1H-NMR detection results of DF-pLNH sulfate II synthesized in Example 2 of the present invention;

[0108] Figure 11 This is a HPLC-ELSD detection result of TF-pLNH sulfate synthesized in Example 3 of the present invention;

[0109] Figure 12 This is a graph showing the MS (negative ion mode) detection results of the TF-pLNH sulfate synthesized in Example 3 of the present invention;

[0110] Figure 13 The image shows the 1H-NMR detection results of the TF-pLNH sulfate synthesized in Example 3 of the present invention. Detailed Implementation

[0111] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0112] The full names of the abbreviations or acronyms used in the following embodiments are as follows:

[0113] UDP-GlcNAc: Uridine diphosphate-N-acetylglucosamine;

[0114] NmLgtA: β-1,3-N-acetylglucosamine transferase (or β-1,3-N-acetylglucosamine transferase).

[0115] PAPS: 3'-Adenosine-5'-phosphate sulfate;

[0116] CHST2: Carbohydrate sulfonyltransferase 2;

[0117] UDP-Gal: uridine diphosphate galactose;

[0118] Hpβ14GalT: β-1,4-galactosyltransferase;

[0119] Hpβ3GlcNAcT: β-1,3-N-acetylglucosamine transferase;

[0120] GDP-Fuc: fucose guanosine diphosphate;

[0121] Hp34FucT: α-1,3 / 4-fucosyltransferase;

[0122] Cvβ13GalT: β-1,3-galactosyltransferase;

[0123] α12FucT: α-1,2-fucosyltransferase;

[0124] CMP-Neu5Ac: Cytidine monophosphate-N-acetylneuraminic acid;

[0125] Pd2,6ST: α-2,6-sialyltransferase;

[0126] AuSialidase S: ​​α-2,6-sialic acid glycosidase;

[0127] DF-pLNH sulfate I: Difucosyl-para-lacto-N-hexaose sulfate I (difucosyl-para-lacto-N-hexaose sulfate I);

[0128] DF-pLNH sulfate II: Difucosyl-para-lacto-N-hexaose sulfate II (difucosyl-para-lacto-N-hexaose sulfate II);

[0129] TF-pLNH sulfate: Trifucosyl-para-lacto-N-hexaose sulfate.

[0130] TLC: Thin-layer chromatography analysis;

[0131] Neisseria meningitidis : Neisseria meningitidis;

[0132] Helicobacter pylori Helicobacter pylori;

[0133] Chromobacterium violaceum : Purple violet bacillus;

[0134] Homo sapiens Homo sapiens, humankind;

[0135] IPTG: Isopropyl-β-D-thiogalactoside.

[0136] The expression and purification methods for various enzymes used in the following examples are as follows:

[0137] Source Neisseria meningitidis NmLgtA (Uniprot No.: Q9JXQ6) is derived from Helicobacter pylori Hpβ14GalT (Genbank No.: AB035971) is derived from Helicobacter pylori The Hpβ3GlcNAcT (Genbank No.: WP_145819987.1) is derived from... Helicobacter pylori The Hp34FucT (Genbank No.: AAF35291.2) is derived from... Chromobacterium violaceum The Cvβ13GalT (Genbank No.: WP_048405302.1) is derived from... Helicobacter pylori The α12FucT (Genbank No.: WP_080473865.1) is derived from... Photobacterium damselae Pd2,6ST (Genbank No.: BAA25316.1), sourced from Arthrobacter ureafaciens AuSialidase S (Genbank No.: WP_069696302.1) was synthesized using the following method: the enzyme gene sequence was optimized according to the codon preferences of *E. coli*, and then the gene was synthesized (by General Biotechnology (Anhui) Co., Ltd.). It was cloned into the pET-22b vector and then transformed into *E. coli* BL21(DE3) for expression. Single clones were picked and cultured overnight in test tubes containing LB liquid medium. The next day, they were inoculated into 200 mL of LB medium at a volume ratio of 1:100 and cultured at 37°C for 6 hours. Then, 200 mL of this medium was inoculated into a 5 L M9 fermenter and cultured at 37°C until OD... 600nm When the temperature approaches 30°C, the temperature is lowered to 20°C, and IPTG is added to bring the final concentration of IPTG in the reaction system to 0.2 mM. After induction for 12 hours, the mixture is transferred to a container. The cells are collected by centrifugation at 7000 g and resuspended in 100 mM Tris-HCl (pH 8.0) buffer. The recombinant protein is then released by high-pressure disruption and purified by nickel affinity chromatography to obtain the target protein.

[0138] Source Homo sapiensThe GlcNAc-6-O-sulfotransferase CHST2 (GenBank No.: BAA34265.2) was synthesized (by General Biotech (Anhui) Co., Ltd.), cloned into the AAV expression vector, and then transfected into HEK-293 cells for expression. The target protein was then purified by nickel column affinity chromatography.

[0139] The HPLC-ELSD (High Performance Liquid Chromatography with Evaporative Light Scattering Detector) detection conditions for DF-pLNH sulfate I in the following examples are as follows:

[0140] High-performance liquid chromatograph: Shanghai Tongwei EasySep®-3030;

[0141] Column: Waters XBridge® BEH Amide (250 × 4.6 mm, 5.0 μm);

[0142] Mobile phase A: 30 mM ammonium acetate aqueous solution;

[0143] Mobile phase B: ACN (acetonitrile);

[0144] Flow rate: 1 mL / min;

[0145] Detection wavelength: 254nm;

[0146] Column temperature: 40℃;

[0147] Detector: ELSD-UM5800;

[0148] Gas flow rate: 2.5 L / min;

[0149] Drift tube temperature: 40℃;

[0150] Injection volume: 10 μL;

[0151] The mobile phase gradient elution program is as follows:

[0152] .

[0153] The detection methods for DF-pLNH sulfate II and TF-pLNH sulfate are the same as those for DF-pLNH sulfate I.

[0154] The ammonia water used in the following examples is concentrated ammonia water, containing 25% to 28% ammonia.

[0155] Example 1

[0156] like Figure 1 As shown, a method for synthesizing human milk oligosaccharide DF-pLNH sulfate I includes the following steps:

[0157] S1, lactose synthetic compounds (1)

[0158] Lactose, UDP-GlcNAc, MgCl2, Tris-HCl buffer at pH 7.5, and NmLgtA were mixed thoroughly to achieve a final concentration of 10 mM for lactose, 15 mM for UDP-GlcNAc, 10 mM for MgCl2, 50 mM for the Tris-HCl buffer at pH 7.5, and 1.0 mg / mL for NmLgtA. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1). The reaction was terminated by boiling in a water bath for 10 minutes when the product concentration stopped increasing. The resulting reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was used for the next reaction.

[0159] S2, Compound (1) Synthesis of Compound (2)

[0160] Compound (1), PAPS, MgCl2, Tris-HCl buffer at pH 7.5, and CHST2 were mixed thoroughly to achieve a final concentration of 5 mM for compound (1), 15 mM for PAPS, 10 mM for MgCl2, 50 mM for Tris-HCl buffer at pH 7.5, and 2 mg / mL for CHST2. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was stopped when the product concentration stopped increasing (see [link to TLC]). Figure 4 The reaction was terminated by boiling in a water bath for 10 minutes. The resulting reaction solution was centrifuged at 12000g for 10 minutes, and the supernatant was purified by using a DEAE ion exchange column (DEAE Beads 6FF, Changzhou Tiandi Renhe Biotechnology Co., Ltd.) to obtain compound (2).

[0161] S3-1, Compound (2) Synthesis of Compound (3)

[0162] Compound (2), UDP-Gal, MgCl2, Tris-HCl at pH 7.5, and Hpβ14GalT were mixed thoroughly to achieve a final concentration of 5 mM for compound (2), 7.5 mM for UDP-Gal, 10 mM for MgCl2, 50 mM for the Tris-HCl buffer at pH 7.5, and 1.0 mg / mL for Hpβ14GalT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was stopped when the product concentration stopped increasing (see [link to TLC]). Figure 4 The reaction was terminated by boiling in a water bath for 10 minutes. The resulting reaction solution was centrifuged at 12000g for 10 minutes, and the supernatant was used for the next reaction.

[0163] S4-1, Compound (3) Synthesizes Compound (4)

[0164] Compound (3), UDP-GlcNAc, MnCl2, Tris-HCl buffer at pH 8.0, and Hpβ3GlcNAcT were mixed thoroughly to achieve a final concentration of 5 mM for compound (3), 7.5 mM for UDP-GlcNAc, 10 mM for MnCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1 mg / mL for Hpβ3GlcNAcT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1) until the product concentration stopped increasing (see [link to TLC]). Figure 4 The reaction was terminated by boiling in a water bath for 10 minutes. The resulting reaction solution was centrifuged at 12000g for 10 minutes, and the supernatant was used for the next reaction.

[0165] S5-1, Compound (4) Synthesize Compound (5)

[0166] Compound (4), GDP-Fuc, MgCl2, Tris-HCl buffer at pH 8.0, and Hp34FucT were mixed thoroughly to achieve a final concentration of 5 mM for compound (4), 7.5 mM for GDP-Fuc, 10 mM for MgCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1.0 mg / mL for Hp34FucT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). When the product no longer increased (see...), the reaction was stopped. Figure 4The reaction was terminated by boiling in a water bath for 10 minutes. The resulting reaction solution was centrifuged at 12000g for 10 minutes, and the supernatant was used for the next reaction.

[0167] S6, Compound (5) Synthesize Compound (6)

[0168] Compound (5), UDP-Gal, MnCl2, Tris-HCl buffer at pH 7.5, and Cvβ13GalT were mixed thoroughly to achieve a final concentration of 5 mM for compound (5), 7.5 mM for UDP-Gal, 10 mM for MnCl2, 50 mM for Tris-HCl buffer at pH 7.5, and 1.0 mg / mL for Cvβ13GalT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was stopped when the product concentration stopped increasing (see [link to TLC]). Figure 4 The reaction was terminated by boiling in a water bath for 10 minutes. The reaction solution was then centrifuged at 12000g for 10 minutes, and the supernatant was used for the next step of the reaction.

[0169] S7, Compound (6) Synthesis of DF-pLNH sulfate I

[0170] Compound (6), GDP-Fuc, MnCl2, Tris-HCl buffer at pH 8.0, and α12FucT were mixed thoroughly to achieve a final concentration of 5 mM for compound (6), 7.5 mM for GDP-Fuc, 10 mM for MnCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1.0 mg / mL for α12FucT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1) as the developing solvent. When the product no longer increased (see [link to TLC]...), the reaction was stopped. Figure 4 The reaction was terminated by boiling in a water bath for 10 minutes. The reaction solution was centrifuged at 12000g for 10 minutes, and the supernatant was purified by a Bio-Gel P2 chromatography column (purchased from Bio-rad, catalog number 1504114), and then purified by an amide column (purchased from Yuexu Technology (Shanghai) Co., Ltd., catalog number 02627-31106) to obtain the DF-pLNH sulfate I product.

[0171] The purified DF-pLNH sulfate I product was analyzed by HPLC-ELSD and the purity was 98.024% (see...). Figure 5This indicates that the purification achieved good results. MS (mass spectrometry) negative ion mode and H-NMR (hydrogen nuclear magnetic resonance) detection results confirmed that the molecular weight and structure were correct (see...). Figure 6 and Figure 7 ).

[0172] Example 2

[0173] like Figure 2 As shown, a method for synthesizing human milk oligosaccharide DF-pLNH sulfate II includes the following steps:

[0174] S1 is the same as step S1 in Example 1.

[0175] S2 is the same as step S2 in Example 1.

[0176] S3-2, Compound (2) Synthesis of Compound (7)

[0177] Compound (2), CMP-Neu5Ac, MgCl2, Tris-HCl buffer at pH 7.5, and Pd2,6ST were mixed thoroughly to achieve a final concentration of 10 mM for compound (2), 15 mM for CMP-Neu5Ac, 10 mM for MgCl2, 50 mM for Tris-HCl buffer at pH 7.5, and 1.0 mg / mL for Pd2,6ST. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The resulting reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was used for the next reaction.

[0178] S4-2, Compound (7) Synthesize Compound (8)

[0179] Compound (7), UDP-Gal, MgCl2, Tris-HCl buffer at pH 7.5, and Hpβ14GalT were mixed thoroughly to achieve a final concentration of 5 mM for compound (7), 7.5 mM for UDP-Gal, 10 mM for MgCl2, 50 mM for Tris-HCl buffer at pH 7.5, and 1 mg / mL for Hpβ14GalT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The resulting reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was purified using a DEAE ion exchange column to obtain compound (8).

[0180] S5-2, Compound (8) Synthesize Compound (9)

[0181] Compound (8), UDP-GlcNAc, MgCl2, Tris-HCl at pH 8.0, and Hpβ3GlcNAcT were mixed thoroughly to achieve a final concentration of 5 mM for compound (8), 7.5 mM for UDP-GlcNAc, 10 mM for MgCl2, 50 mM for the Tris-HCl buffer at pH 8.0, and 1 mg / mL for Hpβ3GlcNAcT. The reaction was carried out overnight at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The resulting reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was used for the next reaction.

[0182] S6-2, Compound (9) Synthesize Compound (10)

[0183] Compound (9), UDP-Gal, MnCl2, Tris-HCl buffer at pH 8.0, and Cvβ13GalT were mixed thoroughly to achieve a final concentration of 5 mM for compound (9), 7.5 mM for UDP-Gal, 10 mM for MnCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1 mg / ml for Cvβ13GalT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1) as the developing solvent. The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The resulting reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was used for the next reaction.

[0184] S7-2', Compound (10) Synthesize Compound (11)

[0185] Compound (10), GDP-Fuc, MgCl2, Tris-HCl buffer at pH 8.0, and Hp34FucT were mixed thoroughly to achieve a final concentration of 5 mM for compound (10), 15 mM for GDP-Fuc, 10 mM for MgCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1 mg / mL for Hp34FucT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The resulting reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was used for the next reaction.

[0186] S8', Compound (11) Synthesis of DF-pLNH sulfate II

[0187] Compound (11), Tris-HCl buffer at pH 7.5, and α-2,6-sialic acid glycosidase were mixed thoroughly to achieve a final concentration of 5 mM for compound (11), 50 mM for Tris-HCl buffer at pH 7.5, and 1 mg / mL for α-2,6-sialic acid glycosidase in the reaction system. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The reaction solution was centrifuged at 12000g for 10 minutes. The supernatant was purified using a Bio-Gel P2 chromatography column (purchased from Bio-rad, catalog number 1504114) and then purified again using an amide column (purchased from Yuexu Technology (Shanghai) Co., Ltd., catalog number 02627-31106) to obtain DF-pLNH sulfate II.

[0188] The purified DF-pLNH sulfate II product was analyzed by HPLC-ELSD and its purity was 98.951% (see...). Figure 8 This indicates that the purification achieved good results. MS (mass spectrometry) negative ion mode and H-NMR (hydrogen nuclear magnetic resonance) results confirm that the molecular weight and structure are correct (see...). Figure 9 and Figure 10 ).

[0189] Example 3

[0190] like Figure 3 As shown, a method for synthesizing the human milk oligosaccharide TF-pLNH sulfate includes the following steps:

[0191] The steps S1 to S6-2 are the same as in Example 2.

[0192] S7-2", Compound (10) Synthesize Compound (12)

[0193] Compound (10), GDP-Fuc, MnCl2, Tris-HCl buffer at pH 8.0, and α12FucT were mixed thoroughly to achieve a final concentration of 5 mM for compound (10), 7.5 mM for GDP-Fuc, 10 mM for MnCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1.0 mg / mL for α12FucT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, ammonia, and water in a volume ratio of 5:4:1 as the developing solvent). The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant was used for the next reaction.

[0194] Compound (12), GDP-Fuc, MgCl2, Tris-HCl buffer at pH 8.0, and Hp34FucT were mixed thoroughly to achieve a final concentration of 5 mM for compound (12), 15 mM for GDP-Fuc, 10 mM for MgCl2, 50 mM for Tris-HCl buffer at pH 8.0, and 1.0 mg / mL for Hp34FucT. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1) as the developing solvent. The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The reaction solution was centrifuged at 12000 g for 10 minutes, and the supernatant (compound (13)) was collected for the next reaction.

[0195] S8”, compound (13) synthesizes TF-pLNH sulfate

[0196] Compound (13), Tris-HCl buffer at pH 7.5, and α-2,6-sialic acid glycosidase were mixed thoroughly to achieve a final concentration of 5 mM for compound (13), a final concentration of 50 mM for Tris-HCl buffer at pH 7.5, and a final concentration of 1 mg / mL for α-2,6-sialic acid glycosidase. The reaction was carried out at 37°C. The reaction progress was monitored by TLC (using a mixture of isopropanol, concentrated ammonia, and water in a volume ratio of 5:4:1) as the developing solvent. The reaction was terminated by boiling in a water bath for 10 minutes when the product no longer increased. The reaction solution was centrifuged at 12000g for 10 minutes. The supernatant was purified using a Bio-Gel P2 chromatography column (purchased from Bio-rad, catalog number 1504114) and then purified again using an amide column (purchased from Yuexu Technology (Shanghai) Co., Ltd., catalog number 02627-31106) to obtain TF-pLNH sulfate.

[0197] The purified TF-pLNH sulfate product was analyzed by HPLC-ELSD and its purity was 99.361% (see...). Figure 11 This indicates that the purification achieved good results. MS (mass spectrometry) negative ion mode and H-NMR (hydrogen nuclear magnetic resonance) results confirm that the molecular weight and structure are correct (see...). Figure 12 and Figure 13 ).

[0198] It should be noted that the pH of the Tirs-HCl buffer in the above embodiments can be between 7.0 and 8.0. In the above embodiments, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer with a pH of 7.0 to 8.0 can also be used instead of the Tirs-HCl buffer with a pH of 7.0 to 8.0. The enzymatic reaction temperature for each step can be between 30℃ and 37℃.

[0199] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of this invention and the content of the specification should be included within the scope of protection of the present invention.

Claims

1. A method for the synthesis of N-acetylglucosamine 6-position sulfonated human milk oligosaccharide, characterized by, The method comprises the following steps: S1, lactose reacts with UDP-GlcNAc under the action of NmLgtA to generate compound (1); S2, compound (1) reacts with PAPS under the action of CHST2 to generate compound (2); S3-1, compound (2) reacts with UDP-Gal under the action of Hpβ14GalT to generate compound (3); S4-1, compound (3) reacts with UDP-GlcNAc under the action of Hpβ3GlcNAcT to generate compound (4); S5-1, compound (4) reacts with GDP-Fuc under the action of Hp34FucT to generate compound (5); S6-1, compound (5) reacts with UDP-Gal under the action of Cvβ13GalT to generate compound (6); S7-1, compound (6) reacts with GDP-Fuc under the action of α12FucT to generate DF-pLNH sulfate I; The method for synthesizing the N-acetylglucosamine 6 sulfated human milk oligosaccharide comprises the following steps: S1, lactose reacts with UDP-GlcNAc under the action of NmLgtA to generate compound (1); S2, compound (1) reacts with PAPS under the action of CHST2 to generate compound (2); S3-2, compound (2) reacts with CMP-Neu5Ac under the action of Pd2,6ST to generate compound (7); S4-2, compound (7) reacts with UDP-Gal under the action of Hpβ14GalT to generate compound (8); S5-2, compound (8) reacts with UDP-GlcNAc under the action of Hpβ3GlcNAcT to generate compound (9); S6-2, compound (9) reacts with UDP-Gal under the action of Cvβ13GalT to generate compound (10); S7-2, compound (10) reacts with GDP-Fuc under the action of Hp34FucT to generate compound (11); or, compound (10) reacts with GDP-Fuc under the action of α12FucT to generate compound (12), and compound (12) reacts with GDP-Fuc under the action of Hp34FucT to generate compound (13); S8, compound (11) is hydrolyzed under the action of α-2,6-sialidase to obtain DF-pLNH sulfate II; or compound (13) is hydrolyzed under the action of α-2,6-sialidase to obtain TF-pLNH sulfate; The structural formulas of compounds (1) to (13) and DF-pLNH sulfate I, DF-pLNH sulfate II, and TF-pLNH sulfate are as follows: , , ， ， ， ， ， ， ， ， ， ， ， ， ， 。 2. The method of synthesis of claim 1, wherein, Step S1 includes the following steps: mixing lactose, UDP-GlcNAc, Mg 2+ containing solution and NmLgtA uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37℃ until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of lactose, Mg 2+ and UDP-GlcNAc in the reaction system is 1:1:1.5, and the final concentration of NmLgtA is 1 mg / mL; Or / and, Step S2 includes the following steps: mixing compound (1), PAPS, a solution containing Mg 2+ and CHST2 uniformly, adjusting the pH of the reaction system to 7.0-8.0, reacting the reaction system at 30°C-37°C until the TLC detection result shows that the reaction product no longer increases, and stopping the reaction in a boiling water bath; the molar ratio of compound (1), PAPS and Mg 2+ in the reaction system is 1:3:2, and the final concentration of CHST2 is 2 mg / mL.

3. The method of synthesis of claim 1, wherein, Step S3-1 includes the following steps: mixing compound (2), UDP-Gal, Mg 2+ containing solution and Hpβ14GalT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (2), UDP-Gal and Mg 2+ in the reaction system is 1:1.5:2, and the final concentration of Hpβ14GalT is 1 mg / mL; or / and, step S4-1 includes the following steps: mixing compound (3), UDP-GlcNAc, Mn 2+ containing solution and Hpβ3GlcNAcT uniformly, adjusting the pH of the reaction system to 7.0~8.0, and reacting the reaction system at 30℃~37℃ until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (3), UDP-GlcNAc and Mn 2+ in the reaction system is 1:1.5:2, and the final concentration of Hpβ3GlcNAcT is 1 mg / mL; Or / and, Step S5-1 includes the following steps: mixing compound (4), GDP-Fuc, a solution containing Mg 2+ and Hp34FucT uniformly, adjusting the pH of the reaction system to 7.0~8.0, and reacting the reaction system at 30℃~37℃ until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (4), GDP-Fuc and Mg 2+ in the reaction system is 1:1.5:2, and the final concentration of Hp34FucT is 1 mg / mL.

4. The method of synthesis of claim 1, wherein, Step S6-1 includes the following steps: mixing compound (5), UDP-Gal, a solution containing Mn 2+ and Cvβ13GalT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (5), UDP-Gal and Mn 2+ in the reaction system is 1:1.5:2, and the final concentration of Cvβ13GalT is 1 mg / mL; Or / and, Step S7-1 includes the following steps: mixing compound (6), GDP-Fuc, a solution containing Mn 2+ and a12FucT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (6), GDP-Fuc and Mn 2+ in the reaction system is 1:1.5:2, and the final concentration of a12FucT is 1 mg / mL.

5. The method of synthesis of claim 1, wherein, Step S3-2 includes the following steps: mixing compound (2), CMP-Neu5Ac, a solution containing Mg 2+ and Pd2,6ST uniformly, adjusting the pH of the reaction system to 7.0~8.0, and reacting the reaction system at 30℃~37℃ until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (2), CMP-Neu5Ac and Mg 2+ in the reaction system is 1:1.5:2, and the final concentration of Pd2,6ST is 1 mg / mL; Or / and, Step S4-2 includes the following steps: mixing compound (7), a solution of UDP-Gal containing Mg 2+ and Hpβ14GalT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (7), UDP-Gal and Mg 2+ in the reaction system is 1:1.5:2, and the final concentration of Hpβ14GalT is 1 mg / mL.

6. The method of synthesis of claim 1, wherein, Step S5-2 includes the following steps: mixing compound (8), UDP-GlcNAc, Mg 2+ containing solution and Hpβ3GlcNAcT uniformly, adjusting the pH of the reaction system to 7.0~8.0, and reacting the reaction system at 30℃~37℃ until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (8), UDP-GlcNAc and Mg 2+ in the reaction system is 1:1.5:2, and the final concentration of Hpβ3GlcNAcT is 1 mg / mL; Or / and, Step S6-2 includes the following steps: mixing compound (9), a solution of UDP-Gal containing Mn 2+ and Cvβ13GalT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (9), UDP-Gal and Mn 2+ in the reaction system is 1:1.5:2, and the final concentration of Cvβ13GalT is 1 mg / mL.

7. The method of synthesis of claim 1, wherein, Step S7-2 includes the following steps: mixing compound (10), GDP-Fuc, a solution containing Mg 2+ and Hp34FucT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, and the reaction is terminated in a boiling water bath; the molar ratio of compound (10), GDP-Fuc and Mg 2+ in the reaction system is 1:3:2, and the final concentration of Hp34FucT is 1 mg / mL; or, step S7-2 comprises the following steps: mixing compound (10), GDP-Fuc, a solution containing Mn 2+ and a12FucT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases, the reaction is terminated in a boiling water bath, and the supernatant obtained by centrifugation is compound (12); the molar ratio of compound (10), GDP-Fuc and Mn 2+ in the reaction system for synthesizing compound (12) is 1:1.5:2, and the final concentration of a12FucT is 1 mg / mL; mixing compound (12), GDP-Fuc, a solution containing Mg 2+ and Hp34FucT uniformly, adjusting the pH of the reaction system to 7.0-8.0, and reacting the reaction system at 30-37°C until the TLC detection result shows that the reaction product no longer increases; the molar ratio of compound (12), GDP-Fuc and Mg 2+ in the reaction system is 1:3:2, and the final concentration of Hp34FucT is 1 mg / mL.

8. The method of synthesis of claim 1, wherein, In the reaction system for synthesizing DF-pLNH sulfate II in step S8, the final concentration of compound (11) is 5 mmol / L, and the final concentration of α-2,6-sialidase is 1 mg / mL; Or in step S8, the final concentration of compound (13) in the reaction system for synthesizing TF-pLNH sulfate is 5 mmol / L, and the final concentration of α-2,6-sialidase is 1 mg / mL.

9. The method of synthesis according to any one of claims 2 to 8, wherein, The developing agent for TLC detection in each step is a mixture of isopropyl alcohol, concentrated ammonia water and water in a volume ratio of 5:4:1; or / and the buffer used for adjusting the pH to 7.0-8.0 in each step is Tris-HCl buffer or HEPES buffer.

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