A method for the synthesis of beta 1,6-glcnaclyated chitinous oligosaccharide derivatives

Abstract

The application discloses a synthesis method of a beta 1,6-GlcNAc glycosylation chitin oligosaccharide derivative, and belongs to the technical field of enzymatic synthesis of marine saccharides. Chitin tetrasaccharide is used as a starting acceptor, and fucose is introduced as a temporary protective group to reduce the beta 1,6-GlcNAc glycosylation reactivity of a reducing end LacNAc unit. A newly generated linear chitin pentasaccharide containing alpha 1,3-fucosylation continues to extend a skeleton sugar chain, that is, a GCNT2 optimal poly-LacNAc tetrasaccharide structure is constructed at a non-reducing end. By controlling reaction conditions, the beta 1,6-GlcNAc residue is introduced at a site only on the internal galactose of the non-reducing end. Finally, fucosidase E1-10125 is used to remove the temporary protective group, and the target product is released. In the application, the intermediate compound generated by using fucose as the temporary protective group is itself a potential target sugar chain, and is beneficial to construction of a complex natural sugar library with a definite structure.

Description

Technical Field

[0001] This invention belongs to the field of enzymatic synthesis technology of marine carbohydrates, specifically relating to a method for synthesizing β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives. Background Technology

[0002] Chitin oligosaccharide derivatives, due to their excellent biocompatibility and well-defined molecular structure, serve as important vehicles for exploring glycobiological functions. Among them, the linear poly-LacNAc backbone composed of tandem LacNAc (Galβ1,4GlcNAc) units can be converted by β1,6- N GCNT2 modification generates I-antigen epitopes with important physiological functions. However, GCNT2 enzymes can recognize multiple internal galactose residues in the linear poly-LacNAc backbone, lacking site selectivity. This results in a mixture of isomers with varying branch positions, severely hindering the construction of a homogeneous chitin-associated I-antigen oligosaccharide library and limiting its application in studies of mechanisms such as immune recognition.

[0003] To address the aforementioned site selectivity challenges, existing strategies often employ chemical protecting groups (such as TFA and Boc) or introduce non-natural human orthogonal protecting groups (such as C6-aldehyde galactose and KDN) to shield non-target sites. However, these chemical-enzymatic routes involve cumbersome protecting group operations, and the introduction of non-natural human glycan intermediates increases the risk of synthetic steps and byproduct generation, hindering the efficient construction of natural oligosaccharide libraries. Although the fucosidase-assisted substrate engineering strategy (using enzyme E1-10125 to remove the temporary fucosylate protecting group) has been successfully applied to the sulfonation and sialylation modification of linear poly-LacNAc, it remains unclear whether this strategy can overcome the site selectivity challenge of GCNT2 and achieve specific introduction of the I-antigen at the non-reducing end of chitin oligosaccharide derivatives. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows.

[0006] In a first aspect, the present invention provides a method for synthesizing β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives, comprising the following steps: Using chitin tetrasaccharides GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc as glycosyl acceptors and fucose as a glycosyl donor, chitin pentasaccharides were obtained via enzymatic assembly module one. The structure of the chitin pentasaccharide is as follows: ; Chitin hexasaccharide was obtained via enzymatic assembly module II catalysis using chitin pentose as a glycosyl acceptor and galactose as a glycosyl donor; the structure of the chitin hexasaccharide is as follows: ; Using chitin hexasaccharide as a glycosyl acceptor, N - Acetaminoglycine is used as a glycosyl donor, and chitin heptaose is obtained through a three-catalyzed enzymatic assembly module; the structure of the chitin heptaose is as follows: ; Using chitin heptaose as a glycosyl acceptor and galactose as a glycosyl donor, chitin octaose was obtained via enzymatic assembly module II catalysis; the structure of the chitin octaose is as follows: ; Using chitin octasaccharide as a glycosyl acceptor, N - Acetaminoglycine is used as a glycosyl donor, and chitinine nonaose is obtained by enzymatic assembly module four-catalysis; the structure of chitinine nonaose is as follows: ; Using chitin nonaose as a glycosyl acceptor, fucosidase E1-10125 catalyzes the generation of site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives; the structure of the β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives is as follows: ; R is any one of hydroxyl, azide-substituted alkyl, alkynyl-substituted alkyl, mercapto-substituted alkyl, α- or β-configuration substituted alkyl, α- or β-configuration serine residue, or α- or β-configuration threonine residue.

[0007] The beneficial effects of this invention are as follows: This invention provides a method for synthesizing β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives, successfully applying a fucosidase-assisted substrate engineering strategy to the chitin oligosaccharide system, solving the key problem of disordered I-antigen branching caused by the lack of site selectivity of GCNT2 enzyme. The core lies in utilizing the fuc (Fuc) site-blocking mechanism. First, using chitin tetrasaccharide as the initiating acceptor, Fuc is introduced as a temporary protecting group via fucosyltransferase catalysis, reducing the GCNT2 enzyme recognition activity of the reducing-terminal LacNAc unit. Subsequently, through the tandem use of multi-step enzymatic assembly (alternating introduction of Gal and GlcNAc modules), the optimal recognition backbone of GCNT2 enzyme—the non-reducing-terminal poly-LacNAc tetrasaccharide—is introduced. Since the non-target LacNAc unit at the reducing end is occupied by Fuc, the GCNT2 enzyme is forced to recognize and act on the galactose residues inside the non-reducing end, thereby achieving site-specific introduction of β1,6-GlcNAc residues. Finally, the specific fucosidase E1-10125 was used to precisely cleave the temporary protecting group of Fuc under mild conditions, releasing the target product. This strategy completely eliminates the cumbersome operation of traditional chemical protecting groups and the waste of intermediates caused by non-natural human modifications. All enzyme preparations are derived from recombinant expression in *E. coli*, and combined with a multi-enzyme cascade system, the synthesis efficiency and product purity are significantly improved. Importantly, in this enzymatic assembly route, fucose can not only serve as a structural unit but also as a highly efficient dynamic protecting group, providing a universal synthetic paradigm for constructing structurally homogeneous complex branched sugar libraries and exploring their biological functions in immune recognition. Attached Figure Description

[0008] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0009] Figure 1 This is a schematic diagram of the mechanism of the enzymatic reaction catalyzing the formation of α1,3-Fuc glycosidic bonds in Example 1 of the present invention.

[0010] Figure 2 This is a schematic diagram of the mechanism of the enzymatic reaction catalyzing the formation of β1,4-Gal glycosidic bonds in Example 1 of the present invention.

[0011] Figure 3 This is a schematic diagram of the mechanism of the enzymatic reaction catalyzing the formation of β1,3-GlcNAc glycosidic bonds in Example 1 of the present invention.

[0012] Figure 4 This is a schematic diagram of the mechanism of the enzyme-catalyzed reaction in Example 1 of the present invention to form β1,6-GlcNAc glycosidic bonds.

[0013] Figure 5This is the synthetic route for chitin pentasaccharide in Example 1 of the present invention.

[0014] Figure 6 This is the synthetic route for chitin hexasaccharide in Example 1 of the present invention.

[0015] Figure 7 This is the synthetic route for chitin heptaose in Example 1 of the present invention.

[0016] Figure 8 This is the synthetic route for chitin octasaccharide in Example 1 of the present invention.

[0017] Figure 9 This is the synthetic route for chitin nonasugar in Example 1 of the present invention.

[0018] Figure 10 This is the synthetic route for the β1,6-GlcNAc glycosylated chitin oligosaccharide derivative in Example 1 of the present invention. Detailed Implementation

[0019] Fucose is a common component of human glycans, and the resulting α1,3-fucosylated Lewis x (Lex), α1,4-fucosylated Lewis a (Le a), and their derivatives are common antigenic epitopes in human glycans. Meanwhile, a specific fucosidase, E1-10125, has been discovered and used in the synthesis of linear keratin sulfate-related glycans KS-1 and KS-II. This fucosidase-assisted substrate engineering strategy has been successfully applied to the site-selective sulfonation or sialylation modification of linear poly-LacNAc. However, it remains unclear whether this strategy can achieve site-specific introduction of β1,6-GlcNAc residues from I-antigens into the non-reducing ends of chitin oligosaccharide derivatives.

[0020] Among chitin oligosaccharide derivatives, poly-LacNAc is a common functional backbone. However, the GCNT2 enzymes widely used for in vitro synthesis currently lack site selectivity for multiple internal galactose residues in the poly-LacNAc backbone, resulting in a mixture of isomers with indistinct branching positions. Previous studies have shown that linear poly-LacNAc tetrasaccharides are the optimal catalytic substrates for GCNT2 enzymes; however, their catalytic activity significantly decreases when α1,3-Fuc is introduced into the reducing end of the tetrasaccharide. Based on this, this invention proposes to further extend the LacNAc unit to a hexasaccharide backbone based on the linear poly-LacNAc tetrasaccharide containing α1,3-Fuc at the reducing end, i.e., to construct the optimal catalytic poly-LacNAc tetrasaccharide structure for GCNT2 at the non-reducing end. By controlling the reaction conditions, the site-specific introduction of β1,6-GlcNAc residues can be achieved only at the internal galactose of the non-reducing end.

[0021] To address this issue, this invention proposes a method for synthesizing β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives. This invention employs a fucosidase-assisted substrate engineering strategy, achieving mutual exclusivity between β1,6-GlcNAc residues and α1,3-Fuc residues on the same LacNAc unit by controlling reaction conditions. This allows for the site-specific introduction of β1,6-GlcNAc residues into the chitin oligosaccharide derivative, resulting in the efficient synthesis of β1,6-GlcNAc chitin oligosaccharide derivatives. Specifically, fucose is used as a "temporary protecting group" to selectively block non-target LacNAc units on the poly-LacNAc grafted onto the chitin backbone. Subsequently, by controlling the enzyme reaction conditions and sequence, after the target site completes the I-branch, the fucose is gently and efficiently cleaved by a specific fucosidase (E1-10125), ultimately achieving site-specific β1,6-GlcNAc glycosylation modification of the chitin oligosaccharide derivative. This provides an important molecular tool and synthetic method for exploring the role of related glycans in organismal development, immune regulation, and other aspects.

[0022] In a first aspect, the present invention provides a method for synthesizing β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives, comprising the following steps: Using chitin tetrasaccharides GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc as glycosyl acceptors and fucose as a glycosyl donor, chitin pentasaccharides were obtained via enzymatic assembly module one. The structure of the chitin pentasaccharide is as follows: ; Chitin hexasaccharide was obtained via enzymatic assembly module II catalysis using chitin pentose as a glycosyl acceptor and galactose as a glycosyl donor; the structure of the chitin hexasaccharide is as follows: ; Using chitin hexasaccharide as a glycosyl acceptor, N - Acetaminoglycine is used as a glycosyl donor, and chitin heptaose is obtained through a three-catalyzed enzymatic assembly module; the structure of the chitin heptaose is as follows: ; Using chitin heptaose as a glycosyl acceptor and galactose as a glycosyl donor, chitin octaose was obtained via enzymatic assembly module II catalysis; the structure of the chitin octaose is as follows: ; Using chitin octasaccharide as a glycosyl acceptor, N- Acetaminoglycine is used as a glycosyl donor, and chitinine nonaose is obtained by enzymatic assembly module four-catalysis; the structure of chitinine nonaose is as follows: ; Using chitin nonaose as a glycosyl acceptor, fucosidase E1-10125 catalyzes the generation of site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives; the structure of the β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives is as follows: ; R is any one of hydroxyl, azide-substituted alkyl, alkynyl-substituted alkyl, mercapto-substituted alkyl, α- or β-configuration substituted alkyl, α- or β-configuration serine residue, or α- or β-configuration threonine residue.

[0023] To achieve site-specific β1,6-GlcNAc glycosylation modification on chitin oligosaccharide derivatives containing multiple β1,6-GlcNAc glycosylation modification sites, this invention employs a fucosidase-assisted strategy to achieve the efficient enzymatic synthesis of a β1,6-GlcNAc glycosylated chitin oligosaccharide derivative. This provides technical support for the systematic construction of a library of site-specific β1,6-GlcNAc glycosylated lactosamine-grafted chitin oligosaccharides. This invention uses the chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc as the starting substrate. It should be noted that the preparation method of this tetrasaccharide is referenced in the article (…). Chin. J. Chem. 2023, 41 (1299-1304), which will not be elaborated upon here.

[0024] Enzymes are proteins by nature, and their catalytic activity is affected by factors such as metal ions, substrate concentration, temperature, pH, and time. Specifically: metal ions, acting as coenzymes, are crucial for enzymes to perform their catalytic function; temperature and pH primarily regulate enzyme activity by affecting the enzyme's structural stability; substrate concentration influences the collision frequency between the enzyme and substrate, thus altering the reaction rate; and reaction time directly affects whether the reaction can be fully completed. This invention designs optimal reaction condition systems for different enzymes required for the synthesis of site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives, ensuring the high efficiency of enzymes in the enzymatic module.

[0025] In this invention, the enzymes used in the enzymatic assembly module one are glycoside synthase FKP and α1,3-fucotransferase Hpα1,3FucT. Specifically, glycoside synthase FKP is a bifunctional enzyme that first converts Fucose to Fuc-1-P, and then further converts Fuc-1-P to GDP-Fuc. Figure 1 This is a schematic diagram of the mechanism by which the enzyme assembly module catalyzes the formation of α1,3-fucoside bonds.

[0026] In this invention, the process of obtaining chitin pentasaccharide by enzymatic assembly module one using GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc chitin tetrasaccharide as a glycosyl acceptor and fucose as a glycosyl donor includes the following steps: Chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc, fucose, adenine nucleoside triphosphate, and guanine nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-9.5. Finally, glycosyl synthase FKP and α1,3-fucosyltransferase Hpα1,3FucT were added to react and obtain chitin pentasaccharide.

[0027] The molar ratio of chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc, fucose, adenine nucleoside triphosphate, and guanine nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0), preferably 1:(1.3-2.0):(1.3-2.0):(1.3-2.0), and more preferably 1:1.5:1.5:1.5.

[0028] The mass ratio of chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc, glycosyl synthase FKP, and α1,3-fucosyltransferase Hpα1,3FucT is 10:(0.01-100):(0.01-100), preferably 10:(0.01-5):(0.01-5), and more preferably 10:(1.5-2):(1.5-2).

[0029] The buffer solution may be a Tris buffer solution containing MgCl2, wherein the molar ratio of MgCl2 to Tris is 1:2.5-40, the volume of the buffer solution is 10-500 mmol, and the pH is 5.0-10.0.

[0030] In this invention, the enzymes used in the second enzymatic assembly module are galactokinase (GalK), nucleotide synthase (BLUSP), and β1,4-galactosyltransferase (HpLgtB). GalK is a Gal-1-P generating enzyme, and BLUSP is a UDP-Gal generating enzyme. Figure 2 This is a schematic diagram of the mechanism for the formation of β1,4-galactosidic bonds by the enzymatic assembly module II.

[0031] In this invention, the process of obtaining chitin hexasaccharide by enzymatic assembly module II using chitin pentasaccharide as a glycosyl acceptor and galactose as a glycosyl donor includes the following steps: Chitin pentasaccharide, galactose, adenine nucleoside triphosphate, and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.5, and galactokinase GalK, glycosyl synthase BLUSP, and β1,4-galactosyltransferase HpLgtB were added. The reaction was carried out to obtain chitin hexasaccharide.

[0032] The molar ratio of chitin pentasaccharide, galactose, adenine nucleoside triphosphate and uracil nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0), preferably 1:(1.3-2.0):(1.3-2.0):(1.3-2.0), and more preferably 1:1.5:1.5:1.5.

[0033] The mass ratio of chitin pentasaccharide, galactokinase GalK, glycosyl synthase BLUSP, and β1,4-galactosyltransferase HpLgtB is 10:(0.01-100):(0.01-100):(0.01-100), preferably 10:(0.01-2):(0.01-2):(0.01-5), and more preferably 10:(0.5-1.5):(0.5-1.5):(1-2).

[0034] The buffer solution may be a Tris buffer solution, and in the buffer solution containing MgCl2, the molar ratio of MgCl2 to Tris buffer solution is 1:2.5-40, the volume of the buffer solution is 10-500 mmol, and the pH is 5.0-10.0.

[0035] In this invention, the enzymes used in the enzymatic assembly module three are nucleotide synthase NahK-GlmU and β1,3- N - Acetylglucosamine transferase HpLgtA. In the fusion enzyme NahK-GlmU, NahK is a GlcNAc-1-P generating enzyme, and GlmU is a UDP-GlcNAc generating enzyme. Figure 3 For the enzymatic assembly module, three catalytic reactions form β1,3- N Mechanism diagram of α-acetylglucosinolate bond.

[0036] In this invention, chitin hexasaccharide is used as a glycosyl acceptor, with N - Using acetylglucosamine as a glycosyl donor, the enzymatic assembly module catalyzes the production of chitin heptaose, which includes the following steps: Chitin hexasaccharide, N Acetaminophen, adenine nucleoside triphosphate, and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.5, and glyconucleotide synthase NahK-GlmU and β1,3-N The reaction of acetylglucosamine transferase HpLgtA yields chitin heptaose.

[0037] Among them, chitin hexasaccharide, N The molar ratio of acetylglucosamine, adenine nucleoside triphosphate and uracil nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0), preferably 1:(1.3-2.0):(1.3-2.0):(1.3-2.0), and more preferably 1:1.5:1.5:1.5.

[0038] Among them, chitin hexasaccharide, nucleotide synthase NahK-GlmU, and β1,3- N The mass ratio of α-acetylglucosamine transferase HpLgtA is 10:(0.01-100):(0.01-100), preferably 10:(0.01-2):(0.01-5), and more preferably 10:(0.5-1.5):(1-2).

[0039] The buffer solution may be a Tris buffer solution, and in the buffer solution containing MgCl2, the molar ratio of MgCl2 to Tris buffer solution is 1:2.5-40, the volume of the buffer solution is 10-500 mmol, and the pH is 5.0-10.0.

[0040] In this invention, the process of obtaining chitin octasaccharide by enzymatic assembly module II using chitin heptasaccharide as a glycosyl acceptor and galactose as a glycosyl donor includes the following steps: Chitin heptaose, galactose, adenine nucleoside triphosphate, and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.5, and galactokinase GalK, glycosyl synthase BLUSP, and β1,4-galactosyltransferase HpLgtB were added. The reaction was carried out to obtain chitin octaose.

[0041] The molar ratio of chitin heptaose, galactose, adenine nucleoside triphosphate and uracil nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0), preferably 1:(1.3-2.0):(1.3-2.0):(1.3-2.0), and more preferably 1:1.5:1.5:1.5.

[0042] The mass ratio of chitin heptaose, galactokinase GalK, glycosyl synthase BLUSP, and β1,4-galactosyltransferase HpLgtB is 10:(0.01-100):(0.01-100):(0.01-100), preferably 10:(0.01-10):(0.01-10):(0.01-10), and more preferably 10:(1-3):(1-3):(1-5).

[0043] The buffer solution may be a Tris buffer solution, and in the buffer solution containing MgCl2, the molar ratio of MgCl2 to Tris buffer solution is 1:2.5-40, the volume of the buffer solution is 10-500 mmol, and the pH is 5.0-10.0.

[0044] In this invention, the enzymes used in the enzymatic assembly module four are nucleotide synthase NahK-GlmU and β1,6- N - Acetaminoglycopene transferase GCNT2. In the fusion enzyme NahK-GlmU, NahK is a GlcNAc-1-P generating enzyme, and GlmU is a UDP-GlcNAc generating enzyme. Figure 4 For the enzymatic assembly module, four catalytic reactions form β1,6- N Mechanism diagram of α-acetylglucosinolate bond.

[0045] In this invention, the chitin octasaccharide is a glycosyl acceptor, used to... N - Using acetylglucosamine as a glycosyl donor, the enzymatic assembly of module four catalyzes the production of chitinine nonasugar, which includes the following steps: Chitin octaose, N Acetaminophen, adenine nucleoside triphosphate, and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.5, and glyconucleotide synthase NahK-GlmU and β1,6- N The reaction of acetylglucosamine transferase GCNT2 yields chitin nonaose.

[0046] Among them, chitin octasaccharide, N The molar ratio of acetylglucosamine, adenine nucleoside triphosphate and uracil nucleoside triphosphate is 1:(1.0-1.5):(1.0-1.5):(1.0-1.5).

[0047] Among them, chitin octasaccharide, glyconucleotide synthase NahK-GlmU, and β1,6- NThe mass ratio of γ-acetylglucosamine transferase GCNT2 is 10:(0.01-100):(0.01-100), preferably 10:(0.01-40):(0.01-50), and more preferably 10:(1-5):(1-10).

[0048] The buffer solution may be a Tris buffer solution containing MgCl2, wherein the molar ratio of MgCl2 to Tris buffer solution is 1:2.5-40, the volume of the buffer solution is 10-500 mmol, and the pH is 5.0-10.0.

[0049] In this invention, the reaction temperature of the enzymatic reaction is preferably 0-37℃, more preferably 30-37℃, such as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, and preferably within the range of any of the above values ​​as the upper or lower limit; the rotation speed is preferably 0-240 r / min, more preferably 100-150 r / min, such as 100 r / min, 105 r / min, 110 r / min, 120 r / min, 130 r / min, 140 r / min, 145 r / min, 150 r / min, and preferably within the range of any of the above values ​​as the upper or lower limit; the reaction time is preferably 0.5-96 h, more preferably 40-50 h, such as 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, and preferably within the range of any of the above values ​​as the upper or lower limit.

[0050] In this invention, the enzyme-catalyzed reaction is stopped by adding anhydrous ethanol of the same volume as the reaction solution to the reaction system and incubating at -20°C for 0-6 h.

[0051] In this invention, the step of using chitin nonaose as a glycosyl acceptor and catalyzing the generation of β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives by fucosidase E1-10125 includes the following steps: Chitin nonaglycosylation and fucosidase E1-10125 were dispersed in buffer solution, and the pH of the reaction system was adjusted to 3.0-9.5. The mixture was incubated at 0-37℃ and 110 r / min for 0.1-96 h to obtain β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives.

[0052] The mass ratio of chitin nonaose to fucosidase E1-10125 is 10:(0.01-100), preferably 10:(1-10).

[0053] In this invention, after incubation, anhydrous ethanol and 1,4-dioxane of equal volume to the reaction solution are added to the reaction system, and the mixture is incubated at -20°C for 0-6 h.

[0054] The enzymatic assembly module constructed in this invention is based on a "one-pot, multi-enzyme" system. Specifically, under the action of a specific glyconucleotide synthase, free monosaccharides (Gal, GlcNAc, Fucose) are converted into specific nucleotide-activated glycosyl donors (UDP-Gal, UDP-GlcNAc, GDP-Fucose). Subsequently, the monosaccharide units on the nucleotide-activated glycosyl donors are covalently linked to the hydroxyl groups at specific positions on the glycosyl acceptors under the action of a specific glycosyltransferase. In the experimental process, the enzymes used in this invention underwent multiple optimizations and screenings, and the results showed that: Escherichia coli galactokinase (GalK) Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP) and Helicobacter pylori β1,4-galactosyltransferase (HpLgtB); Bifidobacterium longum NahK source and Escheerichia coli The fusion enzyme composed of GlmU from the source (NahK-GlmU) and Helicobacter pylori β1,3- N -acetyl-glucosaminyltransferase (HpLgtA); Bifidobacterium longum NahK source and Escheerichia coli The fusion enzyme composed of GlmU from the source (NahK-GlmU) and Human β1,6- N -acetyl-glucosaminyltransferase (GCNT2); Bacteroides fragilis bifunctional L-fucokinase / GDP-fucose pyrophosphorylase (FKP) and Helicobacter pylori α 1,3-fucosyltransferase (Hpα1,3FucT), used as the enzyme in the enzymatic module of this invention, exhibits superior catalytic performance, particularly suitable for the site-specific β1,6-GlcNAc glycosylation modification of lactosamine-grafted chitin oligosaccharides. It boasts high synthesis efficiency, virtually no byproduct generation, and facilitates purification. To achieve site-specific β1,6-GlcNAc glycosylation modification on the poly-LacNAc backbone, this invention introduces a fucosidase: Ruminococcus gnavusE1 α-fucosidases (E1-10125). All of the above enzymes are of bacterial origin and can be easily expressed and purified using standard E. coli expression systems.

[0055] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0056] The raw materials used in the following examples are all conventional products and can be purchased. The enzymes selected were all recombinantly expressed in a conventional E. coli expression system according to existing literature reports, and were obtained by conventional purification using nickel ion exchange resin.

[0057] Example 1 A method for synthesizing a β1,6-GlcNAc glycosylated chitin oligosaccharide derivative, comprising the following steps: (1) Synthesis of chitin pentasaccharide.

[0058] The structure of chitin pentasaccharide is: .

[0059] Synthetic routes such as Figure 5 As shown.

[0060] Chitosan tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc (110 mg, 1.0 equivalent), fucose (34 mg, 1.5 equivalent), ATP (115 mg, 1.5 equivalent), GTP (126 mg, 1.5 equivalent), Tris-HCl buffer (100 mmol, pH 7.5, 121 mg), and MgCl2 (20 mmol, 40 mg) were dissolved in a 50 mL centrifuge tube. FKP (20 mg) and Hpα1,3FucT (20 mg) were added, and double-distilled water was added to a total volume of 10 mL. The tube was incubated on a shaker at 37 °C and 110 r / min for 48 h. Thin-layer chromatography (TLC) (ethyl acetate:ethanol:water:ammonia = 5:5:4:0.3) was used to detect the reaction. After the reaction was complete, an equal volume of anhydrous ethanol was added, and the reaction was terminated by standing at -20 °C for 30 min. The reaction system was then centrifuged at 4°C and 8000 r / min for 20 min. The supernatant was collected, concentrated, and dried. It was then separated and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain 120 mg of chitin pentasaccharide, with a yield of 93%.

[0061] 1 H NMR (400 MHz, D2O) δ 5.21 (d, J= 2.3 Hz, 0.50H), 5.12 (d, J = 4.1 Hz,1H), 4.88 – 4.79 (m, 1H), 4.71 (d, J = 8.5 Hz, 1H), 4.71 (d, J = 8.5 Hz, 0.50H), 4.68 – 4.62 (m, 1H), 4.45 (d, J = 7.8 Hz, 1H), 4.11 (d, J = 3.3 Hz, 1H), 4.05 –3.42 (m, 26H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.16 (d, J = 6.5 Hz, 3H); 13 C NMR (150 MHz, D2O) δ 175.0, 174.8, 174.6, 174.4, 102.9, 101.9, 101.1,98.9, 94.9, 90.5, 81.5, 79.7, 79.3, 75.7, 75.4, 74.9, 74.6, 74.5, 73.6, 73.0,72.6, 72.0, 70.7, 70.1, 69.8, 69.4, 69.3, 68.4, 67.7, 66.8, 61.6, 60.6, 60.3,60.2, 59.8, 56.2, 56.0, 55.8, 53.7, 22.4, 22.4, 22.1, 15.4; HRMS (ESI) m / z calcd for C 36 H 61 N3O 25 Cl [M+Cl] - 970.3288, found 970.3301.

[0062] (2) Synthesis of chitin hexasaccharide.

[0063] The structure of chitin hexasaccharide is: .

[0064] Synthetic routes such as Figure 6 As shown.

[0065] Chitin pentasaccharide (110 mg, 1.0 equivalent), galactose (32 mg, 1.5 equivalent), ATP (97 mg, 1.5 equivalent), UTP (103 mg, 1.5 equivalent), Tris-HCl buffer (100 mmol, pH 7.5, 121 mg), and MgCl2 (20 mmol, 40 mg) were dissolved in a 50 mL centrifuge tube. GalK (10 mg), BLUSP (10 mg), and HpLgtB (15 mg) were added, and double-distilled water was added to a total volume of 10 mL. The tube was incubated on a shaker at 37 °C and 110 r / min for 48 h. After the reaction was detected by thin-layer chromatography (TLC) (ethyl acetate:ethanol:water:ammonia = 5:5:4:0.3), an equal volume of anhydrous ethanol was added, and the reaction was terminated by standing at -20 °C for 30 min. The reaction system was then centrifuged at 4°C and 8000 r / min for 20 min. The supernatant was collected, concentrated, and dried. It was then separated and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain 118 mg of chitin hexasaccharide, with a yield of 92%.

[0066] 1 H NMR (400 MHz, D2O) δ 5.17 (d, J = 2.5 Hz, 0.56H), 5.09 (d, J = 4.1 Hz,1H), 4.85 – 4.79 (m, 1H), 4.68 (d, J = 8.1 Hz, 1H), 4.68 (d, J = 8.1 Hz, 0.44H), 4.64 – 4.57 (m, 1H), 4.46 (d, J = 7.8 Hz, 1H), 4.41 (d, J = 7.8 Hz, 1H), 4.08 (d, J = 3.3 Hz, 1H), 4.03 – 3.44 (m, 33H), 2.04 (s, 3H), 2.02 (s, 3H), 2.01 (s,3H), 1.13 (d, J = 6.5 Hz, 3H); 13C NMR (151 MHz, D2O) δ 174.9, 174.8, 174.5,174.3, 102.9, 102.8, 101.8, 101.1, 98.9, 94.9, 90.5, 81.6, 79.7, 79.2, 78.2,75.4, 75.4, 74.8, 74.6, 74.6, 74.5, 73.0, 72.9, 72.6, 72.2, 71.9, 71.0, 70.5,70.0, 69.3, 69.2, 68.6, 68.3, 67.7, 66.8, 61.5, 61.1, 60.1, 59.9, 59.7, 56.1,55.9, 55.2, 53.7, 22.3, 22.2, 21.9, 15.3; HRMS (ESI) m / z calcd for C 42 H 71 N3O 30 Cl[M+Cl] - 1132.3816, found 1132.3801.

[0067] (3) Synthesis of chitin heptaose.

[0068] The structure of chitin heptaose is: .

[0069] Synthetic routes such as Figure 7 As shown.

[0070] Chitin hexasaccharide (105 mg, 1.0 equivalent) NAcetylglucosamine (32 mg, 1.5 equivalents), ATP (79 mg, 1.5 equivalents), UTP (84 mg, 1.5 equivalents), Tris-HCl buffer (100 mmol, pH 7.5, 121 mg), and MgCl2 (20 mmol, 40 mg) were dissolved in a 50 mL centrifuge tube. NahK-GlmU (10 mg) and HpLgtA (15 mg) were added, and double-distilled water was added to a total volume of 10 mL. The tube was placed on a shaker and incubated at 37 °C and 110 r / min for 48 h. After the reaction was detected by thin-layer chromatography (TLC) (ethyl acetate:ethanol:water:ammonia = 5:5:4:0.3), an equal volume of anhydrous ethanol was added, and the reaction was terminated by standing at -20 °C for 30 min. The reaction system was then centrifuged at 4°C and 8000 r / min for 20 min. The supernatant was collected, concentrated, and dried. It was then separated and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain 112 mg of chitin heptaose, with a yield of 90%.

[0071] 1 H NMR (400 MHz, D2O) δ 5.19 (d, J = 2.3 Hz, 0.56H), 5.10 (d, J = 4.1 Hz,1H), 4.88 – 4.79 (m, 1H), 4.71 (d, J = 7.4 Hz, 1H), 4.71 (d, J = 7.4 Hz, 0.44H), 4.69 (d, J = 8.2 Hz, 1H), 4.66 – 4.60 (m, 1H), 4.47 (d, J = 7.9 Hz, 1H), 4.43 (d, J = 7.8 Hz, 1H), 4.16 (d, J = 3.2 Hz, 1H), 4.10 (d, J = 3.3 Hz, 1H), 4.04 – 3.38(m, 37H), 2.06 (s, 3H), 2.04 (s, 6H), 2.03 (s, 3H), 1.15 (d, J = 6.6 Hz, 3H); 13CNMR (150 MHz, D2O) δ 175.0, 174.9, 174.8, 174.5, 174.3, 102.9, 102.7, 101.8,101.1, 98.9, 94.9, 90.5, 82.0, 81.6, 79.7, 79.2, 78.2, 75.7, 75.4, 74.9,74.8, 74.6, 74.6, 74.5, 73.6, 73.0, 72.5, 72.2, 71.9, 70.6, 70.1, 70.0, 69.7,69.3, 69.2, 68.4, 68.3, 67.7, 66.8, 61.6, 61.1, 60.5, 60.2, 60.1, 59.9, 59.7,56.1, 55.9, 55.7, 55.2, 53.7, 22.3, 22.0, 15.4; HRMS (ESI) m / z calcd forC 50 H 84 N4O 35 Cl [M+Cl] - 1335.4610, found 1335.4586.

[0072] (4) Synthesis of chitin octasaccharide.

[0073] The structure of chitin octasaccharide is: .

[0074] Synthetic routes such as Figure 8 As shown.

[0075] Chitin heptaose (50 mg, 1.0 equivalent), galactose (10 mg, 1.5 equivalent), ATP (32 mg, 1.5 equivalent), UTP (34 mg, 1.5 equivalent), Tris-HCl buffer (100 mmol, pH 7.5, 121 mg), and MgCl2 (20 mmol, 40 mg) were dissolved in a 50 mL centrifuge tube. GalK (10 mg), BLUSP (10 mg), and HpLgtB (15 mg) were added, and double-distilled water was added to a total volume of 10 mL. The tube was then incubated on a shaker at 37 °C and 110 r / min for 48 h. After the reaction was detected by thin-layer chromatography (TLC) (ethyl acetate:ethanol:water:ammonia = 4:5:5:0.3), an equal volume of anhydrous ethanol was added, and the reaction was terminated by standing at -20 °C for 30 min. The reaction system was then centrifuged at 4°C and 8000 r / min for 20 min. The supernatant was collected, concentrated, and dried. It was then separated and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain 61 mg of chitin octasaccharide, with a yield of 92%.

[0076] 1 H NMR (400 MHz, D2O) δ 5.15 (d, J = 2.3 Hz, 0.45H, 1α-H), 5.06 (d, J =4.1 Hz, 1H), 4.88 – 4.79 (m, 1H), 4.68 – 4.62 (m, 2.5H), 4.61 – 4.55 (m, 1H), 4.35 (d, J = 7.5 Hz, 1H), 4.42 (d, J = 7.5 Hz, 1H), 4.38 (d, J = 7.9 Hz, 1H), 4.11(d, J = 3.2 Hz, 1H), 4.05 (d, J = 3.2 Hz, 1H), 4.00 – 3.40 (m, 43H), 2.01 (s,3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H), 1.10 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, D2O) δ 174.8, 174.8, 174.6, 174.4, 174.2, 102.8, 102.7, 102.6,101.7, 100.9, 98.7, 94.7, 90.3, 82.0, 81.5, 79.5, 79.1, 78.1, 78.1, 75.3,75.2, 74.8, 74.7, 74.5, 74.4, 74.4, 72.9, 72.4, 72.1, 72.0, 71.8, 70.9, 70.4,69.9, 69.9, 69.2, 69.1, 68.5, 68.2, 68.2, 67.5, 66.6, 61.4, 61.0, 60.9, 60.1,60.0, 59.8, 59.6, 56.0, 55.8, 55.1, 55.0, 53.5, 22.1, 22.1, 21.8, 15.2; HRMS(ESI) m / z calcd for C 56 H 94 N4O 40 Na [M+Na] + 1485.5337, found 1485.5463.

[0077] (5) Synthesis of chitin-nonose.

[0078] The structure of chitin-nonose is: .

[0079] Synthetic routes such as Figure 9 As shown.

[0080] Chitin octasaccharide (30 mg, 1.0 equivalent) NAcetylglucosamine (5 mg, 1.1 equivalents), ATP (12 mg, 1.1 equivalents), UTP (13 mg, 1.1 equivalents), Tris-HCl buffer (100 mmol, pH 7.5, 121 mg), and MgCl2 (20 mmol, 40 mg) were dissolved in a 50 mL centrifuge tube. NahK-GlmU (10 mg) and GCNT2 (15 mg) were added, and double-distilled water was added to a total volume of 10 mL. The tube was then incubated on a shaker at 37 °C and 110 r / min for 48 h. After the reaction was detected by thin-layer chromatography (TLC) (ethyl acetate:ethanol:water:ammonia = 4:5:5:0.3), an equal volume of anhydrous ethanol was added, and the reaction was terminated by standing at -20 °C for 30 min. The reaction system was then centrifuged at 4°C and 8000 r / min for 20 min. The supernatant was collected, concentrated, and dried. It was then separated and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain 38 mg of chitin nonaose, with a yield of 90%.

[0081] 1 H NMR (500 MHz, D2O) δ 5.16 (d, J = 2.6 Hz, 0.45H), 5.07 (d, J = 4.5 Hz,1H), 4.88 – 4.75 (m, 1H), 4.70 – 4.64 (m, 2.5H), 4.63 – 4.55 (m, 2H), 4.45(d, J = 8.0 Hz, 1H), 4.43 (d, J = 8.6 Hz, 1H), 4.40 (d, J = 7.7 Hz, 1H), 4.12 (d, J =3.3 Hz, 1H), 4.07 (d, J = 3.3 Hz, 1H), 4.02 – 3.32 (m, 49H), 2.03 (s, 3H), 2.02(s, 3H), 2.01 (s, 3H), 2.00 (s, 6H), 1.12 (d, J = 6.3 Hz, 3H); 13C NMR (125 MHz, D2O) δ 174.9, 174.8, 174.6, 174.5, 174.3, 103.0, 102.9, 102.7, 102.7, 101.8,101.0, 98.8, 94.8, 90.4, 81.8, 81.5, 79.6, 79.2, 78.8, 78.1, 75.8, 75.3,75.3, 74.5, 74.4, 73.8, 73.6, 72.5, 72.2, 72.1, 71.9, 71.0, 70.5, 69.9, 69.9,69.8, 69.3, 69.2, 68.7, 68.5, 68.5, 68.3, 67.6, 66.7, 61.5, 61.0, 60.7, 60.0,59.8, 59.7, 55.5, 55.2, 55.1, 53.6, 22.4, 22.2, 21.9, 15.3; HRMS (ESI) m / z calcd for C 64 H 107 N5O 45 Na [M+Na] + 1688.6130, found 1688.6259.

[0082] (6) Synthesis of β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives.

[0083] The structure of the β1,6-GlcNAc glycosylated chitin oligosaccharide derivative is as follows: .

[0084] Synthetic routes such as Figure 10 As shown.

[0085] Chitin nonaglycosylation (25 mg) was dissolved in double-distilled water (2 mL), and Tris-HCl buffer (100 mmol, pH 9.0, 121 mg) was added to a 10 mL EP tube. Fucosidase E1-10125 (20 mg) was added, and the tube was incubated on a shaker at 37 °C and 110 r / min for 0.5 h. After the reaction was detected by thin-layer chromatography (TLC) (ethyl acetate:ethanol:water:ammonia = 4:5:5:0.3), anhydrous ethanol of equal volume to the reaction mixture was added, and the reaction was terminated by standing at -20 °C for 12 h. Subsequently, the reaction mixture was centrifuged at 4 °C and 8000 r / min for 20 min, and the supernatant was collected, concentrated, and dried. The supernatant was then purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain 22 mg of β1,6-GlcNAc glycosylated chitin oligosaccharide derivative, with a yield of 96%.

[0086] 1 H NMR (400 MHz, D2O) δ 5.16 (d, J = 2.5 Hz, 0.5H), 4.69 – 4.62 (m, 2.5H), 4.61 – 4.55 (m, 2H), 4.45 (d, J = 8.2 Hz, 1H), 4.42 (d, J = 7.8 Hz, 2H), 4.12 (d, J = 3.2 Hz, 2H), 4.00 – 3.31 (m, 46H), 2.04 (m, 3H), 2.02 (s, 3H), 2.00 (s, 9H); 13 C NMR (125 MHz, D2O) δ 174.9, 174.7, 174.5, 174.5, 174.4,102.9, 102.7, 101.3, 100.8, 94.8, 90.4, 81.9, 81.7, 79.7, 79.2, 78.6, 78.3,78.1, 75.6, 75.3, 74.9, 74.7, 74.5, 73.8, 73.6, 72.5, 72.5, 72.3, 72.2, 72.1,71.0, 70.1, 70.0, 69.8, 69.7, 69.2, 68.6, 68.6, 68.4, 61.0, 61.0, 60.5, 60.0,59.9, 59.8, 55.7, 55.2, 55.1, 55.0, 53.6, 22.4, 22.2, 21.9; HRMS (ESI)m / z calcd for C 58 H 97 N5O 41 Na [M+Na] + 1542.5551, found 1542.5685.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing a β1,6-GlcNAc glycosylated chitin oligosaccharide derivative, characterized in that, The steps include the following: Using chitin tetrasaccharides GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc as glycosyl acceptors and fucose as a glycosyl donor, chitin pentasaccharides were obtained via enzymatic assembly module one. The structure of the chitin pentasaccharide is as follows: ； Chitin hexasaccharide was obtained via enzymatic assembly module II catalysis using chitin pentose as a glycosyl acceptor and galactose as a glycosyl donor; the structure of the chitin hexasaccharide is as follows: ； Using chitin hexasaccharide as a glycosyl acceptor, N - Acetaminoglycine is used as a glycosyl donor, and chitin heptaose is obtained through a three-catalyzed enzymatic assembly module; the structure of the chitin heptaose is as follows: ； Using chitin heptaose as a glycosyl acceptor and galactose as a glycosyl donor, chitin octaose was obtained via enzymatic assembly module II catalysis; the structure of the chitin octaose is as follows: ； Using chitin octasaccharide as a glycosyl acceptor, N - Acetaminoglycine is used as a glycosyl donor, and chitinine nonaose is obtained by enzymatic assembly module four-catalysis; the structure of chitinine nonaose is as follows: ； Using chitin nonaose as a glycosyl acceptor, fucosidase E1-10125 catalyzes the generation of site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives; the structure of the β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives is as follows: ； R is any one of hydroxyl, azide-substituted alkyl, alkynyl-substituted alkyl, mercapto-substituted alkyl, α- or β-configuration substituted alkyl, α- or β-configuration serine residue, or α- or β-configuration threonine residue.

2. The synthesis method according to claim 1, characterized in that, The enzymes used in the first reaction of the enzymatic assembly module are nucleotide synthase FKP and α1,3-fucosyltransferase Hpα1,3FucT. The process of obtaining chitin pentasaccharide via enzymatic assembly module one, using chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc as a glycosyl acceptor and fucose as a glycosyl donor, comprises the following steps: Chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc, fucose, adenine nucleoside triphosphate, and guanine nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-9.

5. Finally, glycosyl synthase FKP and α1,3-fucosyltransferase Hpα1,3FucT were added, and the reaction was carried out to obtain chitin pentasaccharide. The molar ratio of chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc, fucose, adenine nucleoside triphosphate, and guanine nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0). The mass ratio of chitin tetrasaccharide GlcNAcβ1,3Galβ1,4GlcNAcβ1,4GlcNAc, glycosyl synthase FKP, and α1,3-fucosyltransferase Hpα1,3FucT was 10:(0.01-100):(0.01-100).

3. The synthesis method as described in claim 1, characterized in that, The enzymes used in the second enzymatic assembly module are galactokinase GalK, nucleotide synthase BLUSP, and β1,4-galactosyltransferase HpLgtB. The process of obtaining chitin hexasaccharide via enzymatic assembly module II catalysis using chitin pentasaccharide as a glycosyl acceptor and galactose as a glycosyl donor includes the following steps: Chitin pentasaccharide, galactose, adenine nucleoside triphosphate and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.

5. Galactokinase GalK, glycosyl synthase BLUSP and β1,4-galactosyltransferase HpLgtB were added and reacted to obtain chitin hexasaccharide. The molar ratio of chitin pentasaccharide, galactose, adenine nucleoside triphosphate, and uracil nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0). The mass ratio of chitin pentasaccharide, galactokinase GalK, glycosyl synthase BLUSP, and β1,4-galactosyltransferase HpLgtB was 10:(0.01-100):(0.01-100):(0.01-100).

4. The synthesis method according to claim 1, characterized in that, The enzymes used in the enzymatic assembly module three are nucleotide synthase NahK-GlmU and β1,3- N - Acetaminoglycine transferase HpLgtA; The use of chitin hexasaccharide as a glycosyl acceptor, N - Using acetylglucosamine as a glycosyl donor, the enzymatic assembly module catalyzes the production of chitin heptaose, which includes the following steps: Chitin hexasaccharide, N Acetaminophen, adenine nucleoside triphosphate, and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.5, and glyconucleotide synthase NahK-GlmU and β1,3- N -Acetaminoglycyltransferase HpLgtA, reaction, to obtain chitin heptaose; Chitin hexasaccharide, N The molar ratio of acetylglucosamine, adenine nucleoside triphosphate, and uracil nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0); The chitin hexasaccharide, glyconucleotide synthase NahK-GlmU, and β1,3- N- The mass ratio of acetylglucosamine transferase HpLgtA is 10:(0.01-100):(0.01-100).

5. The synthesis method according to claim 1, characterized in that, The process of obtaining chitin octasaccharide via enzymatic assembly module II catalysis, using chitin heptasaccharide as a glycosyl acceptor and galactose as a glycosyl donor, includes the following steps: Chitin heptaose, galactose, adenine nucleoside triphosphate and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.

5. Galactokinase GalK, glycosyl synthase BLUSP and β1,4-galactosyltransferase HpLgtB were added and reacted to obtain chitin octaose. The molar ratio of chitin heptaose, galactose, adenine nucleoside triphosphate, and uracil nucleoside triphosphate is 1:(1.3-3.0):(1.3-5.0):(1.3-5.0). The mass ratio of chitin heptaose, galactokinase GalK, glycosyl synthase BLUSP, and β1,4-galactosyltransferase HpLgtB was 10:(0.01-100):(0.01-100):(0.01-100).

6. The synthesis method according to claim 1, characterized in that, The enzymes used in the enzymatic assembly module four are nucleotide synthase NahK-GlmU and β1,6- N -Acetaminoglycine transferase GCNT2; The use of chitin octasaccharide as a glycosyl acceptor, N - Using acetylglucosamine as a glycosyl donor, the enzymatic assembly of module four reactions yields chitinine nonasugar, including the following steps: Chitin octaose, N Acetaminophen, adenine nucleoside triphosphate, and uracil nucleoside triphosphate were dispersed in a buffer solution containing MgCl2. The pH of the reaction system was adjusted to 4.5-8.5, and glyconucleotide synthase NahK-GlmU and β1,6- N -Acetaminoglycyltransferase GCNT2, reaction, to obtain chitin nonaose; Chitin octasaccharide, N The molar ratio of acetylglucosamine, adenine nucleoside triphosphate, and uracil nucleoside triphosphate is 1:(1.0-1.5):(1.0-1.5):(1.0-1.5); Chitin octasaccharide, glyconucleotide synthase NahK-GlmU, and β1,6- N The mass ratio of γ-acetylglucosamine transferase GCNT2 is 10:(0.01-100):(0.01-100).

7. The synthesis method according to any one of claims 2-6, characterized in that, The reaction temperature for the enzymatic reaction is 0-37℃, the rotation speed is 0-240 r / min, and the reaction time is 3-96 h.

8. The synthesis method according to any one of claims 2-6, characterized in that, The enzyme-catalyzed reaction can be stopped by adding an equal volume of anhydrous ethanol to the reaction system and incubating at -20°C for 0-6 h.

9. The synthesis method according to claim 1, characterized in that, The process of generating site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives using chitin nonaose as a glycosyl acceptor and catalyzed by fucosidase E1-10125 includes the following steps: Chitin nonaglycosylation and fucosidase E1-10125 were dispersed in buffer solution, and the pH of the reaction system was adjusted to 3.0-9.

5. The reaction system was incubated at 0-37℃ and 110 r / min for 0.1-96 h to obtain site-specific β1,6-GlcNAc glycosylated chitin oligosaccharide derivatives. The mass ratio of chitin nonaose to fucosidase E1-10125 is 10:(0.01-100).

10. The synthesis method according to claim 9, characterized in that, After incubation, add anhydrous ethanol and 1,4-dioxane of equal volume to the reaction solution to the reaction system and incubate at -20°C for 0-6 h.

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