Enzymatic synthesis method and application of Gb3-chitosan oligosaccharide conjugate

Abstract

The invention relates to an enzymatic synthesis method and application of a marine carbohydrate Gb3-chitosan oligosaccharide conjugate, in particular to a synthesis method and application of Gb3 and two Gb3-chitosan oligosaccharide conjugates with clear structures. According to the invention, Gb3 trisaccharide as shown in a formula (I) is efficiently prepared by utilizing an enzyme method, and then Gb3-chitosan oligosaccharide conjugate pentasaccharide as shown in a marine carbohydrate substance formula (II) and Gb3-chitosan oligosaccharide conjugate pentasaccharide as shown in a marine carbohydrate substance formula (III) are respectively synthesized through a one-step reduction reaction and an amidation reaction. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to an enzymatic synthesis method and application of marine carbohydrate substances, and in particular to a synthesis method and application of a Gb3-chitosan oligosaccharide conjugate with a clear structure. Background Art

[0002] Chitin (also known as chitosan) polysaccharide, widely found in the shells of marine crustaceans, such as shrimp and crab shells, is a component of the cell walls of most fungi, algae and insects. It is the second largest natural polysaccharide in nature after cellulose. It is an insoluble biomass polysaccharide formed by N-acetyl-D-glucosamine (GlcNAc) connected by β1,4-glycosidic bonds. Chitin can be obtained by deacetylation to obtain chitosan oligosaccharide (CS), which is composed of N-acetylglucosamine (GlcNAc) and D-glucosamine (GlcNH 2 ) are randomly arranged and connected by β-1,4-glycosidic bonds. Chitin and its derived oligosaccharides are marine polysaccharides with the characteristics of wide distribution and easy acquisition. Many studies have shown that the marine sugar substance oligosaccharides have anti-inflammatory, anti-cancer, and anti-tumor activities. In addition, the active groups in the structure of oligosaccharides, such as amino groups, can be coupled with important active biological molecules through simple amidation reactions or condensation reactions to improve the physical and chemical properties of oligosaccharides, give them unique properties, and expand the application of oligosaccharides in the biomedical field.

[0003] Globotriaosylceramide (Gb3), also known as CD77 or P k Blood group antigen is an important cell surface molecule. Its sugar antigen epitope is Galα1,4Galβ1,4Glc, which can be recognized by a variety of pathogens, such as Shiga toxin, Escherichia coli P-type pili, etc., and plays an important role in the infection process of various bacteria.

[0004] In recent years, some chitosan oligosaccharide derivatives have been widely used as drug delivery carriers due to their good biological properties such as biocompatibility, degradability, low immunogenicity, and mucosal adhesion. However, most of the chitosan oligosaccharides currently used in bioactivity research are obtained by enzymatic, chemical, or physical degradation, resulting in chitosan oligosaccharide conjugates being mostly mixtures, making it difficult to clearly reveal the structure-activity relationship of a specific structure. Therefore, the efficient preparation of chitosan oligosaccharide conjugates with clear structures, a marine carbohydrate substance, is of great significance for the elucidation and development of biological molecules with important biological activities. Summary of the invention

[0005] In order to overcome the above problems, the present invention grafts lactotriose Gb3 onto homogeneous chitosan oligosaccharides to obtain homogeneous marine carbohydrate substance Gb3-chitosan oligosaccharide conjugates, so as to improve the physicochemical properties of chitosan oligosaccharides and enhance the biological activity of Gb3, thereby expanding the application scope of marine chitosan oligosaccharides and Gb3.

[0006] The present invention provides a method for synthesizing a marine carbohydrate substance Gb3-chitosan oligosaccharide conjugate, which comprises a Gb3 trisaccharide shown in formula (I), and a Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (II) and formula (III). ; The enzymatic assembly module constructed by the present invention is based on a "one-pot multi-enzyme" system, that is, under the action of a specific sugar nucleotide synthetase, free monosaccharides (Gal) are converted into specific nucleotide-activated glycosyl donors (UDP-Gal). Subsequently, the monosaccharide units on the nucleoside-activated glycosyl donors are covalently linked to the hydroxyl groups at specific positions of the glycosyl acceptor under the action of specific glycosyltransferases. The present invention has optimized and screened the enzymes used for many times during the experiment, and the results show that: Escherichia coli galactokinase (GalK), Bifidobacterium longum UDP-sugarpyrophosphorylase (BLUSP) and Neisseria meningitides α1,4-galactosyltransferase (NmLgtC) is the enzyme used in the enzymatic module of the present invention, and its catalytic effect is the best, and it is particularly suitable for the enzymatic synthesis of Gb3 trisaccharide, with high synthesis efficiency, basically no by-products, and convenient purification. Moreover, the above enzymes are all derived from bacteria and can be simply expressed and purified in a conventional Escherichia coli expression system.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides a method for synthesizing Gb3 and Gb3-chitosan oligosaccharide conjugates, comprising: Using lactoside with an azide linker at the reducing end as a glycosyl acceptor and galactose as a glycosyl donor, the Gb3 trisaccharide shown in formula (I) is generated by catalysis of the enzymatic assembly module; The Gb3 trisaccharide shown in formula (I) is catalyzed by a one-step chemical method (Pd / C, H 2 ), to obtain Gb3 trisaccharide with an amino group at the reducing end. Subsequently, the Gb3 trisaccharide with an amino group at the reducing end was coupled with DSG (disuccinimidyl glutarate) to obtain a Gb3-DSG conjugate, and the newly generated Gb3-DSG conjugate was further coupled with two marine sugar substances, chitosan oligosaccharides, to obtain Gb3-chitosan oligosaccharide conjugate pentasaccharides shown in formula (II) and formula (III), respectively. Among them, the two chitosan oligosaccharides are GlcNH 2β1,4GlcNHAc and GlcNHAcβ1,4GlcNH 2 They are obtained on the basis of homogeneous chitin disaccharide GlcNHAcβ1,4GlcNHAc, catalyzed by chitin oligosaccharide non-reducing end deacetylase Rhizobium NodB and chitin oligosaccharide non-reducing end adjacent position deacetylase Vibrio cholerae COD. In our research group, this part of the content has been patented and no duplicate protection is sought here.

[0008] In the compounds represented by formula (I) to (III), R 1 is hydroxyl, azido-substituted alkyl, alkynyl-substituted alkyl, thiol-substituted alkyl, α- or β-substituted alkyl, α- or β-serine residue, α- or β-threonine residue; R 2 It is poly-N-acetylglucosamine and its derivatives, azide-substituted alkyl, alkynyl-substituted alkyl, thiol-substituted alkyl, α- or β-configuration substituted alkyl, α- or β-configuration serine residue, α- or β-configuration threonine residue.

[0009] The beneficial effects of the present invention are: (1) Using bacterial sugar nucleotide synthase and glycosyltransferase, we constructed the enzymatic assembly module required for the synthesis of α1,4-galactose glycosidic bonds, which greatly increased the synthesis efficiency of Gb3 trisaccharide and reduced the synthesis cost, thus making it possible for the subsequent large-scale synthesis of Gb3 trisaccharide.

[0010] (2) The present invention uses two amino-containing marine carbohydrate substances, chitosan oligosaccharides, which were maturely prepared by the research group, as substrates, and prepares structurally uniform Gb3-chitosan oligosaccharide conjugates through DSG coupling with enzymatically synthesized Gb3 trisaccharide, providing a structurally clear molecular tool for the study of the biological activity of chitosan oligosaccharide derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The schematic implementation examples of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0012] Figure 1 The diagram is a mechanism diagram of the enzymatic assembly module 1 catalyzing the formation of α1,4-galactosyl glycosidic bonds; Figure 2 This is the synthetic route of compound 1; Figure 3 This is the synthetic route of compound 4; Figure 4 The synthetic route of compound 5; Figure 5 This is the synthetic route of compound 2; Figure 6 This is the synthetic route of compound 3; Figure 7 is the nuclear magnetic resonance spectrum of compound 1, wherein (a) is the 1 H spectrum, (b) is compound 1 13 C spectrum; Figure 8 is the NMR spectrum of compound 4, wherein (a) is the 1 H spectrum, (b) is compound 4 13 C spectrum; Fig. 9 is the NMR spectrum of compound 2, wherein (a) is the 1 H spectrum, (b) is compound 2 13 C spectrum; Fig.10 is the NMR spectrum of compound 3, wherein (a) is the 1 H spectrum, (b) is compound 3 13 C spectrum; DETAILED DESCRIPTION

[0013] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0014] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0015] The structural formula of the compound represented by formula (I) to formula (III) is: ; In the compounds represented by formula (I) to (III), R 1 is hydroxyl, azido-substituted alkyl, alkynyl-substituted alkyl, thiol-substituted alkyl, α- or β-substituted alkyl, α- or β-serine residue, α- or β-threonine residue; R 2 It is poly-N-acetylglucosamine and its derivatives, azide-substituted alkyl, alkynyl-substituted alkyl, thiol-substituted alkyl, α- or β-configuration substituted alkyl, α- or β-configuration serine residue, α- or β-configuration threonine residue.

[0016] In one or more embodiments, in the enzymatic assembly module 1, the enzymes used in sequence are: galactokinase (GalK), sugar nucleoside synthetase (BLUSP) and α1,4-galactosyltransferase (NmLgtC). GalK is a Gal-1-P generating enzyme, and BLUSP is a UDP-Gal generating enzyme. Figure 1 This is a diagram showing the mechanism of the enzymatic assembly module 1 catalyzing the formation of α1,4-galactosyl glycosidic bonds.

[0017] In one or more embodiments, lactoside is used as a glycosyl acceptor and galactose is used as a glycosyl donor, and a Gb3 trisaccharide shown in formula (I) is generated by an enzymatic assembly module 1 catalytic reaction. The specific method includes: Lactose (1.0 equivalent), galactose (1.3-3.0 equivalent), adenosine triphosphate (ATP) (1.3-5.0 equivalent) and uridine triphosphate (UTP) (1.3-5.0 equivalent) were dispersed in a solution containing MgCl 2 (5~100 mmol) of Tris-HCl buffer (10~500 mmol, pH 5.0~10.0), the pH value of the reaction system is adjusted to 4.5~8.5, and finally galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP) and α1,4-galactosyltransferase (NmLgtC) are added, and the reaction is carried out for 3~72 hours. After purification, the Gb3 trisaccharide shown in formula (I) is obtained.

[0018] In one or more embodiments, the trisaccharide Gb3 of formula (I) is subjected to a one-step reduction reaction to reduce the azide group in its linker arm to an amino group, and the specific method includes: The Gb3 trisaccharide (1.0 equivalent) represented by formula (I) was dissolved in an appropriate amount of double distilled water, and Pd / C (0.5-8.0 equivalent), formic acid (0.01-8.0 equivalent), H 2 Under the above conditions, the reaction was stirred for 0.1-72 h, and the Gb3 trisaccharide containing an amino group at the reducing end was obtained after filtration and purification.

[0019] In one or more embodiments, the Gb3 trisaccharide containing an amino group at the reducing end is subjected to a one-step amidation reaction to obtain a Gb3-DSG conjugate intermediate, and the specific method includes: The trisaccharide Gb3 (1.0 equivalent) containing an amino group at the reducing end is dissolved in a double distilled aqueous solution, and then the DSG DMF (N,N-dimethylformamide) (5.0-50.0 equivalent) solution prepared in advance is quickly added, stirred rapidly, and reacted for 0.1-24 hours. In order to make the amino group in Gb3 only undergo amidation reaction with the active ester on one side of DSG, a large excess of DSG needs to be added to prevent the Gb3 trisaccharide from being coupled to both sides of DSG at the same time. After the reaction is complete by TLC detection, the dimethylformamide / water extraction is performed several times, and the upper aqueous solution is concentrated and dried to obtain the Gb3-DSG conjugate intermediate.

[0020] In one or more embodiments, the Gb3-DSG conjugate is combined with the marine carbohydrate chitosan oligobiose GlcNH 2 β1,4GlcNAc, and undergo a one-step amidation reaction to obtain a Gb3-chitosan oligosaccharide conjugate pentasaccharide represented by formula (II). The specific method comprises: The Gb3-DSG conjugate was dissolved in PBS buffer (pH = 7.0-8.0) and the marine sugar substance chitosan oligobiose GlcNH 2 β1,4GlcNAc (0.5-5.0 equivalents) was placed on a shaker (0-250 rpm) for reaction for 3-120 h. After purification by Bio-Gel P2, the Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (II) was obtained.

[0021] In one or more embodiments, the Gb3-DSG conjugate is combined with the marine carbohydrate chitosan oligosaccharide GlcNAcβ1,4GlcNH 2 , performing an amidation reaction to obtain a Gb3-chitosan oligosaccharide conjugate pentasaccharide represented by formula (III), the specific method comprising: The Gb3-DSG conjugate was dissolved in PBS buffer (pH = 7.0-8.0), and the marine carbohydrate chitobiose GlcNAcβ1,4GlcNH 2 (0.5-5.0 equivalents), placed on a shaker (0-250 rpm) for reaction for 3-120 h, and purified by Bio-Gel P2 to obtain the Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (III).

[0022] In one or more embodiments, in the enzymatic assembly module used to synthesize the polysaccharide represented by formula (I), the reaction temperature is 0-37°C and the rotation speed is 0-240 r / min; the method for stopping the enzyme reaction is to add an equal volume of 4°C anhydrous ethanol to the reaction solution and incubate at -20°C for 0-50 min.

[0023] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific implementation examples.

[0024] The chemical nature of enzymes is protein, and their catalytic activity is affected by many factors, such as metal ions, substrate concentration, temperature, time, pH, etc. of the reaction system. The present invention designs the most suitable reaction condition system for different enzymes used in the synthesis of Gb3 trisaccharide, thereby ensuring the high efficiency of the enzymes in the enzymatic module of the system of the present invention.

[0025] The compounds 1 to 3 mentioned in the following examples correspond to R 1 is a propyl azide linker and R 2 The compound described in formula I~III is hydroxyl.

[0026] Example 1 Synthesis of Compound 1 by chemical method.

[0027] refer to Figure 2 , a synthetic method of compound 1.

[0028] LactoseβproN 3 6 (100 mg, 1.0 eq.), galactose (64 mg, 1.5 eq.), ATP (195 mg, 1.5 eq.), UTP (207 mg, 1.5 eq.), Tris-HCl buffer (100 mmol, pH 7.5, 182 mg), and MgCl 2 (20 mmol, 60 mg) was dissolved in a 50 mL centrifuge tube, and GalK (1.0-3.0 mg), BLUSP (1.0-3.0 mg) and NmLgtC (2.0-4.0 mg) were added. Double distilled water was added to a total volume of 15 mL, and the tube was placed on a shaker and incubated at 37 °C and 110 r / min for 48 h. Thin layer chromatography (TLC) (EtOAc: MeOH: H 2 After the reaction was completed, an equal volume of anhydrous ethanol was added to the reaction system and the reaction was stopped in a -20 °C refrigerator 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, and then separated and purified by Bio-gel P2 gel molecular exclusion chromatography and anion exchange column DEAE to obtain compound 1 (125 mg, 90%). 1 H NMR (400 MHz, D 2 O) δ 4.91 (d, J = 3.9 Hz,1H), 4.47 (d, J= 7.5 Hz, 1H), 4.45 (d, J = 7.8 Hz, 1H), 4.35 – 4.29 (m, 1H), 4.02 – 3.50 (m, 18H), 3.42 (t, J = 6.7 Hz, 2H), 3.30 – 3.24 (m, 1H), 1.92 –1.83 (m, 2H); 13 C NMR (150 MHz, D 2 O) δ 103.3, 102.1, 100.3, 78.6, 77.4, 75.4,74.8, 74.4, 72.9, 72.2, 70.9, 70.8, 69.1, 68.9, 68.6, 67.4, 60.5, 60.4, 60.0,47.9, 28.3; HRMS (ESI) m / z Calculate for C 21 H 38 N 3 O 16 [M+H] + 588.2247, found588.2257. Example 1 Synthesis of Compound 4 by chemical method.

[0029] refer to Figure 3 , the synthesis method of compound 4.

[0030] Compound 1 (100 mg) was dissolved in double distilled water (10 mL), and Pd / C (200 mg) (the amount of Pd / C is the mass ratio) was added, and a small amount of formic acid (100 μL) was added. A long needle was used to introduce the hydrogen in the hydrogen balloon below the liquid surface of the reaction solution, and a short needle was inserted into the bottle stopper to achieve gas replacement. During this process, hydrogen was continuously introduced into the reaction system to keep the solution in a bubbling state. The reaction continued for about 45 minutes until the hydrogen was completely consumed. TLC spot plate detection (EtOAc: MeOH: H 2 O: EtOH = 2: 1: 1: 0.5), after the reaction is complete, use a 0.22 micron water filter membrane to slowly dropwise filter, the resulting solution is concentrated and directly used in the next reaction without purification; 1 H NMR (400 MHz, D 2 O) δ 4.91 (d, J =3.9 Hz, 1H), 4.47 (d, J = 7.7 Hz, 1H), 4.46 (d,J = 8.0 Hz, 1H), 4.35 – 4.29(m, 1H), 4.03 – 3.48 (m, 20H), 3.29 – 3.23 (m, 1H), 1.89 – 1.78 (m, 2H); 13 CNMR (150 MHz, D 2 O) δ 103.3, 102.1, 100.3, 78.6, 77.3, 75.4, 74.8, 74.4, 72.9,72.1, 70.9, 70.8, 69.1, 68.9, 68.6, 67.3, 60.5, 60.4, 60.0, 58.5, 31.3; HRMS(ESI) m / z Calculate for C 21 H 40 NO 16 [M+H] + 562.2342, found 562.2350. Example 1 Synthesis of Compound 5 by chemical method.

[0031] refer to Figure 4 , the synthesis method of compound 5.

[0032] Compound 4 (80 mg, 1.0 equivalent) was dissolved in 1 mL of double distilled water, and DSG (100 mg, 15 equivalent) was dissolved in 5 mL of DMF solution in advance. Subsequently, the DSG-containing DMF solution was quickly poured into the aqueous solution of compound 4, and the volume ratio of water to DMF was 1:5. After rapid stirring, TLC detection (EtOAc:MeOH:H 2 O:EtOH=2:1:1:0.5), no raw material remained, and it was completely converted into product points. Subsequently, the reaction system was transferred to a separatory funnel until the volume of the aqueous solution was about 10 ml, and 5 times the equivalent of dichloromethane solution was added. After extracting 10 times in the separatory funnel, the aqueous phase was taken, concentrated and dried to obtain compound 5 (64 mg, 58%). The lower yield this time was mainly due to the large loss during the extraction process. In view of the instability of the NHS activated ester contained in compound 5, no subsequent purification and NMR identification were carried out, and the next step of reaction was carried out directly; Example 1 Synthesis of Compound 2 by chemical method.

[0033] refer to Figure 5 , the synthesis method of compound 2.

[0034] Chitosan oligobiose GlcNH 2β1,4GlcNAc (10 mg, 1.0 equivalent) was dissolved in 1 mL of PBS buffer (pH = 7.4), and compound 5 (20 mg, 1.0 equivalent) was added, and the mixture was placed on a shaker and incubated at 37 °C and 225 r / min for 96 h. Thin layer chromatography (EtOAc:MeOH:H 2 O:EtOH=2:1:1:0.5) to detect the reaction progress. After the reaction was completed, the pentasaccharide compound 2 (16 mg, 60%) was obtained directly by Bio-Gel P2 purification; 1 H NMR (400 MHz, D 2 O) δ 5.14 (d, J = 2.3 Hz,0.6H, 1α-H ), 4.90 (d, J = 3.9 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.4H, 1β-H ), 4.57– 4.51 (m, 1H), 4.46 (d, J = 8.1 Hz, 1H), 4.44 (d, J = 8.1 Hz, 1H), 4.32 (t, J = 6.5 Hz, 1H), 4.03 – 3.19 (m, 33H), 2.33 – 2.19 (m, 4H), 1.99 (s, 3H), 1.90 – 1.72 (m, 4H); 13 C NMR (125 MHz, D 2 O) δ 176.3, 175.8, 174.7, 174.5,103.3, 102.0, 101.2, 100.3, 94.8 ( 1β-C ), 90.4 ( 1α-C ), 79.2, 78.8, 78.7, 77.3,75.9, 75.4, 74.8, 74.6, 74.4, 73.5, 72.9, 72.4, 72.2, 70.9, 70.8, 70.0, 69.8,69.2, 69.1, 68.9, 68.6, 67.8, 60.5, 60.4, 60.1, 60.0, 56.1, 55.5, 53.6, 36.3,35.1, 35.0, 28.4, 22.2, 21.9, 21.7; HRMS (ESI) m / z Calculate for C 40 H70 N 3 O 28 [M+H] + 1040.4140, found 1040.4171. Example 1 Synthesis of Compound 3 by chemical method.

[0035] refer to Figure 6 , the synthesis method of compound 3.

[0036] Chitosan oligobiose GlcNAcβ1,4GlcNH 2 (10 mg, 1.0 equivalent) was dissolved in 1 mL of PBS buffer (pH = 7.4), compound 5 (20 mg, 1.0 equivalent) was added, and the mixture was placed on a shaker and incubated at 37 °C and 225 r / min for 96 h. Thin layer chromatography (EtOAc:MeOH:H 2 O:EtOH=2:1:1:0.5) to detect the reaction progress. After the reaction was completed, the pentasaccharide compound 3 (18 mg, 65%) was obtained directly by Bio-Gel P2 purification; 1 H NMR (400 MHz, D 2 O) δ 5.16 (d, J = 2.3 Hz,0.6H, 1α-H ), 4.90 (d, J = 3.9 Hz, 1H), 4.66 (d, J = 7.7 Hz, 0.4H, 1β-H ), 4.57– 4.52 (m, 1H), 4.46 (d, J = 7.9 Hz, 1H), 4.44 (d, J = 8.1 Hz, 1H), 4.32 (t, J = 6.4 Hz, 1H), 4.03 – 3.20 (m, 33H), 2.32 – 2.19 (m, 4H), 2.03 (s, 3H), 1.91 – 1.73 (m, 4H); 13 C NMR (125 MHz, D 2 O) δ 176.4, 176.2, 175.9, 175.8,174.6, 103.3, 102.0, 101.5, 100.3, 94.8 ( 1β-C ), 90.5 ( 1α-C), 79.9, 79.5,78.7, 77.4, 75.9, 75.4, 74.8, 74.5, 74.4, 73.5, 72.9, 72.5, 72.2, 70.9, 70.8,69.9, 69.7, 69.3, 69.1, 68.9, 68.6, 67.8, 62.9, 60.6, 60.5, 60.4, 60.2, 60.1,56.0, 55.6, 55.6, 53.5, 36.3, 35.1, 34.8, 34.8, 34.7, 28.4, 22.1, 21.8; HRMS(ESI) m / z Calculate for C 40 H 70 N 3 O 28 [M+H] + 1040.4140, found 1040.4171. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The enzymatic synthesis method of Gb3-chitosan oligosaccharide conjugate is characterized by: Using lactoside as a glycosyl acceptor and galactose as a glycosyl donor, the Gb3 trisaccharide shown in formula (I) is generated by enzymatic assembly module 1; The Gb3 trisaccharide shown in formula (I) is subjected to a one-step Pd / C and H2 reduction reaction, and the azide group on the reducing end linker is reduced to an amino group; the newly generated Gb3 trisaccharide containing an amino group at the reducing end is coupled with DSG through a one-step amidation reaction to obtain a Gb3-DSG conjugate intermediate containing an NHS activated ester at the reducing end; The activated ester at the reducing end of the Gb3-DSG conjugate is coupled with the marine sugar substance chitosan oligosaccharide GlcNH2β1,4GlcNAc to generate the marine sugar substance Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (II); The activated ester at the reducing end of the Gb3-DSG conjugate is coupled with the marine sugar substance chitosan oligosaccharide GlcNAcβ1,4GlcNH2 to generate the marine sugar substance Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (III); The structural formula of the compound represented by formula (I) to formula (III) is: ； In the compounds represented by formula (I) to (III), R1 is hydroxyl, azido-substituted alkyl, alkynyl-substituted alkyl, thiol-substituted alkyl, α- or β-substituted alkyl, α- or β-configuration serine residue, α- or β-configuration threonine residue; R2 is poly-N-acetylglucosamine and its derivatives, azido-substituted alkyl, alkynyl-substituted alkyl, thiol-substituted alkyl, α- or β-configuration substituted alkyl, α- or β-configuration serine residue, α- or β-configuration threonine residue.

2. The synthesis method according to claim 1, characterized in that In the enzymatic assembly module 1, the enzymes used successively are: galactokinase GalK, sugar nucleoside synthetase BLUSP and α1,4-galactosyltransferase NmLgtC.

3. The synthesis method according to claim 1, characterized in that Using lactoside as a glycosyl acceptor and galactose as a glycosyl donor, the Gb3 trisaccharide shown in formula (I) is generated by enzymatic assembly module 1, and the specific method includes: 1.0 equivalent of lactosidase, 1.0-3.0 equivalents of galactose, 1.0-5.0 equivalents of adenosine triphosphate ATP and 1.0-5.0 equivalents of uridine triphosphate UTP are dispersed in 10-500 mmol of Tris-HCl buffer containing 5-100 mmol of MgCl2 and pH 5.0-10.0, and the pH value of the reaction system is adjusted to 4.5-8.

5. Finally, galactokinase GalK, sugar nucleoside generating enzyme BLUSP and α1,4-galactosyltransferase NmLgtC are added, and the reaction is carried out for 3-96 hours. After purification, the Gb3 trisaccharide shown in formula (I) is obtained.

4. The synthesis method according to claim 1, characterized in that The Gb3 trisaccharide shown in formula (I) is used as a substrate, and a Gb3-DSG conjugate intermediate containing an NHS activated ester at the reducing end is generated through a one-step reduction reaction and a one-step amidation reaction. The specific method includes: 1.0 equivalent of Gb3 trisaccharide shown in formula (I) is dissolved in double distilled water solution, 1.0~10.0 equivalent of Pd / C and 0.01~8.0 equivalent of formic acid are added, and Gb3 trisaccharide with an amino group at the reducing end is obtained under H2 conditions; the newly generated Gb3 trisaccharide is coupled with an excess of 5.0~50.0 equivalents of DSG. After the reaction is completed, dichloromethane solution is added for multiple extractions, the aqueous phase is collected, concentrated and dried, and a Gb3-DSG conjugate intermediate containing an NHS activated ester at the reducing end can be obtained.

5. The synthesis method according to claim 1, characterized in that The Gb3-DSG conjugate intermediate containing NHS activated ester at the reducing end is coupled with the marine sugar substance chitosan oligosaccharide GlcNH2β1,4GlcNAc to generate the Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (II), and the specific method includes: The Gb3-DSG conjugate was dissolved in PBS buffer pH = 7.0-8.0, 0.5-5.0 equivalents of marine sugar substance chitosan oligosaccharide GlcNH2β1,4GlcNAc were added, and the mixture was placed on a shaker at 0-250 rpm for reaction for 3-120 h. After purification by Bio-Gel P2, the Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (II) was obtained.

6. The synthesis method according to claim 1, characterized in that The Gb3-DSG conjugate intermediate containing NHS activated ester at the reducing end is coupled with the marine sugar substance chitosan oligosaccharide GlcNAcβ1,4GlcNH2 to generate the Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (III), and the specific method includes: The Gb3-DSG conjugate was dissolved in PBS buffer pH = 7.0-8.0, and 0.5-5.0 equivalents of marine sugar substance chitosan oligosaccharide GlcNAcβ1,4GlcNH2 were added, and the mixture was placed on a shaker at 0-250 rpm for reaction for 3-120 h. After purification by Bio-Gel P2, the Gb3-chitosan oligosaccharide conjugate pentasaccharide shown in formula (III) was obtained.

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