A kind of fucosylated chondroitin and its preparation method and application

Abstract

The present invention discloses a fucosylated chondroitin and a preparation method and application thereof. The present invention utilizes the substrate broadness of the enzyme, screens fucosyltransferases from different sources for substrate adaptability research, constructs a multi-enzyme cascade catalytic reaction system, and develops an efficient enzymatic method for synthesizing fucosylated chondroitin. The method of the present invention can achieve different degrees of fucosylation modification at different sites of the chondroitin sugar chain, laying a foundation for the industrial production of fucosyl sulfate chondroitin and promoting the development of carbohydrate anticoagulant drugs.

Description

Technical Field

[0001] The invention relates to the fields of enzyme engineering technology and synthetic biology, and in particular to a fucosylated chondroitin and a preparation method and application thereof. Background Art

[0002] With the general improvement of human living standards and the acceleration of the aging process of the population, thrombotic diseases are increasing day by day and have become a serious problem affecting human life and health. Heparin and low molecular weight heparins (LMWHs) are widely used in the treatment of thrombotic diseases as anticoagulants. Heparin and low molecular weight heparins have the risk of severe bleeding during treatment and cannot be taken orally. Therefore, researchers have continued to look for more effective and safer anticoagulant drugs. Fucosylated chondroitin sulfate (FuCS) is a uniquely structured active polysaccharide isolated from sea cucumbers. It has significant anticoagulant and antithrombotic activities and can be used as a potential candidate drug. Its synthesis and preparation as well as biological function research have become a hot topic of concern for scientists at home and abroad. However, the lack of homogeneous oligosaccharides has hindered further structure-activity relationship research.

[0003] At present, there are two main methods for obtaining FuCS: sea cucumber extraction and chemical synthesis. The extraction method is limited by the quality and source of sea cucumbers, and it is impossible to obtain FuCS sugar chains with clear structures, which limits the structure-activity relationship and biological research (such as ACS ChemBiol 2020, 15 (8), 2232-2246. or CarbohydrPolym 2017, 165, 7-12. or CarbohydrPolym 2021, 262, 117969.); the chemical synthesis of FuCS has made great progress in recent years, but due to the complexity of the sugar chain, there are still problems such as cumbersome steps and low yield, and it is impossible to achieve economical and efficient synthesis (such as Angew Chem Int Ed Engl 2018, 57 (39), 12880-12885. or J Org Chem 2020, 85, 24, 15908–15919.).

[0004] The sugar chain structure of FuCS is composed of a sulfated modified disaccharide repeating unit and a GlcA O-3 branched modified fucose residue, wherein the disaccharide repeating unit is mainly composed of uronic acid (glucuronic acid GlcA) and N-acetylgalactosamine (GalNAc) connected by a β-glycosidic bond. FuCS from different species differ in the number of sulfated sites and fucosylated branches. There is no report on the enzymatic synthesis of FuCS oligosaccharide fragments in the prior art. In view of this, the present invention is proposed. Summary of the invention

[0005] The present invention overcomes the deficiencies in the prior art and provides a fucosylated chondroitin and a preparation method and application thereof.

[0006] The first aspect of the present invention provides a method for preparing fucosylated chondroitin, the method comprising the following steps:

[0007] The L-fucosyltransferase is mixed with the substrate GDP-L-fucose and chondroitin oligosaccharide to undergo an enzymatic reaction to obtain fucosylated chondroitin.

[0008] Furthermore, the molar ratio of fucosylated GlcA to GDP-L-fucose (guanosine diphosphate-fucose) in the chondroitin oligosaccharide is 0.5-2:1-4, preferably, the molar ratio is 0.5-1.5:1-3.5, more preferably, the molar ratio is 1:1-3.5 (such as 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5).

[0009] In a specific embodiment of the present invention, the molar ratio of fucosylated GlcA to GDP-L-fucose (guanosine diphosphate-fucose) in the chondroitin oligosaccharide is 1:2.

[0010] Further, the pH of the reaction is 6.5-8.5, preferably, the pH of the reaction is 7-8, and more preferably, the pH of the reaction is 7.5.

[0011] Further, the reaction temperature is 30-45°C, preferably, the reaction temperature is 35-40°C, and more preferably, the reaction temperature is 37°C.

[0012] Furthermore, the reaction time is 10-30 hours, preferably, the reaction time is 10-25 hours, and more preferably, the reaction time is 12-24 hours.

[0013] Further, the concentration of the L-fucosyltransferase is 0.1-2 mg / mL, preferably, the concentration of the L-fucosyltransferase is 0.5-1.5 mg / mL, and more preferably, the concentration of the L-fucosyltransferase is 1 mg / mL.

[0014] Furthermore, the chondroitin oligosaccharide has the following structure:

[0015]

[0016] Furthermore, n1 is an integer selected from 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0017] Preferably, the n1 is an integer selected from 1-5, and more preferably, the n1 is an integer selected from 1-3.

[0018] In a specific embodiment of the present invention, the chondroitin oligosaccharide is selected from the following structures:

[0019]

[0020]

[0021] Furthermore, the nucleotide sequence of the L-fucosyltransferase is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0022] Furthermore, the nucleotide sequence of the L-fucosyltransferase Bfα1,3FT is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 3;

[0023] Furthermore, the nucleotide sequence of the L-fucosyltransferase Hpα1,3FT S45F V30G is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 4.

[0024] Furthermore, the L-fucosyltransferase is selected from one of α-1,2-fucosyltransferase and α-1,3-fucosyltransferase.

[0025] Preferably, the L-fucosyltransferase is α-1,3-fucosyltransferase.

[0026] Preferably, the L-fucosyltransferase is selected from: a polypeptide represented by any one of SEQ ID NOs: 3-4, a functional variant of a polypeptide represented by any one of SEQ ID NOs: 3-4, a functional fragment of a polypeptide represented by any one of SEQ ID NOs: 3-4, and a functional variant of a functional fragment of a polypeptide represented by any one of SEQ ID NOs: 3-4.

[0027] Further, the source of the L-fucosyltransferase is: one of Bacteroides fragilis, Helicobacter pylori, Vibrio cholerae, H.bilis, H.cinaede, Bacteroides ovatus and Bacteroides vulgatus. Preferably, the source of the L-fucosyltransferase is Bacteroides fragilis and / or Helicobacter pylori.

[0028] In a specific embodiment of the present invention, the L-fucosyltransferase is Bfα1,3FT (derived from Bacteroides fragilis).

[0029] In a specific embodiment of the present invention, the L-fucosyltransferase is Hpα1,3FT (derived from Helicobacter pylori).

[0030] In a specific embodiment of the present invention, the L-fucosyltransferase is a Hpα1,3FT S45FV30G mutant, and its catalytic activity is improved.

[0031] Bfα1,3FT can recognize all D-GalNAc1-4-β-D-GlcA disaccharide units in oligosaccharides and perform fucosylation modification on the GlcA O-3 position, while Hpα1,3FT can only recognize oligosaccharides with D-GalNAc1-4-β-D-GlcA structure at the non-reducing end and only perform single-site fucosylation modification on the GlcA O-3 position at the non-reducing end. When Bfα1,3FT catalyzes the recognition of oligosaccharides with D-GlcA1-3-β-D-GalNAc structure at the non-reducing end, the non-reducing end has two or more sugars that are not fucosylated and can be further extended by chondroitin synthase and further fucosylated at specific positions as needed.

[0032] In a specific embodiment of the present invention, the chondroitin oligosaccharide structure that can be recognized by the Hpα1,3FT S45F V30G mutant is shown in formula (I):

[0033]

[0034] In a specific embodiment of the present invention, the structures of the chondroitin oligosaccharides that can be recognized by the Hpα1,3FT S45F V30G mutant are shown in formulas (IV), (VI), and (VIII), respectively:

[0035]

[0036] In a specific embodiment of the present invention, the structures of the single-site fucosylated chondroitin oligosaccharides synthesized by the Hpα1,3FT S45F V30G mutant are shown in formulas (IX), (X), and (XI), respectively:

[0037]

[0038] In a specific embodiment of the present invention, the chondroitin oligosaccharide structures recognizable by Bfα1,3FT are respectively shown in formula (I) and (II):

[0039]

[0040] In a specific embodiment of the present invention, the chondroitin oligosaccharide structures recognizable by Bfα1,3FT are respectively represented by formula (IV), (V), (VI), (VII), and (VIII):

[0041]

[0042]

[0043] In a specific embodiment of the present invention, the structures of the fucosylated chondroitin oligosaccharides synthesized by Bfα1,3FT are shown in formulas (IX), (XII), (XIII), (XIV), and (XV), respectively:

[0044]

[0045]

[0046] Furthermore, the preparation method also includes separation and purification steps.

[0047] Furthermore, when the non-reducing end of the fucosylated chondroitin oligosaccharide has more than or equal to two unfucosylated sugars, the chondroitin synthase PmCS can catalyze further extension of the main chain and further fucosylation modification can be catalyzed by Bfα1,3FT or Hpα1,3FTS45F V30G at specific positions as needed to synthesize chondroitin oligosaccharides with a defined structure and a defined degree of fucosylation.

[0048] Furthermore, the L-fucosyltransferase Hpα1,3FT S45F V30G catalyzes single-site fucosylation modification at the O-3 position of GlcA whose non-reducing end structure is D-GalNAc1-4-β-D-GlcA.

[0049] Furthermore, the L-fucosyltransferase Bfα1,3FT catalyzes the fucosylation modification of all GlcA O-3 positions of the structure D-GalNAc1-4-β-D-GlcA. Furthermore, the step of extending the fucosylated chondroitin backbone comprises mixing the backbone with chondroitin synthase PmCS and sugar nucleotides for reaction.

[0050] Furthermore, the sugar nucleotide is selected from: UDP-GalNAc, UDP-GlcA.

[0051] In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: firstly, the main chain of fucosylated chondroitin is extended using chondroitin synthase, and then L-fucosyltransferase Hpα1,3FT S45FV30G or Bfα1,3FT is used to catalyze the fucosylation modification of a specific site.

[0052] In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: firstly, the main chain of fucosylated chondroitin is extended using chondroitin synthase, and then, L-fucosyltransferase Hpα1,3FT S45F V30G is used to catalyze the further single-site fucosylation modification at the GlcA O-3 position whose non-reducing end structure is D-GalNAc1-4-β-D-GlcA.

[0053] In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: firstly, the main chain of fucosylated chondroitin is extended using chondroitin synthase, and then further fucosylation modification is catalyzed by L-fucosyltransferase Bfα1,3FT at all GlcA O-3 positions of the structure D-GalNAc1-4-β-D-GlcA.

[0054] In a specific embodiment of the present invention, the structures of the fucosylated chondroitin main chain that can be further extended by chondroitin synthase PmCS are shown in (XII) and (XIV) respectively:

[0055]

[0056] In a specific embodiment of the present invention, the structures of the fucosylated chondroitin after main chain extension are shown in (XVI), (XVII), (XVIII), and (XIX), respectively:

[0057]

[0058]

[0059] In a specific embodiment of the present invention, the method of extending the main chain of fucosylated chondroitin using chondroitin synthase comprises the following steps: using chondroitin synthase PmCS to catalyze further fucosylation modification of fucosylated chondroitin (XII) to synthesize fucosylated chondroitin (XVI).

[0060] In a specific embodiment of the present invention, the method of extending the main chain of fucosylated chondroitin using chondroitin synthase comprises the following steps: using chondroitin synthase PmCS to catalyze further fucosylation modification of fucosylated chondroitin (XVI) to synthesize fucosylated chondroitin (XVIII).

[0061] In a specific embodiment of the present invention, the method of extending the main chain of fucosylated chondroitin using chondroitin synthase comprises the following steps: using chondroitin synthase PmCS to catalyze further fucosylation modification of fucosylated chondroitin (XVIII) to synthesize fucosylated chondroitin (XIX).

[0062] In a specific embodiment of the present invention, the method of extending the main chain of fucosylated chondroitin using chondroitin synthase comprises the following steps: using chondroitin synthase PmCS to catalyze further fucosylation modification of fucosylated chondroitin (XIV) to synthesize fucosylated chondroitin (XVII).

[0063] Furthermore, the L-fucosyltransferase Hpα1,3FTS45F V30G can recognize the extended fucosylated chondroitin and catalyze its further fucosylation modification.

[0064] In a specific embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: using L-fucosyltransferase Hpα1,3FTS45F V30G to catalyze further fucosylation modification of fucosylated chondroitin (XVI) to synthesize fucosylated chondroitin (XIII).

[0065] In a specific embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: using L-fucosyltransferase Hpα1,3FTS45F V30G to catalyze further fucosylation modification of fucosylated chondroitin (XVII) to synthesize fucosylated chondroitin (XV).

[0066] In a specific embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: using L-fucosyltransferase Hpα1,3FTS45F V30G to catalyze further fucosylation modification of fucosylated chondroitin (XIX) to synthesize fucosylated chondroitin (XX).

[0067] Furthermore, the L-fucosyltransferase Bfα1,3FT can recognize the extended fucosylated chondroitin and catalyze its further fucosylation modification.

[0068] In a specific embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: using L-fucosyltransferase Bfα1,3FT to catalyze further fucosylation modification of fucosylated chondroitin (XVIII) to synthesize fucosylated chondroitin (XIV).

[0069] In a specific embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: using L-fucosyltransferase Bfα1,3FT to catalyze further fucosylation modification of fucosylated chondroitin (XIX) to synthesize fucosylated chondroitin (XV).

[0070] Furthermore, the method for preparing chondroitin oligosaccharides comprises the following steps: mixing chondroitin synthase with a donor substrate and an acceptor substrate, and reacting them to obtain chondroitin oligosaccharides.

[0071] Furthermore, the donor substrate (ie, sugar nucleotide) is selected from: UDP-GalNAc, UDP-GlcA.

[0072] Furthermore, the receptor substrate is selected from: GalNAc.

[0073] The UDP-GalNAc described in the present invention has the Chinese full name UDP-N-acetylgalactosamine, which functions to provide an activated acetylgalactosamine donor (donor substrate) for the synthesis of chondroitin oligosaccharides or chondroitin polysaccharides.

[0074] The UDP-GlcA described in the present invention has the Chinese full name of UDP-glucuronic acid, and its function is to provide an activated glucuronic acid donor (donor substrate) for the synthesis of chondroitin oligosaccharides or chondroitin polysaccharides.

[0075] Further, the molar ratio of the monosaccharide GalNAc to the sugar nucleotide is 0.5-2:1-5, preferably, the molar ratio of the chondroitin oligosaccharide to the sugar nucleotide is 0.5-1.5:1-5 (such as 0.5:1-5, 0.6:1-5, 0.7:1-5, 0.8:1-5, 0.9:1-5, 1:1-5, 1.1:1-5, 1.2:1-5, 1.3:1-5, 1.4:1-5, 1.5:1-5), more preferably, the molar ratio of the chondroitin oligosaccharide to the sugar nucleotide is 1:1-5 (such as 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:5).

[0076] In a specific embodiment of the present invention, the molar ratio of the chondroitin oligosaccharide receptor to the sugar nucleotide is 1:1.5-1:2.

[0077] Furthermore, the chondroitin synthase is derived from: Pasteurella multocida, Escherichia coli, Actinobacillus Ureae.

[0078] Furthermore, the method also includes a separation step.

[0079] Further, the separation is selected from: ion exchange resin, molecular sieve, clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high efficiency filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration and ligand exchange chromatography.

[0080] Furthermore, the molecular sieve is selected from: polyacrylamide molecular sieve (such as Bio-Gel P2 to P300), agarose molecular sieve, dextran molecular sieve.

[0081] Furthermore, the ion exchange resin is an anion exchange resin, and the anion exchange resin is a strongly basic anion exchange resin or a weakly basic anion exchange resin. Preferably, the anion exchange resin is a strongly basic anion exchange resin.

[0082] Furthermore, the strong basic anion exchange resin is a gel-type strong basic anion exchange resin.

[0083] In a specific embodiment of the present invention, the separation is performed by an anion exchange resin.

[0084] In a specific embodiment of the present invention, the separation is performed using a strongly basic anion exchange resin.

[0085] In a specific embodiment of the present invention, the separation is molecular sieve.

[0086] In a specific embodiment of the present invention, the separation is performed by polyacrylamide molecular sieve.

[0087] In a specific embodiment of the present invention, the separation is Bio-Gel P2 molecular sieve.

[0088] Furthermore, the method also includes a purification step.

[0089] Furthermore, the purification includes the step of washing the chondroitin oligosaccharide using an appropriately diluted solution of alkali, salt, buffer solution or acid.

[0090] Furthermore, the purification also includes the steps of using activated charcoal or carbon, using charcoal, nanofiltration, ultrafiltration or ion exchange, using alcohol, using a water-alcohol mixture for crystallization, evaporation, concentration, precipitation, and drying.

[0091] Furthermore, the concentration is selected from: atmospheric pressure concentration, reduced pressure concentration, thin film concentration, and multi-effect concentration; and the drying is selected from: freeze drying, atmospheric pressure drying, reduced pressure drying, boiling drying, spray drying, infrared drying, and microwave drying.

[0092] In a specific embodiment of the present invention, the drying is freeze-drying.

[0093] Furthermore, the fucosylated chondroitin has the following structure:

[0094]

[0095] In a specific embodiment of the present invention, the fucosylated chondroitin has the following structure:

[0096]

[0097]

[0098]

[0099] The second aspect of the present invention provides a fucosylated chondroitin having the following structure:

[0100]

[0101] In a specific embodiment of the present invention, the fucosylated chondroitin has the following structure:

[0102]

[0103]

[0104]

[0105] The third aspect of the present invention provides two fucosyltransferases, wherein the nucleotide sequence of the fucosyltransferase is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0106] The fourth aspect of the present invention provides a recombinant vector, which is constructed by inserting the nucleotide sequence of the fucosyltransferase as shown in SEQ ID NO: 1 or SEQ ID NO: 2 described in the third aspect into a plasmid vector.

[0107] Furthermore, the vector is a viral vector or a non-viral vector. In one embodiment of the present invention, the vector is a plasmid vector.

[0108] In one embodiment of the present invention, the vector is PMAL-C2X.

[0109] In one embodiment of the present invention, the vector is pET-41a.

[0110] A fifth aspect of the present invention provides a recombinant cell.

[0111] Further, the recombinant cell is selected from: Gram-positive bacteria, Gram-negative bacteria, actinomycetes, and fungi;

[0112] Preferably, the Gram-positive bacteria is selected from: Bacillus subtilis;

[0113] Preferably, the Gram-negative bacteria are selected from: Escherichia coli; the actinomycetes are Streptomyces;

[0114] Preferably, the fungus is yeast or Aspergillus niger.

[0115] The sixth aspect of the present invention provides a pharmaceutical composition comprising the fucosylated chondroitin according to the second aspect.

[0116] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0117] Furthermore, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an antibacterial agent, a solute that makes the preparation isotonic with the recipient's blood, a suspending agent, a suspending aid, a solubilizer, a thickener, a stabilizer, a sweetener and a flavor.

[0118] The seventh aspect of the present invention provides a use of the fucosylated chondroitin according to the second aspect or the pharmaceutical composition according to the sixth aspect in the preparation of an anticoagulant drug or fucosylated chondroitin sulfate.

[0119] The eighth aspect of the present invention provides a use of the fucosyltransferase as described in the third aspect in synthesizing fucosylated chondroitin.

[0120] The ninth aspect of the present invention provides use of the fucosylated chondroitin as described in the second aspect in the preparation of fucosylated chondroitin sulfate.

[0121] The biological enzymes (fucosyltransferase and chondroitin synthase) described in the present invention are from prokaryotes, and have the advantages of high protein expression, wide substrate adaptability, high catalytic efficiency, etc. The enzymatic synthesis process has high efficiency and high regional selectivity, is simple and safe to operate, and lays a foundation for the industrial production of fucosyl sulfate chondroitin, solves the bottleneck problem of enzymatic synthesis of fucosylated chondroitin, and provides a method for preparing fucosylated chondroitin by multi-enzyme cascade in vitro. The present invention utilizes the substrate breadth of the enzyme, screens fucosyltransferases from different sources for substrate adaptability research, and develops an efficient enzymatic method for synthesizing fucosylated chondroitin. The method of the present invention can be used to perform different degrees of fucosylation modification on the substrate chondroitin oligosaccharide, and the oligosaccharide non-reducing end lays a foundation for the industrial production of fucosyl sulfate chondroitin, promoting the development of anticoagulant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Schematic diagram of the preparation of fucosylated chondroitin. DETAILED DESCRIPTION

[0123] In order to more clearly understand the technical content of the present invention, the following embodiments are given in detail, the purpose of which is only to better understand the content of the present invention but not to limit the protection scope of the present invention.

[0124] The structures of formula (IX) to formula (XX) described in the present invention are shown below:

[0125]

[0126]

[0127]

[0128] Example 1 Synthesis of single-site fucosylated chondroitin oligosaccharides catalyzed by Hpα1,3FT S45F V30G

[0129] (1) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), chondroitin trisaccharide (IV), MgCl2, and Tris-HCl, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, the final concentration of chondroitin trisaccharide (IV) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase, reacting for 12 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product IX, with a conversion rate of 90%. 1 H NMR,13 The \(^{13}\)C NMR and MS data are as follows:

[0130] 1 \(^{1}\)H NMR (600 MHz, D\(_2\)O) δ 5.40 (d, J = 4.0 Hz, 2H, H-1 Fuc ), 5.22 (d, J = 3.8 Hz, 1H, H-1 GalNAc-A ), 4.69 (d, J = 8.5 Hz, 1H, H-1 GalNAc-A ), 4.57 (d, J = 8.0 Hz, 1H), 4.52 (d, J = 7.9 Hz, 1H), 4.46 (dd, J = 8.4, 2.5 Hz, 2H), 4.29 (dd, J = 11.1, 3.8 Hz, 1H), 4.22 (d, J = 3.1 Hz, 1H), 4.16 - 4.11 (m, 2H), 4.02 - 3.89 (m, 11H), 3.83 (d, J = 3.3 Hz, 2H), 3.81 - 3.64 (m, 19H), 3.61 - 3.56 (m, 4H), 2.05 (dd, J = 26.0, 2.3 Hz, 12H, COOCH\(_3\), COOCH\(_3\)), 1.25 (d, J = 6.6 Hz, 6H, CH\(_3\) of Fucose).

[0131] 13 \(^{13}\)C NMR (151 MHz, D\(_2\)O) δ 175.42, 175.40, 175.06, 174.92, 174.62, 104.01, 103.85, 100.00, 98.31, 95.17, 91.19, 80.77, 77.73, 76.73, 76.67, 76.62, 75.22, 75.18, 74.95, 74.91, 74.22, 74.19, 74.16, 72.06, 71.54, 70.25, 69.25, 68.42, 68.13, 67.71, 67.53, 66.66, 61.50, 61.26, 61.05, 52.24, 48.83, 22.47, 22.22, 22.01, 15.37.

[0132] ESI-MS (negative mode) calcd for C 28 H 45 N\(_2\)O 21 - [M - H] - m / z 745.2520, found 745.2527.;

[0133] (2) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), chondroitin pentasaccharide (VI), MgCl2, and Tris-HCl, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, the final concentration of chondroitin pentasaccharide (VI) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase, reacting for 12 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product X, with a conversion rate of 86%. 1 H NMR, 13 C NMR and MS data are as follows,

[0134] 1 H NMR(600MHz,D2O)δ5.39(d,J=3.9Hz,2H,H-1 Fuc ),5.22(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.68(d,J=8.5Hz,1H,H-1 GalNAc-A ),4.58-4.43(m,8H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA -B ,H-1 GalNAc-C ),4.29(dd,J=11.1,3.7Hz,1H),4.20(d,J=3.1Hz,1H),4.15-4.11(m,4H),4.03-3.89(m,12H),3.83-3.68(m,31 H),3.67-3.54(m,9H),3.40-3.36(m,2H),2.09-2.00(m,18H,COOCH3,COOCH3,COOCH3),1.24(d,J=6.5Hz,6H,CH3 of Fucose).

[0135] 13C NMR(151MHz,D2O)δ175.40,175.06,174.98,174.93,174.67,174.58,104.28,104.10,104.03,100.8 4,99.95,98.28,95.18,91.18,80.70,80.34,79.71,77.32,76.59,76.52,76.38,75.13,74.95,74.91 ,74.20,73.79,73.75,72.49,72.42,72.07,71.57,70.30,69.24,68.51,68.11,67.83,67.60,67.53,66.62,61.50,61.26,61.08,61.04,52.31,52.24,50.88,48.91,22.48,22.45,22.22,21.99,15.35.

[0136] ESI-MS(negative mode)calcd for C 42 H 66 N3O 32 - [MH] - m / z1124.3635,found1124.3399;calcd for C 42 H 65 N3O 32 2- [M-2H] 2- m / z561.6781, found 561.6675.;

[0137] (3) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), chondroitin heptasaccharide (VIII), MgCl2, and Tris-HCl, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, the final concentration of chondroitin heptasaccharide (VIII) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase, reacting for 12 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XI, with a conversion rate of 90%. 1 H NMR, 13 C NMR and MS data are as follows,

[0138] 1H NMR(600MHz,D2O)δ5.39(d,J=4.0Hz,2H,H-1 Fuc ),5.22(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.68(d,J=8.5Hz,1H,H-1 GalNAc-A ),4.58-4.43(m,12H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA -B ,H-1 GalNAc-C ,H-1 GlcA-C ,H-1 GalNAc-D ),4.29(dd,J=11.1,3.8Hz,1H),4.20(d,J=3.1Hz,1H),4.14-4.12(m,6H),4.02-3.88(m,13H),3.84-3.67(m,43H),3.67-3.63(m,2H),3.62-3.54(m,8H),3.41-3.33(m,4H),2.08-2.00(m,24H,COOCH3,COOCH3,COOCH3,COOCH3),1.24(d,J=6.5Hz,6H,CH3 of Fucose).

[0139] 13 C NMR(151MHz,D2O)δ175.38,175.07,175.00,174.98,174.93,174.67,174.50,104.31,104.29,104.11,104.04,100.87,100.81,99.94,98.28,95.18,91.18,80.72,80.32,79.75,79.66,77.30,76.57,76.48,76.32,76.26,75.13,74.95,74.91,74.20,73.79,73.76,73.68,72.48,72.44,72.07,72.04,71.58,70.31,69.24,68.52,68.37,68.11,67.84,67.71,67.64,67.61,67.53,66.61,61.49,61.26,61.07,52.32,52.25,50.97,50.89,48.92,22.45,22.21,22.13,21.99,15.35.

[0140] ESI-MS(negative mode)calcd for C 56 H86 N4O 43 2- [M-2H] 2- m / z751.2338, found751.2213.

[0141] Example 2 Synthesis of Fucosylated Chondroitin Oligosaccharides Catalyzed by Bfα1,3FT

[0142] (1) Guanosine diphosphate fucose (GDP-Fuc), chondroitin tetrasaccharide (V), MgCl2, and Tris-HCl are prepared into an aqueous solution, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, the final concentration of chondroitin tetrasaccharide (V) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, and then L-fucosyltransferase Bfα1,3FT is added, and the reaction is carried out for 12 hours. After the detection reaction is completed, ice ethanol is added to terminate the reaction; the reaction solution is centrifuged for 10-20 minutes, the supernatant is desalted by Bio-Gel P2 molecular sieve, and then separated and purified by strong alkaline anion exchange resin medium, and desalted by Bio-Gel P2 molecular sieve again to obtain product XII, which 1 H NMR, 13 C NMR and MS data are as follows,

[0143] 1 H NMR(600MHz,D2O)δ5.36(d,J=3.9Hz,2H,H-1 Fuc ),5.17(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.63(d,J=8.5Hz,1H,H-1 GalNAc-A ),4.53-4.41(m,6H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA -B ),4.24(dd,J=11.2,3.8Hz,1H),4.17(d,J=3.0Hz,1H),4.11-4.06(m,4H ,),4.03-3.98(m,2H),3.97-3.86(m,6H),3.79(d,J=3.3Hz,2H),3.76-3. 62(m,22H),3.55-3.51(m,4H),3.47-3.41(m,4H),3.28(t,J=8.4Hz,2H),1.99(dd,J=13.5,2.3Hz,12H,COOCH3,COOCH3),1.21(d,J=6.6Hz,6H,CH3 of Fucose).

[0144] 13 C NMR(151MHz,D2O)δ175.85,175.20,175.18,174.83,174.78,174.49,103.96,103.81,99.6 9,98.18,95.04,91.06,80.66,80.52,80.50,77.61,76.48,76.46,76.40,76.08,75.14,74. 84,74.80,74.58,74.11,74.07,72.62,71.93,71.71,70.14,69.06,68.31,67.97,67.59,67.47,66.55,61.47,61.15,60.93,59.17,52.08,50.78,48.70,22.39,22.09,21.87,15.24.

[0145] ESI-MS(negative mode)calcd for C 34 H 53 N2O 27 - [MH] - m / z921.2841, found921.2821.;

[0146] (2) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), chondroitin pentasaccharide (VI), MgCl2, and Tris-HCl, wherein the final concentration of GDP-Fuc in the reaction system is 40 mM, the final concentration of MgCl2 is 20 mM, the final concentration of chondroitin pentasaccharide (VI) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Bfα1,3FT, reacting for 24 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XIII, which 1 H NMR, 13 C NMR and MS data are as follows,

[0147] 1 H NMR(600MHz,D2O)δ5.36(t,J=3.6Hz,4H,,H-1 Fuc-A ,H-1 Fuc-B ),5.18(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.65(d,J=8.5Hz,1H,H-1 GalNAc-A),4.53-4.38(m,8H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA-B ,H-1 GalNAc-C ),4.25(dd,J=11.0,3.9Hz,1H),4.18(d,J=3.1Hz,1H),4.12-4.04(m,4H),4.03-3.97(m,2H),3.97-3.85(m,15H),3.8-3.78(m,4H),3.77-3.65(m,30H),3.62(dd,J=10.7,3.2Hz,2H),3.57-3.50(m,9H),2.05-1.98(m,18H,COOCH3,COOCH3,COOCH3),1.21(dd,J=6.6,1.9Hz,12H,CH3 of Fucose-A,CH3 of Fucose-B).

[0148] 13 C NMR(151MHz,D2O)δ175.33,175.19,175.17,174.95,174.75,174.50,103.96,103.86,103.81,99.83,99.65,98.19,98.15,95.07,91.07,81.05,80.66,77.60,76.47,76.42,75.02,74.85,74.83,74.51,74.12,71.96,71.93,71.44,70.16,69.13,69.08,68.32,68.00,67.61,67.42,67.39,66.56,66.52,61.49,61.41,61.17,60.94,52.13,50.66,48.72,22.39,22.35,22.11,21.88,15.25.

[0149] ESI-MS(negative mode)calcd for C 48 H 76 N3O 36 - [M-H] - m / z 1270.4214,found1270.3972;calcd for C 48 H 75 N3O 36 2- [M-2H] 2- m / z 634.7071,found 634.6962.;

[0150] (3) preparing an aqueous solution of guanosine diphosphate-fucose (GDP-Fuc), chondroitin hexasaccharide (VII), MgCl2, and Tris-HCl, wherein the final concentration of GDP-Fuc in the reaction system is 40 mM, the final concentration of MgCl2 is 20 mM, the final concentration of chondroitin hexasaccharide (VII) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Bfα1,3FT, reacting for 48 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XIV, which 1 H NMR and MS data are as follows:

[0151] 1 H NMR(600MHz,D2O)δ5.40(d,J=4.0Hz,4H,H-1 Fuc-A ,H-1 Fuc-B ),5.22(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.69(d,J=8.5Hz,1H,H-1 GalNAc-A ),4.57-4.44(m,10H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA-B ,H-1 GalNAc-C ,H-1 GlcA-C ),4.29(dd,J=10.7,3.9Hz,1H),4.22(d,J=2.1Hz,1H),4.16-4.12(m,4H),4.10-3.91(m,14H),3.84(s,4H),3.80-3.68(m,38H),3. 60-3.56(m,8H),3.53-3.46(m,4H),3.33(t,J=8.2Hz,2H),2.07-2.01(m,18H,COOCH3,COOCH3,COOCH3),1.25(d,J=6.1Hz,12H,CH3 of Fucose-A,CH3 of Fucose-B).

[0152] ESI-MS(negative mode)calcd for C 54 H 83 N3O 42 2- [M-2H] 2- m / z 722.7231,found722.7092.;

[0153] (4) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), chondroitin heptasaccharide (VIII), MgCl2, and Tris-HCl, wherein the final concentration of GDP-Fuc in the reaction system is 60 mM, the final concentration of MgCl2 is 20 mM, the final concentration of chondroitin heptasaccharide (VIII) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Bfα1,3FT, reacting for 48 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XV, which 1 H NMR and MS data are as follows:

[0154] 1 H NMR(600MHz,D2O)δ5.36(d,J=3.3Hz,6H,H-1 Fuc-A ,H-1 Fuc-B ,H-1 Fuc-C ),5.18(d,J=3.5Hz,1H),4.65(d,J=8.3Hz,1H,H-1 GalNAc-A ),4.53(d,J=8.1Hz,1H,H-1 GalNAc-A ),4.54-4.40(m,12H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA-B ,H-1 GalNAc-C ,H-1 GlcA-C ,H-1 GalNAc-D ),4.25(d,J=11.6Hz,1H),4.18(d,J=2.7Hz,1H),4.12-3.85(m,21H),3.80(m,6H),3.75-3.61(m,53H) ,3.55-3.52(m,14H),2.04-1.99(m,24H,COOCH3,COOCH3,COOCH3,COOCH3),1.21(d,J=6.5Hz,18H,CH3 of Fucose-A,CH3 of Fucose-B,CH3 of Fucose-C).

[0155] ESI-MS(negative mode)calcd for C 68 H 106 N4O 51 2- [M-2H]2- m / z 897.2917, found897.2679.

[0156] Example 3 Main chain extension of fucosylated chondroitin oligosaccharide

[0157] (1) A reaction system of 20 mM uridine diphosphate N-acetylgalactosamine (UDP-GalNAc), 10 mM fucosylated chondroitin oligosaccharide receptor (XII), 20 mM MgCl2, 50 mM Tris-HCl buffer (pH=7.5), and 1 mg / mL chondroitin synthase was reacted at 30°C for 12 h. After the detection reaction was completed, ice ethanol was added to terminate the reaction; the reaction solution was centrifuged for 10-20 min, and separated and purified using a strong alkaline anion exchange resin medium to obtain product XVI, which 1 H NMR, 13 C NMR and MS data are as follows,

[0158] 1 H NMR(600MHz,D2O)δ5.38(d,J=3.9Hz,2H,H-1 Fuc ),5.20(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.66(d,J=8.5Hz,1H,H-1 GalNAc-A ),4.56-4.44(m,8H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA -B ,H-1 GalNAc-C ),4.27(dd,J=11.0,3.8Hz,1H),4.20(d,J=3.1Hz,1H),4.14-4.09(m,2H),4.07-4.02(m,3H),3.98-3.85(m,10H),3.83-3 .66(m,35H),3.58-3.54(m,6H),3.37-3.33(m,2H),2.05-1.98(m,18H,COOCH3,COOCH3,COOCH3),1.23(d,J=6.6Hz,6H,CH3 ofFucose).

[0159] 13C NMR(151MHz,D2O)δ175.21,175.19,174.97,174.87,174.81,174.63,174.52,104.16,103.97,1 03.82,100.87,99.69,98.21,95.08,91.09,80.66,79.47,77.62,76.54,76.27,75.14,74.87,7 4.84,74.56,74.14,74.11,73.55,72.43,71.96,71.00,70.17,69.11,68.34,68.01,67.70,67.62,67.53,66.57,61.49,61.18,60.95,52.22,50.77,48.74,22.42,22.37,22.13,21.91,15.27.

[0160] ESI-MS(negative mode)calcd for C 42 H 66 N3O 32 - [MH] - m / z1124.3635,found1124.3574;calcd for C 42 H 65 N3O 32 2- [M-2H] 2- m / z561.6781, found 561.6755.;

[0161] (2) A reaction system of 20 mM uridine diphosphate N-acetylgalactosamine (UDP-GalNAc), 10 mM fucosylated chondroitin oligosaccharide receptor (XIV), 20 mM MgCl2, 50 mM Tris-HCl buffer (pH = 7.5), and 1 mg / mL chondroitin synthase was reacted at 30°C for 12 h. After the detection reaction was completed, ice ethanol was added to terminate the reaction; the reaction solution was centrifuged for 10-20 min, and separated and purified using a strong alkaline anion exchange resin medium to obtain product XVII, which 1 H NMR and MS data are as follows:

[0162] 1 H NMR(600MHz,D2O)δ5.40(d,J=3.7Hz,4H,H-1 Fuc-A ,H-1 Fuc-B ),5.22(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.69(d,J=8.6Hz,1H,H-1GalNAc-A ),4.59-4.46(m,12H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA-B ,H-1 GalNAc-C ,H-1 GlcA-C ,H-1 GalNAc-D ),4.29(dd,J=11.1,3.8Hz,1H),4.22(d,J=3.1Hz,1H),4.15-3.88(m,24H),3.85-3.69(m,50H),3.61-3.54(m,10H ),3.37(dd,J=9.5,7.9Hz,2H),2.06-2.01(m,24H,COOCH3,COOCH3,COOCH3,COOCH3),1.25(d,J=6.6Hz,12H,CH3of Fucose-A,CH3 of Fucose-B).

[0163] ESI-MS(negative mode)calcd for C 62 H 96 N4O 47 2- [M-2H] 2- m / z824.2628, found824.2469.;

[0164] (3) A reaction system of 20 mM uridine diphosphoglucuronic acid (UDP-GlcA), 10 mM fucosylated chondroitin oligosaccharide receptor (XVI), 20 mM MgCl2, 50 mM Tris-HCl buffer (pH = 7.5), and 1 mg / mL chondroitin synthase was reacted at 30°C for 12 h. After the detection reaction was completed, ice ethanol was added to terminate the reaction; the reaction solution was centrifuged for 10-20 min, and separated and purified using a strong alkaline anion exchange resin medium to obtain product XVIII, which 1 H NMR, 13 C NMR and MS data are as follows,

[0165] 1 H NMR(600MHz,D2O)δ5.38(d,J=4.0Hz,2H,H-1 Fuc ),5.19(d,J=3.8Hz,1H,H-1 GalNAc-A ),4.66(d,J=8.5Hz,1H,H-1 GalNAc-A ),4.54-4.44(m,10H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA -B ,H-1GalNAc-C ,H-1 GlcA-C ),4.26(dd,J=11.1,3.8Hz,1H),4.20-4.18(m,1H),4.15(d,J=3.1Hz,2H),4.13-4.08(m,2H),4.06-4.02(m,2H),4.02-3.89(m,9H),3.82-3.65(m,37H),3.58-3.53(m,6H),3.49-3.43(m,4H),3.36-3.28(m,4H),2.03-1.99(m,18H,COOCH3,COOCH3,COOCH3),1.23(d,J=6.6Hz,6H,CH3 of Fucose).

[0166] 13 C NMR(151MHz,D2O)δ175.79,175.14,175.13,174.87,174.85,174.80,174.50,174.46,104.16,104.05,103.97,103.82,100.73,99.68,98.19,95.07,91.06,80.65,80.21,79.59,77.61,76.49,76.44,76.18,76.17,75.99,75.17,74.88,74.83,74.55,74.12,73.55,72.60,72.37,71.94,71.69,70.16,69.08,68.33,67.99,67.58,67.51,66.57,62.36,61.48,61.17,60.98,52.11,50.91,50.76,48.72,22.39,22.11,21.88,15.25.

[0167] ESI-MS(negative mode)calcd for C 48 H 74 N3O 38 - [M-H] - m / z1300.3956,found;calcd for C 48 H 73 N3O 38 2- [M-2H] 2- m / z649.6942,found 649.6843.;

[0168] (4) A reaction system of 20 mM uridine diphosphoglucuronic acid (UDP-GlcA), 10 mM fucosylated chondroitin oligosaccharide receptor (XVIII), 20 mM MgCl2, 50 mM Tris-HCl buffer (pH = 7.5), and 1 mg / mL chondroitin synthase was reacted at 30°C for 12 h. After the detection reaction was completed, ice ethanol was added to terminate the reaction; the reaction solution was centrifuged for 10-20 min, and separated and purified using a strong alkaline anion exchange resin medium to obtain product XIX, which 1 H NMR, 13 C NMR and MS data are as follows,

[0169] 1 H NMR(600MHz,D2O)δ5.36(d,J=3.5Hz,2H,H-1 Fuc ),5.18(d,J=3.8Hz,1H,H-1 GalNAc-A ),4.64(d,J=8.3Hz,1H,H-1 GalNAc-A ),4.54-4.42(m,12H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA -B ,H-1 GalNAc-C ,H-1 GlcA-C ,H-1 GalNAc-D ),4.25(dd,J=11.1,3.7Hz,1H),4.19-4.14(m,2H),4.12-4.08(m,4H),4.05-3.83(m,15H),3.80-3.65(m,46H),3. 57-3.52(m,8H),3.35-3.31(m,4H),2.03-1.98(m,24H,COOCH3,COOCH3,COOCH3,COOCH3),1.21(d,J=6.6Hz,6H,CH3 of Fucose).

[0170] 13C NMR(151MHz,D2O)δ174.95,174.90,174.86,174.82,174.52,174.37,174.20,104.23,104.17,100.92,100.77,99 .71,98.21,95.09,91.09,80.66,80.60,80.23,79.63,79.46,77.61,76.44,76.06,75.98,75.14,74.85,74.56,7 4.13,74.09,73.56,72.36,71.95,70.97,70.18,69.10,68.34,68.01,67.73,67.69,67.64,67.51,66.58,62.37,61.48,61.18,60.96,60.94,52.23,52.14,50.89,50.77,48.74,45.86,22.40,22.38,22.36,22.13,21.90,15.26.

[0171] ESI-MS(negative mode)calcd for C 56 H 86 N4O 43 2- [M-2H] 2- m / z751.2338, found751.2199.

[0172] Example 4 On-demand fucosylation modification after backbone extension of fucosylated chondroitin oligosaccharide

[0173] (1) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XVI), MgCl2, and Tris-HCl, wherein the final concentration of GDP-Fuc in the reaction system is 20 mM, the final concentration of MgCl2 is 20 mM, the final concentration of fucosylated chondroitin oligosaccharide (XVI) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Hpα1,3FT S45F V30G, reacting for 12 hours, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 minutes, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XIII;

[0174] (2) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XVII), MgCl2, and Tris-HCl, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, 10 mM, and 50 mM, respectively, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Hpα1,3FT S45F V30G, reacting for 12 h, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 min, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XV;

[0175] (3) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XVIII), MgCl2, and Tris-HCl, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, 10 mM, and 50 mM, respectively, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Bfα1,3FT, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 min, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XIV;

[0176] (4) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XIX), MgCl2, and Tris-HCl, wherein the final concentration of GDP-Fuc in the reaction system is 40 mM, the final concentration of MgCl2 is 20 mM, the final concentration of fucosylated chondroitin oligosaccharide (XIX) is 10 mM, the final concentration of Tris-HCl is 50 mM, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Bfα1,3FT, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 min, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XV;

[0177] (5) preparing an aqueous solution of guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XIX), MgCl2, and Tris-HCl, wherein the final concentrations of GDP-Fuc and MgCl2 in the reaction system are 20 mM, 10 mM, and 50 mM, respectively, and the pH of the reaction system is 7.5, then adding L-fucosyltransferase Hpα1,3FT S45F V30G, reacting for 12 h, and after the detection reaction is completed, adding ice ethanol to terminate the reaction; centrifuging the reaction solution for 10-20 min, desalting the supernatant through Bio-Gel P2 molecular sieve, separating and purifying using a strong alkaline anion exchange resin medium, and desalting again using Bio-Gel P2 molecular sieve to obtain product XX, which 1 H NMR and MS data are as follows,

[0178] 1 H NMR(600MHz,D2O)δ5.40(d,J=3.7Hz,4H,H-1 Fuc-A ,H-1 Fuc-B ),5.22(d,J=3.7Hz,1H,H-1 GalNAc-A ),4.69(d,J=8.6Hz,1H,H-1 GalNAc-A ),4.59-4.46(m,12H,H-1 GlcA-A ,H-1 GalNAc-B ,H-1 GlcA-B ,H-1 GalNAc-C ,H-1 GlcA-C ,H-1 GalNAc-D ),4.29(dd,J=11.1,3.8Hz,1H),4.22(d,J=3.1Hz,1H),4.15-3.88(m,24H),3.85-3.69(m,50H),3.61-3.54(m,10H ),3.37(dd,J=9.5,7.9Hz,2H),2.06-2.01(m,24H,COOCH3,COOCH3,COOCH3,COOCH3),1.25(d,J=6.6Hz,12H,CH3of Fucose-A,CH3 of Fucose-B).

[0179] ESI-MS(negative mode)calcd for C 62 H 96 N4O 47 2- [M-2H] 2- m / z 824.2628, found824.2469. Sequence Listing <110> Shandong University <120> A kind of fucosylated chondroitin and its preparation method and application <130> 1 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 996 <212> DNA <213> Artificial Sequence <400> 1 atgtgtgatt gtctgagcat tattctgctg gttaaaatga agaagatcta cctgaagttc 60 gtggacttct gggacggctt tgataccatc agcaatttta tcgtggacgc cctgagcatc 120 cagtatgaag ttgttctgag caatgaaccg gattatctgt tttatagctg cttcggcacc 180 agccatctgg aatatgattg tattaaaatc atgtttatcg gcgagaacat cgtgccggat 240 ttcaatgttt gtgattacgc aattggcttc aactacatcg acttcggcga tcgttatctg 300 cgtctgccgc tgtatgcaat ttatgatggt tttagcaatc tgcagaacaa gaagatcgac 360 gttaataagg cactggatcg taagttctgt agcatcgttg ttagcaataa caagtgggca 420 gatccgattc gtgaaacctt ttttaaactg ctgagcagct ataagaaggt tgacagcggt 480 ggtcgtgcat ggaataatat tggtggtccg gttgataata agctggactt tattagccag 540 tacaagttca acatcgcctt cgagaacagc cgcgttctgg gttataccac cgaaaaaatt 600 atggagccga tgcaggttaa tagcatcccg gtttattggg gtaatccgct ggttggtaaa 660 gattttaatg tggacagctt cgtgaatgcc cacgactttg acagcctgga gcgtctggtt 720 gaatatatta ttgaactgga cagcagcaag gacaagtacc tggaaatgct ggagaagccg 780 tggctgctgg ataaaaccta tctggattgg aaacagctgc tgctgaattt tattaacaac 840 atcatgatga agagctacaa ggacgccaaa tacctggtta actacggtca tgcaggtaaa 900 tatcgtaatg agcagcgttt ttggggtcgt tgtgaacgta aatttaaact gcagcgtatt 960 atcgagtact acagccagct gtttgatcgc aaataa 996 <210> 2 <211> 1120 <212> DNA <213> Artificial Sequence <400> 2 atgtttcagc cgctgttgga tgcgtatgtg gaaagcgcga gcattgaaaa aatggcgagc 60 aaatctccgc cgccgctgaa aattgcgggg gcgaactggt ggggtgatga agaaatcaaa 120 gaattcaaaa acttcgtgct gtattttatt ctgagccagc gctataccat taccctgcat 180 cagaatccga acgaatttag cgatctggtg tttggcaatc cgctgggcag cgcgcgtaaa 240 attctgagct atcagaacgc gaaacgtgtg ttttataccg gcgaaaacga aagcccgaac 300 tttaacctgt ttgattatgc gatcggcttt gatgaactgg atttcaacga tcgttatctg 360 cgtatgccgc tgtattatga tcgtctgcat cataaagcgg aaagcgtgaa cgataccacc 420 gcgccgtata aactgaaaga taacagcctg tacgcgctga aaaaaccgag ccattgcttt 480 aaagaaaaac acccgaacct gtgcgcggtg gtgaacgatg aaagcgatcc gctgaaacgt 540 ggctttgcga gctttgtggc gagcaatccg aacgcgccga ttcgtaacgc gttttatgat 600 gcgctgaaca gcattgaacc ggtgaccggc ggtggcagcg tgcgtaacac cctgggctat 660 aacgtgaaaa acaaaaacga attcctgagc cagtataaat ttaacctgtg cttcgaaaac 720 acccagggct atggctatgt gaccgaaaaa atcatcgatg cgtatttcag ccataccatc 780 ccgatttatt ggggcagccc gagcgtggcg aaagatttca atccgaaaag cttcgtgaac 840 gtgcacgatt tcaaaaactt cgatgaagcg atcgattaca tcaaatatct gcatacccac 900 aaaaacgcgt atctggatat gctgtatgaa aatccgctga acaccctgga tggcaaagcg 960 tatttttatc agaacctgag cttcaaaaaa atcctggcct tcttcaaaac cattctggaa 1020 aacgatacca tctatcacga taatccgttt atcttttgcc gtgatctgaa cgaaccgctg 1080 gtgaccattg atgatctgcg tgttaactac ctcgagcacc 1120 <210> 3 <211> 331 <212> PRT <213> Artificial Sequence <400> 3 Met Cys Asp Cys Leu Ser Ile Ile Leu Leu Val Lys Met Lys Lys Ile 1 5 10 15 Tyr Leu Lys Phe Val Asp Phe Trp Asp Gly Phe Asp Thr Ile Ser Asn 20 25 30 Phe Ile Val Asp Ala Leu Ser Ile Gln Tyr Glu Val Val Leu Ser Asn 35 40 45 Glu Pro Asp Tyr Leu Phe Tyr Ser Cys Phe Gly Thr Ser His Leu Glu 50 55 60 Tyr Asp Cys Ile Lys Ile Met Phe Ile Gly Glu Asn Ile Val Pro Asp 65 70 75 80 Phe Asn Val Cys Asp Tyr Ala Ile Gly Phe Asn Tyr Ile Asp Phe Gly 85 90 95 Asp Arg Tyr Leu Arg Leu Pro Leu Tyr Ala Ile Tyr Asp Gly Phe Ser 100 105 110 Asn Leu Gln Asn Lys Lys Ile Asp Val Asn Lys Ala Leu Asp Arg Lys 115 120 125 Phe Cys Ser Ile Val Val Ser Asn Asn Lys Trp Ala Asp Pro Ile Arg 130 135 140 Glu Thr Phe Phe Lys Leu Leu Ser Ser Tyr Lys Lys Val Asp Ser Gly 145 150 155 160 Gly Arg Ala Trp Asn Asn Ile Gly Gly Pro Val Asp Asn Lys Leu Asp 165 170 175 Phe Ile Ser Gln Tyr Lys Phe Asn Ile Ala Phe Glu Asn Ser Arg Val 180 185 190 Leu Gly Tyr Thr Thr Glu Lys Ile Met Glu Pro Met Gln Val Asn Ser 195 200 205 Ile Pro Val Tyr Trp Gly Asn Pro Leu Val Gly Lys Asp Phe Asn Val 210 215 220 Asp Ser Phe Val Asn Ala His Asp Phe Asp Ser Leu Glu Arg Leu Val 225 230 235 240 Glu Tyr Ile Ile Glu Leu Asp Ser Ser Lys Asp Lys Tyr Leu Glu Met 245 250 255 Leu Glu Lys Pro Trp Leu Leu Asp Lys Thr Tyr Leu Asp Trp Lys Gln 260 265 270 Leu Leu Leu Asn Phe Ile Asn Asn Ile Met Met Lys Ser Tyr Lys Asp 275 280 285 Ala Lys Tyr Leu Val Asn Tyr Gly His Ala Gly Lys Tyr Arg Asn Glu 290 295 300 Gln Arg Phe Trp Gly Arg Cys Glu Arg Lys Phe Lys Leu Gln Arg Ile 305 310 315 320 Ile Glu Tyr Tyr Ser Gln Leu Phe Asp Arg Lys 325 330 <210> 4 <211> 373 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 4 Met Phe Gln Pro Leu Leu Asp Ala Tyr Val Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro Pro Pro Leu Lys Ile Ala Gly Ala Asn 20 25 30 Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Asn Phe Val Leu Tyr 35 40 45 Phe Has Been Gln Arg Tyr Thr Thr Has Gln Asn Pro Asn 50 55 60 Glu Phe Ser Asp Leu Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys 65 70 75 80 Tyr Gln Asn Is Contained With Lys Arg Val Phe Tyr Gly Glu Asn 85 90 95 Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110 Leu Asp Phe Asn Asp Arg Tyr Leu Arg Pro Leu Tyr Tyr Asp Arg 115 120 125 Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys 130 135 140 Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Pro Ser His Cys Phe 145 150 155 160 Lys Glu Lys His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp 165 170 175 Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala 180 185 190 Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val 195 200 205 Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn 210 215 220 Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn 225 230 235 240 Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp Ala Tyr Phe 245 250 255 Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp 260 265 270 Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Lys Asn Phe Asp 275 280 285 Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Lys Asn Ala Tyr 290 295 300 Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala 305 310 315 320 Tyr Phe Tyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Ala Phe Phe Lys 325 330 335 Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asp Asn Pro Phe Ile Phe 340 345 350 Cys Arg Asp Leu Asn Glu Pro Leu Val Thr Ile Asp Asp Leu Arg Val 355 360 365 Asn Tyr Leu Glu His 370

Claims

1. A method for preparing fucosylated chondroitin, the method comprising the following steps: The L-fucosyltransferase is mixed with the substrate GDP-L-fucose and chondroitin oligosaccharide to undergo an enzymatic reaction to obtain fucosylated chondroitin; The L-fucosyltransferase is Hpα1,3FT S45F V30G; The nucleotide sequence of the L-fucosyltransferase Hpα1,3FT S45F V30G is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 4; The preparation method further comprises the following steps: using chondroitin oligosaccharide, and performing single-site fucosylation modification at the O-3 position of GlcA with a non-reducing end structure of D-GalNAc1-4-β-D-GlcA using L-fucosyltransferase Hpα1,3FT S45F V30G as the catalysis; The GDP-L-fucose is guanosine diphosphate fucose; The chondroitin oligosaccharide is selected from the following structures: The fucosylated chondroitin is selected from the following structures: 。 2. The preparation method according to claim 1, characterized in that The preparation method further comprises the following steps: when the non-reducing end of the fucosylated chondroitin oligosaccharide is greater than or equal to two sugars that are not fucosylated, firstly, the main chain of the fucosylated chondroitin is extended by catalysis of chondroitin synthase PmCS, and then further fucosylated by L-fucosyltransferase Hpα1,3FT S45F V30G at the GlcAO-3 position of the structure D-GalNAc1-4-β-D-GlcA.

3. The preparation method according to claim 1, characterized in that: The molar ratio of fucosylated GlcA to GDP-L-fucose in the chondroitin oligosaccharide is 0.5-2:1-4.

4. The preparation method according to claim 1, characterized in that: The preparation method of chondroitin oligosaccharide comprises the following steps: mixing chondroitin synthase with a donor substrate and an acceptor substrate, and reacting them to obtain chondroitin oligosaccharide.

5. The preparation method according to claim 4, characterized in that: The donor substrate is selected from: UDP-GalNAc, UDP-GlcA.

6. The preparation method according to claim 4, characterized in that: The receptor substrate is selected from: GalNAc.

7. The preparation method according to claim 4, characterized in that: The molar ratio of the acceptor substrate to the donor substrate is 0.5-2:1-5.

8. The preparation method according to claim 4, characterized in that: The concentration of the chondroitin synthase is 0.1-2 mg / mL.

9. The preparation method according to claim 1, characterized in that: The method further comprises a step of separating.

10. The preparation method according to claim 1, characterized in that: The method further comprises a purification step.

11. A fucosyltransferase, characterized in that The amino acid sequence of the fucosyltransferase is shown in SEQ ID NO:4.

Images