CN115058465A - Fucosylated chondroitin and preparation method and application thereof - Google Patents

Fucosylated chondroitin and preparation method and application thereof Download PDF

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CN115058465A
CN115058465A CN202210763300.XA CN202210763300A CN115058465A CN 115058465 A CN115058465 A CN 115058465A CN 202210763300 A CN202210763300 A CN 202210763300A CN 115058465 A CN115058465 A CN 115058465A
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chondroitin
glca
fucosyltransferase
fucosylated
galnac
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房俊强
杨林
李爽
凌沛学
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Shandong University
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Abstract

The invention discloses fucosylated chondroitin and a preparation method and application thereof. The invention utilizes the substrate universality of enzyme, screens fucosyltransferase from different sources for substrate adaptability research, constructs a multi-enzyme cascade catalytic reaction system, develops a high-efficiency method for synthesizing fucosylated chondroitin by an enzyme method, can realize fucosylation modification of different degrees at different sites of chondroitin sugar chains by adopting the method, lays a foundation for the industrial production of fucosylated chondroitin sulfate, and promotes the development of carbohydrate anticoagulant drugs.

Description

Fucosylated chondroitin and preparation method and application thereof
Technical Field
The invention relates to the field of enzyme engineering technology and synthetic biology, in particular to fucosylated chondroitin and a preparation method and application thereof.
Background
With the general improvement of the living standard of human beings and the acceleration of the aging process of the population, the increasing number of thrombotic diseases becomes a problem which seriously affects the life health of human beings. Heparin and Low Molecular Weight Heparin (LMWHs) are widely used as anticoagulants in the treatment of thrombotic diseases. Heparin and low molecular heparin risk severe bleeding during treatment and cannot be taken orally. Therefore, researchers continue to search for more effective and safer anticoagulant drugs. Fucosylated chondroitin sulfate (FuCS) is an active polysaccharide with a unique structure separated from sea cucumber, has obvious anticoagulation and antithrombotic activities, can be used as a potential candidate drug, and has been a focus of domestic and foreign scientists for attention in synthesis preparation and biological function research. However, further structure-activity relationship studies are hampered by the lack of homogeneous oligosaccharides.
Currently, two methods, namely sea cucumber extraction and chemical synthesis, are mainly used for obtaining FuCS. The extraction method is limited by the quality and source of the sea cucumber, FuCS sugar chains with clear structures cannot be obtained, and the structure-activity relationship and biological research are limited (such as ACS Chem Biol 2020,15(8),2232-2246, or CarbohydrylPolym 2017,165,7-12, or CarbohydrylPolym 2021,262, 117969); the chemical synthesis of FuCS has been developed greatly in recent years, but the problems of complicated steps, low yield and the like still exist due to the influence of sugar chain complexity, and the economic and high-efficiency synthesis cannot be realized (such as Angew Chem Int Ed Engl 2018,57(39), 12880-.
The FuCS sugar chain structure is composed of a sulfated and modified disaccharide repeating unit and a GlcA O-3 branch modified fucose residue, wherein the disaccharide repeating unit is mainly composed of uronic acid (glucuronic acid GlcA) and N-acetylgalactosamine (GalNAc) which are connected through a beta glycosidic bond. FuCS from different species has differences in the number of sulfation sites and fucosylation branches, and no report on enzymatic synthesis of FuCS oligosaccharide fragments exists in the prior art, so that the invention is provided.
Disclosure of Invention
The invention overcomes the defects in the prior art and provides fucosylated chondroitin and a preparation method and application thereof.
In a first aspect of the present invention, there is provided a method for preparing fucosylated chondroitin, comprising the steps of:
mixing L-fucosyltransferase with substrate GDP-L-fucose and chondroitin oligosaccharide, and performing enzymatic reaction to obtain fucosylated chondroitin.
Further, 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 (e.g., 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1: 3.5).
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.
Further, the pH of the reaction is 6.5 to 8.5, preferably, the pH of the reaction is 7 to 8, more preferably, the pH of the reaction is 7.5.
Further, the temperature of the reaction is 30-45 ℃, preferably, the temperature of the reaction is 35-40 ℃, and more preferably, the temperature of the reaction is 37 ℃.
Further, the reaction time is 10-30h, preferably 10-25h, and more preferably 12-24 h.
Further, the concentration of said L-fucosyltransferase is 0.1-2mg/mL, preferably, the concentration of said L-fucosyltransferase is 0.5-1.5mg/mL, more preferably, the concentration of said L-fucosyltransferase is 1 mg/mL.
Further, the chondroitin oligosaccharide has the following structure:
Figure BDA0003724726220000021
further, said n 1 Is an integer selected from 1-10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10).
Preferably, n is 1 Is an integer selected from 1 to 5, more preferablyOptionally, n is 1 Is an integer selected from 1 to 3.
In one embodiment of the present invention, the chondroitin oligosaccharide is selected from the following structures:
Figure BDA0003724726220000022
Figure BDA0003724726220000031
further, the nucleotide sequence of the L-fucosyltransferase is shown as SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 4 is shown in the specification;
further, the nucleotide sequence of the L-fucosyltransferase Bf alpha 1,3FT is shown in SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification;
further, the nucleotide sequence of the L-fucosyltransferase Hp alpha 1,3FT S45F V30G is shown in SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 4, respectively.
Further, the L-fucosyltransferase is selected from one of alpha-1, 2-fucosyltransferase or alpha-1, 3-fucosyltransferase.
Preferably, the L-fucosyltransferase is an alpha-1, 3-fucosyltransferase.
Preferably, said L-fucosyltransferase is selected from the group consisting of: SEQ ID NO: 3-4, a functional variant of the polypeptide represented by any one of SEQ ID NOs 3-4, SEQ ID NOs: 3-4 and a functional fragment of a polypeptide represented by any one of SEQ ID NOs: 3-4.
Further, the sources of the L-fucosyltransferase are: one of Bacteroides fragilis (Bacteroides fragilis), Helicobacter pylori (Helicobacter pylori), vibrio cholerae (vibrio cholera), Helicobacter cholangii (h. bilis), Helicobacter cinnabarini (h. cinaede), Bacteroides ovatus (Bacteroides ovatus) and Bacteroides vulgares, preferably, the source of the L-fucosyltransferase is Bacteroides fragilis (Bacteroides fragilis) and/or Helicobacter pylori (Helicobacter pylori).
In one embodiment of the invention, the L-fucosyltransferase is Bf α 1,3FT (from Bacteroides fragilis).
In one embodiment of the invention, the L-fucosyltransferase is Hp α 1,3FT (from Helicobacter pylori).
In one embodiment of the invention, the L-fucosyltransferase is a Hp α 1,3FT S45F V30G mutant with improved catalytic activity.
Bf alpha 1,3FT can recognize all D-GalNAc 1-4-beta-D-GlcA disaccharide units in the oligosaccharide and carry out fucosylation modification at GlcA O-3 position, and Hp alpha 1,3FT can only recognize the oligosaccharide with the non-reducing end of D-GalNAc 1-4-beta-D-GlcA structure and carry out fucosylation modification at unit site at GlcA O-3 position of the non-reducing end. When Bf alpha 1,3FT catalyzes and identifies the oligosaccharide with the non-reducing end of D-GlcA 1-3-beta-D-GalNAc structure, the non-reducing end is more than or equal to two sugars which are not fucosylated and can be continuously extended by the catalysis of chondroitin synthetase, and fucosylation modification is further carried out at a specific position as required.
In one embodiment of the present invention, the chondroitin oligosaccharide recognized by the Hp α 1,3FT S45F V30G mutant has a structure shown in formula (I):
Figure BDA0003724726220000041
in one embodiment of the present invention, the structures of chondroitin oligosaccharides recognizable by the Hp α 1,3FT S45F V30G mutant are respectively represented by formulas (IV), (VI) and (viii):
Figure BDA0003724726220000042
in one embodiment of the invention, the structure of the single site fucosylation modified chondroitin oligosaccharide catalytically synthesized by the Hp alpha 1,3FT S45F V30G mutant is shown as formulas (IX), (X) and (XI):
Figure BDA0003724726220000051
in one embodiment of the present invention, the chondroitin oligosaccharide recognized by Bf α 1,3FT has the following structures respectively represented by formulas (I) and (II):
Figure BDA0003724726220000052
in one embodiment of the present invention, the chondroitin oligosaccharide recognized by Bf α 1,3FT has a structure represented by formulas (IV), (V), (VI), (VII), and (VIII), respectively:
Figure BDA0003724726220000053
Figure BDA0003724726220000061
in a specific embodiment of the invention, the structures of the fucosylated chondroitin oligosaccharide catalytically synthesized by Bf alpha 1,3FT are respectively shown in formulas (IX), (XII), (XIII), (XIV) and (XV):
Figure BDA0003724726220000062
Figure BDA0003724726220000071
further, the preparation method also comprises the steps of separation and purification.
Further, when the non-reducing end of the fucosylated chondroitin oligosaccharide is not less than two sugars and is not fucosylated, the chondroitin synthetase PmCS can catalyze the further extension of the main chain, and can catalyze the further fucosylation modification by Bf alpha 1,3FT or Hp alpha 1,3FTS45F V30G at a specific position according to requirements, so as to synthesize the chondroitin oligosaccharide with determined structure and fucosylation degree.
Further, the L-fucosyltransferase Hp alpha 1,3FT S45F V30G catalyzes the non-reducing end structure of D-GalNAc 1-4-beta-D-GlcA to carry out single-site fucosylation modification at the GlcA O-3 position.
Further, the L-fucosyltransferase Bf alpha 1,3FT has a catalytic structure of D-GalNAc 1-4-beta-D-GlcA, and all GlcA O-3 positions of the catalytic structure are subjected to fucosylation modification. Further, the step of extending the fucosylated chondroitin backbone comprises mixing the backbone with chondroitin synthase PmCS, sugar nucleotides, and reacting.
Further, the sugar nucleotide is selected from the group consisting of: UDP-GalNAc, UDP-GlcA.
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: the backbone of fucosylated chondroitin is first extended using chondroitin synthase and then fucosylation modifications at specific sites are catalyzed using L-fucosyltransferases Hp α 1,3FT S45F V30G or Bf α 1,3 FT.
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: firstly, a main chain of fucosylated chondroitin is extended by using chondroitin synthetase, and then the GlcA O-3 position of a non-reducing end structure D-GalNAc 1-4-beta-D-GlcA is catalyzed by using L-fucosyltransferase Hp alpha 1,3FT S45F V30G to further carry out single-site fucosylation modification.
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: the backbone of fucosylated chondroitin is first extended using chondroitin synthase and then further fucosylated modification is catalyzed at all GlcA O-3 positions with the structure D-GalNAc1-4- β -D-GlcA using L-fucosyltransferase Bf α 1,3 FT.
In one embodiment of the present invention, the structure of the fucosylated chondroitin backbone further extendable by chondroitin synthase PmCS is shown in (xii), (XIV), respectively:
Figure BDA0003724726220000081
in one embodiment of the present invention, the structures of the fucosylated chondroitin backbone after extension are respectively shown as (XVI), (XVII), (XVIII), (XIX):
Figure BDA0003724726220000082
Figure BDA0003724726220000091
in one embodiment of the present invention, the method for extending the fucosylated chondroitin backbone using a chondroitin synthase comprises the steps of: fucosylated chondroitin (XVI) is synthesized by further performing fucosylation modification by catalyzing fucosylated chondroitin (XII) with chondroitin synthase PmCS.
In one embodiment of the present invention, the method for extending the fucosylated chondroitin backbone using a chondroitin synthase comprises the steps of: fucosylation modification was further performed using chondroitin synthase PmCS to catalyze fucosylation chondroitin (XVI), and fucosylation chondroitin (XVIII) was synthesized.
In one embodiment of the present invention, the method for extending the fucosylated chondroitin backbone using a chondroitin synthase comprises the steps of: fucosylated chondroitin (XIX) is synthesized by further performing fucosylation modification by using chondroitin synthetase PmCS to catalyze fucosylated chondroitin (XVIII).
In one embodiment of the present invention, the method for extending the fucosylated chondroitin backbone using a chondroitin synthase comprises the steps of: fucosylation modification was further performed using chondroitin synthase PmCS to catalyze fucosylation chondroitin (XIV), and fucosylation chondroitin (XVII) was synthesized.
Further, the L-fucosyltransferase Hp alpha 1,3FTS45F V30G can recognize the extended fucosylated chondroitin and catalyze the further fucosylation modification.
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: fucosylation modification was further performed using L-fucosyltransferase Hp α 1,3FTS45F V30G to catalyze fucosylation chondroitin (xvi) to synthesize fucosylation chondroitin (xiii).
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: fucosylation modification was further performed using L-fucosyltransferase Hp α 1,3FTS45F V30G to catalyze fucosylation chondroitin (XVII) to synthesize fucosylation chondroitin (XV).
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: fucosylation modification was further performed using L-fucosyltransferase Hp α 1,3FTS45F V30G to catalyze fucosylation chondroitin (xix), synthesizing fucosylation chondroitin (XX).
Further, the L-fucosyltransferase Bf alpha 1,3FT can recognize the extended fucosylated chondroitin and catalyze the further fucosylation modification.
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: fucosylation modification was further performed using L-fucosyltransferase Bf α 1,3FT to catalyze fucosylation chondroitin (xviii) to synthesize fucosylation chondroitin (XIV).
In one embodiment of the present invention, the method for preparing fucosylated chondroitin comprises the following steps: fucosylation modification was further performed using L-fucosyltransferase Bf α 1,3FT to catalyze fucosylation chondroitin (XIX), resulting in synthesis of fucosylation chondroitin (XV).
Further, the preparation method of the chondroitin oligosaccharide comprises the following steps: mixing chondroitin synthetase with a donor substrate and an acceptor substrate, and reacting to obtain chondroitin oligosaccharide.
Further, the donor substrate (i.e. sugar nucleotide) is selected from: UDP-GalNAc, UDP-GlcA.
Further, the acceptor substrate is selected from the group consisting of: GalNAc.
The UDP-GalNAc provided by the invention: chinese is called UDP-N-acetylgalactosamine, and its function is to provide activated acetylgalactosamine donor (donor substrate) for the synthesis of chondroitin oligosaccharide or chondroitin polysaccharide.
The UDP-GlcA: chinese is collectively called UDP-glucuronic acid, and the role of the Chinese is to provide an activated glucuronic acid donor (donor substrate) for the synthesis of chondroitin oligosaccharide or chondroitin polysaccharide.
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 (e.g., 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 (e.g., 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1: 5).
In one embodiment of the invention, the molar ratio of chondroitin oligosaccharide receptor to sugar nucleotide is 1:1.5-1: 2.
Further, the chondroitin synthase is derived from: pasteurella multocida (Pasteurella multocida), Escherichia coli (Escherichia coli), Actinomyces urensis (Actinobacillus ureae).
Further, the method may further comprise the step of separating.
Further, said isolating is selected from: ion exchange resins, molecular sieves, 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 one of gel filtration and ligand exchange chromatography.
Further, the molecular sieve is selected from: polyacrylamide molecular sieves (e.g., Bio-Gel P2 to P300), agarose molecular sieves, dextran molecular sieves.
Further, the ion exchange resin is anion exchange resin, the anion exchange resin is strong base anion exchange resin or weak base anion exchange resin, preferably, the anion exchange resin is strong base anion exchange resin.
Further, the strong-base anion exchange resin is gel-type strong-base anion exchange resin.
In one embodiment of the invention, the separation is an anion exchange resin.
In one embodiment of the invention, the separation is a strongly basic anion exchange resin.
In one embodiment of the invention, the separation is a molecular sieve.
In one embodiment of the invention, the separation is a polyacrylamide molecular sieve.
In one embodiment of the invention, the separation is a Bio-Gel P2 molecular sieve.
Further, the method also comprises a purification step.
Further, the purification comprises a step of washing the chondroitin oligosaccharides using a suitably diluted solution of a base, a salt, a buffer solution or an acid.
Further, the purification may further comprise the steps of using activated charcoal or carbon, using charcoal, nanofiltration, ultrafiltration or ion exchange, using alcohol, using an aqueous alcohol mixture for crystallization, evaporation, concentration, precipitation, and drying.
Further, the concentration is selected from: atmospheric concentration, reduced pressure concentration, film concentration and multi-effect concentration, wherein the drying is selected from the following steps: freeze drying, drying under normal pressure, drying under reduced pressure, boiling drying, spray drying, infrared drying, and microwave drying.
In one embodiment of the invention, the drying is freeze-drying.
Further, the fucosylated chondroitin has the following structure:
Figure BDA0003724726220000111
in one embodiment of the present invention, the fucosylated chondroitin has the following structure:
Figure BDA0003724726220000112
Figure BDA0003724726220000121
Figure BDA0003724726220000131
a second aspect of the invention provides a fucosylated chondroitin having the structure:
Figure BDA0003724726220000132
in one embodiment of the present invention, the fucosylated chondroitin has the following structure:
Figure BDA0003724726220000133
Figure BDA0003724726220000141
Figure BDA0003724726220000151
in a third aspect of the invention, there are provided two fucosyltransferases, the nucleotide sequences of which are set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 4, respectively.
A fourth aspect of the present invention provides a recombinant vector comprising a plasmid vector having inserted therein the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and (3) constructing the nucleotide sequence of the fucosyltransferase shown in the figure.
Further, the vector is a viral vector or a non-viral vector. In one embodiment of the invention, the vector is a plasmid vector.
In one embodiment of the invention, the carrier is PMAL-C2X.
In one embodiment of the invention, the vector is pET-41 a.
In a fifth aspect of the invention, a recombinant cell is provided.
Further, the recombinant cell is selected from the group consisting of: gram-positive bacteria, gram-negative bacteria, actinomycetes, fungi;
preferably, said gram-positive bacterium is selected from the group consisting of: b, bacillus subtilis;
preferably, said gram-negative bacteria are selected from the group consisting of: e.coli; the actinomycete is streptomycete;
preferably, the fungus is yeast or aspergillus.
A sixth aspect of the invention provides a pharmaceutical composition comprising fucosylated chondroitin as described in the second aspect.
Further, the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.
Further, the pharmaceutically acceptable adjuvant is selected from one or more of carrier, excipient, diluent, lubricant, wetting agent, emulsifier, preservative, antioxidant, buffer, bacteriostatic agent, solute for making the preparation isotonic with blood of recipient, suspending agent, solubilizer, thickener, stabilizer, sweetener and perfume.
A seventh aspect of the invention provides a use of the fucosylated chondroitin of the second aspect or the pharmaceutical composition of the sixth aspect for the preparation of an anticoagulant medicament or a fucosylated chondroitin sulfate.
An eighth aspect of the invention provides a use of the fucosyltransferase of the third aspect in the synthesis of fucosylated chondroitin.
The ninth aspect of the present invention provides the use of a fucosylated chondroitin as described in the second aspect for preparing a fucosylated chondroitin sulfate.
The biological enzymes (fucosyltransferase and chondroitin synthetase) are from prokaryotes and have the advantages of high protein expression level, wide substrate adaptability, high catalytic efficiency and the like. The method for synthesizing the fucosylated chondroitin by the enzyme method has the advantages that the synthetic process by the enzyme method has high efficiency and high regioselectivity, the operation is simple and safe, the foundation is laid for the industrial production of the fucosylated chondroitin sulfate, the bottleneck problem of the synthesis of the fucosylated chondroitin by the enzyme method is solved, the method for preparing the fucosylated chondroitin by the in vitro multi-enzyme cascade is provided, the fucosyltransferase from different sources is screened for carrying out substrate adaptability research by utilizing the substrate universality of the enzyme, the efficient method for synthesizing the fucosylated chondroitin by the enzyme method is developed, the fucosylation modification of different degrees can be carried out on the substrate chondroitin oligosaccharide by adopting the method, the non-reducing end of the oligosaccharide lays the foundation for the industrial production of the fucosylated chondroitin sulfate, and the development of an anticoagulant drug is promoted.
Drawings
FIG. 1 is a schematic diagram of the preparation of fucosylated chondroitin.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following examples are given in detail for the purpose of better understanding the contents of the present invention and are not intended to limit the scope of the present invention.
The structures of formulas (IX) to (XX) described in the present invention are respectively as follows:
Figure BDA0003724726220000171
Figure BDA0003724726220000181
Figure BDA0003724726220000191
example 1Hp alpha 1,3FT S45F V30G catalytic Synthesis of Single site fucosylation modified chondroitin oligosaccharide
(1) Mixing guanosine diphosphate fucose (GDP-Fuc), chondroitin trisaccharide (IV) and MgCl 2 Tris-HCl is prepared into water solution, and GDP-Fuc and MgCl are contained in a reaction system 2 The final concentration of chondroitin trisaccharide (IV) is 20mM, the final concentration of chondroitin trisaccharide (IV) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase is added for reaction for 12 hours, and after the detection reaction is finished, glacial ethanol is added for stopping the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product IX with conversion rate of 90%, and collecting the product IX 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)δ5.40(d,J＝4.0Hz,2H,H-1 Fuc ),5.22(d,J＝3.8Hz,1H, H-1 GalNAc-A ),4.69(d,J＝8.5Hz,1H,H-1 GalNAc-A ),4.57(d,J＝8.0Hz,1H),4.52(d,J＝7.9Hz, 1H),4.46(dd,J＝8.4,2.5Hz,2H),4.29(dd,J＝11.1,3.8Hz,1H),4.22(d,J＝3.1Hz,1H), 4.16-4.11(m,2H),4.02-3.89(m,11H),3.83(d,J＝3.3Hz,2H),3.81-3.64(m,19H),3.61-3.56(m, 4H),2.05(dd,J＝26.0,2.3Hz,12H,COOCH 3 ,COOCH 3 ),1.25(d,J＝6.6Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)δ175.42,175.40,175.06,174.92,174.62,104.01,103.85,100.00, 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.
ESI-MS(negative mode)calcd for C 28 H 45 N 2 O 21 - [M-H] - m/z745.2520,found 745.2527.；
(2) preparing guanosine diphosphate fucose (GDP-Fuc), chondroitin pentasaccharide (VI), MgCl2 and Tris-HCl into an aqueous solution, and preparing the GDP-Fuc and the MgCl into the reaction system 2 The final concentration of the compound is 20mM, the final concentration of chondroitin pentasaccharide (VI) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase is added for reaction for 12 hours, and after the detection reaction is finished, glacial ethanol is added for stopping the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product X with conversion rate of 86%, and collecting the product X 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,31H),3.67-3.54(m,9H),3.40-3.36(m,2H),2.09-2.00(m,18H, COOCH 3 ,COOCH 3 ,COOCH 3 ),1.24(d,J＝6.5Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)δ175.40,175.06,174.98,174.93,174.67,174.58,104.28,104.10, 104.03,100.84,99.95,98.28,95.18,91.18,80.70,80.34,79.71,77.32,76.59,76.52,76.38,75.13, 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.
ESI-MS(negative mode)calcd for C 42 H 66 N 3 O 32 - [M-H] - m/z1124.3635,found 1124.3399； calcd for C 42 H 65 N 3 O 32 2- [M-2H] 2- m/z561.6781,found 561.6675.；
(3) preparing guanosine diphosphate fucose (GDP-Fuc), chondroitin heptasaccharide (VIII), MgCl2 and Tris-HCl into an aqueous solution, and preparing the GDP-Fuc, the MgCl and the MgCl into a reaction system 2 The final concentration of the compound is 20mM, the final concentration of chondroitin heptasaccharide (VIII) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase is added for reaction for 12 hours, and after the detection reaction is finished, glacial ethanol is added for stopping the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product XI with conversion rate of 90%, and collecting the product XI 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.24(d,J＝ 6.5Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)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.
ESI-MS(negative mode)calcd for C 56 H 86 N 4 O 43 2- [M-2H] 2- m/z751.2338,found 751.2213.。
example 2Bf alpha 1,3FT catalyzed Synthesis of fucosylated modified chondroitin oligosaccharides
(1) Mixing guanosine diphosphate fucose (GDP-Fuc), chondroitin tetrasaccharide (V), MgCl 2 Tris-HCl is prepared into water solution, and GDP-Fuc and MgCl are contained in a reaction system 2 The final concentration of the protein is 20mM, the final concentration of chondroitin tetrasaccharide (V) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase Bf alpha 1 and 3FT are added for reaction for 12 hours, and after the detection reaction is finished, the glacial ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product XII, which is purified with ethanol 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ), 1.21(d,J＝6.6Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)δ175.85,175.20,175.18,174.83,174.78,174.49,103.96,103.81, 99.69,98.18,95.04,91.06,80.66,80.52,80.50,77.61,76.48,76.46,76.40,76.08,75.14,74.84, 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.
ESI-MS(negative mode)calcd for C 34 H 53 N 2 O 27 - [M-H] - m/z921.2841,found 921.2821.；
(2) mixing guanosine diphosphate fucose (GDP-Fuc), chondroitin pentasaccharide (VI) and MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 40mM, and MgCl is adopted 2 The final concentration of the compound is 20mM, the final concentration of chondroitin pentasaccharide (VI) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase Bf alpha 1,3FT is added for reaction for 24 hours, and after the detection reaction is finished, the glacial ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product XIII, which is 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.21(dd,J＝ 6.6,1.9Hz,12H,CH 3 of Fucose-A,CH 3 of Fucose-B).
13 C NMR(151MHz,D 2 O)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.
ESI-MS(negative mode)calcd for C 48 H 76 N 3 O 36 - [M-H] - m/z 1270.4214,found 1270.3972； calcd for C 48 H 75 N 3 O 36 2- [M-2H] 2- m/z 634.7071,found 634.6962.；
(3) mixing guanosine diphosphate fucose (GDP-Fuc), chondroitin hexasaccharide (VII), and MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 40mM, and MgCl is adopted 2 The final concentration of the compound is 20mM, the final concentration of chondroitin hexasaccharide (VII) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase Bf alpha 1,3FT is added for reaction for 48 hours, and after the detection reaction is finished, glacial ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product XIV, which is a product of XIV 1 The H NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ), 1.25(d,J＝6.1Hz,12H,CH 3 of Fucose-A,CH 3 of Fucose-B).
ESI-MS(negative mode)calcd for C 54 H 83 N 3 O 42 2- [M-2H] 2- m/z 722.7231,found 722.7092.；
(4) mixing guanosine diphosphate fucose (GDP-Fuc), chondroitin heptasaccharide (VIII), and MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 60mM, and MgCl is adopted 2 Has a final concentration of 20mM, chondroitin heptasaccharide (VIII) at a final concentration of 10mM, Tris-HClThe final concentration is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase Bf alpha 1 and 3FT are added for reaction for 48 hours, and after the detection reaction is finished, glacial ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain XV, which is the product 1 The H NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.21(d,J＝6.5Hz,18H, CH 3 of Fucose-A,CH 3 of Fucose-B,CH 3 of Fucose-C).
ESI-MS(negative mode)calcd for C 68 H 106 N 4 O 51 2- [M-2H] 2- m/z 897.2917,found 897.2679.。
example 3 backbone extension of fucosylated chondroitin oligosaccharides
(1)20mM uridine diphosphate N-acetylgalactosamine (UDP-GalNAc), 10mM fucosylated chondroitin oligosaccharide receptor (XII), 20mM MgCl 2 Reacting the mixture at 30 ℃ for 12 hours in a reaction system of 50mM Tris-HCl buffer solution (pH 7.5) and 1mg/mL chondroitin synthetase, and adding glacial ethanol to stop the reaction after the detection reaction is finished; centrifuging the reaction solution for 10-20min, and separating and purifying with strongly basic anion exchange resin medium to obtain product XVI 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.23(d,J＝6.6Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)δ175.21,175.19,174.97,174.87,174.81,174.63,174.52,104.16, 103.97,103.82,100.87,99.69,98.21,95.08,91.09,80.66,79.47,77.62,76.54,76.27,75.14,74.87, 74.84,74.56,74.14,74.11,73.55,72.43,71.96,71.00,70.17,69.11,68.34,68.01,67.70,67.62, 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.
ESI-MS(negative mode)calcd for C 42 H 66 N 3 O 32 - [M-H] - m/z1124.3635,found 1124.3574； calcd for C 42 H 65 N 3 O 32 2- [M-2H] 2- m/z561.6781,found 561.6755.；
(2)20mM uridine diphosphate N-acetylgalactosamine (UDP-GalNAc), 10mM fucosylated chondroitin oligosaccharide receptor (XIV), 20mM MgCl 2 Reacting the mixture at 30 ℃ for 12 hours in a reaction system of 50mM Tris-HCl buffer solution (pH 7.5) and 1mg/mL chondroitin synthetase, and adding glacial ethanol to stop the reaction after the detection reaction is finished; centrifuging the reaction solution for 10-20min, and separating and purifying with strongly basic anion exchange resin medium to obtain product XVII 1 The H NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.25(d,J＝6.6Hz,12H,CH 3 of Fucose-A,CH 3 of Fucose-B).
ESI-MS(negative mode)calcd for C 62 H 96 N 4 O 47 2- [M-2H] 2- m/z824.2628,found 824.2469.；
(3)20mM uridine diphosphate glucuronate (UDP-GlcA), 10mM fucosylated chondroitin oligosaccharide receptor (XVI), 20mM MgCl 2 Reacting the mixture at 30 ℃ for 12 hours in a reaction system of 50mM Tris-HCl buffer solution (pH 7.5) and 1mg/mL chondroitin synthetase, and adding glacial ethanol to stop the reaction after the detection reaction is finished; centrifuging the reaction solution for 10-20min, and separating and purifying with strongly basic anion exchange resin medium to obtain product XVIII 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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-1 GalNAc-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,COOCH 3 ,COOCH 3 , COOCH 3 ),1.23(d,J＝6.6Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)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.
ESI-MS(negative mode)calcd for C 48 H 74 N 3 O 38 - [M-H] - m/z1300.3956,found；calcd for C 48 H 73 N 3 O 38 2- [M-2H] 2- m/z649.6942,found 649.6843.；
(4)20mM uridine diphosphate glucuronate (UDP-GlcA), 10mM fucosylated chondroitin oligosaccharide receptor (XVIII), 20mM MgCl 2 Reacting the mixture at 30 ℃ for 12 hours in a reaction system of 50mM Tris-HCl buffer solution (pH 7.5) and 1mg/mL chondroitin synthetase, and adding glacial ethanol to stop the reaction after the detection reaction is finished; centrifuging the reaction solution for 10-20min, separating and purifying with strongly basic anion exchange resin medium to obtain product XIX, which is 1 H NMR、 13 The C NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.21(d,J＝6.6Hz,6H,CH 3 of Fucose).
13 C NMR(151MHz,D 2 O)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, 76.06,75.98,75.14,74.85,74.56,74.13,74.09,73.56,72.36,71.95,70.97,70.18,69.10,68.34, 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.
ESI-MS(negative mode)calcd for C 56 H 86 N 4 O 43 2- [M-2H] 2- m/z751.2338,found 751.2199.。
example 4 on-demand fucosylation modification after extension of fucosylation chondroitin oligosaccharide backbone
(1) Mixing guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XVI), and MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 20mM, MgCl 2 The final concentration of (A) is 20mM, the final concentration of fucosylated chondroitin oligosaccharide (XVI) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of the reaction system is 7.5, then L-fucosyltransferase Hp alpha 1,3FT S45F V30G is added for reaction for 12 hours, and after the detection reaction is finished, ice ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant through a Bio-Gel P2 molecular sieve, separating and purifying by using a strong-base anion exchange resin medium, and desalting again by using a Bio-Gel P2 molecular sieve to obtain a product XIII;
(2) mixing guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XVII), MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 20mM, MgCl 2 The final concentration of (A) is 20mM, the final concentration of fucosylated chondroitin oligosaccharide (XVII) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of the reaction system is 7.5, then L-fucosyltransferase Hp alpha 1,3FT S45F V30G is added for reaction for 12 hours, and after the detection reaction is finished, ice ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant through a Bio-Gel P2 molecular sieve, separating and purifying by using a strong basic anion exchange resin medium, and desalting again by using a Bio-Gel P2 molecular sieve to obtain a product XV;
(3) mixing guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XVIII), MgCl 2 Tris-HCl is prepared into water solution, the final concentration of GDP-Fuc in the reaction system is 20mM, MgCl 2 The final concentration of (A) is 20mM, the final concentration of fucosylated chondroitin oligosaccharide (XVIII) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of the reaction system is 7.5, then L-fucosyltransferase Bf alpha 1,3FT is added, and after the detection reaction is finished, the reaction is stopped by adding glacial ethanol; centrifuging the reaction solution for 10-20min, desalting the supernatant through a Bio-Gel P2 molecular sieve, separating and purifying by using a strong basic anion exchange resin medium, and desalting again by using a Bio-Gel P2 molecular sieve to obtain a product XIV;
(4) mixing guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XIX), and MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 40mM, MgCl 2 The final concentration of the compound is 20mM, the final concentration of fucosylated chondroitin oligosaccharide (XIX) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase Bf alpha 1,3FT is added, and after the detection reaction is finished, the reaction is stopped by adding glacial ethanol; centrifuging the reaction solution for 10-20min, desalting the supernatant through a Bio-Gel P2 molecular sieve, separating and purifying by using a strong basic anion exchange resin medium, and desalting again by using a Bio-Gel P2 molecular sieve to obtain a product XV;
(5) mixing guanosine diphosphate fucose (GDP-Fuc), fucosylated chondroitin oligosaccharide receptor (XIX), and MgCl 2 Tris-HCl is prepared into aqueous solution, the final concentration of GDP-Fuc in the reaction system is 20mM, MgCl 2 The final concentration of the compound is 20mM, the final concentration of fucosylated chondroitin oligosaccharide (XIX) is 10mM, the final concentration of Tris-HCl is 50mM, the pH of a reaction system is 7.5, then L-fucosyltransferase Hp alpha 1,3FT S45F V30G is added for reaction for 12 hours, and after the detection reaction is finished, ice ethanol is added to stop the reaction; centrifuging the reaction solution for 10-20min, desalting the supernatant with Bio-Gel P2 molecular sieve, separating and purifying with strongly basic anion exchange resin medium, desalting with Bio-Gel P2 molecular sieve to obtain product XX, which is 1 The H NMR and MS data are as follows,
1 H NMR(600MHz,D 2 O)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,COOCH 3 ,COOCH 3 ,COOCH 3 ,COOCH 3 ),1.25(d,J＝6.6Hz,12H,CH 3 of Fucose-A,CH 3 of Fucose-B).
ESI-MS(negative mode)calcd for C 62 H 96 N 4 O 47 2- [M-2H] 2- m/z 824.2628,found 824.2469.。
sequence listing
<110> Shandong university
<120> fucosylated chondroitin and preparation method and application thereof
<130> 1
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 996
<212> DNA
<213> Artificial Sequence (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 (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 (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 Ile Leu Ser Gln Arg Tyr Thr Ile Thr Leu His 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
Ile Leu Ser Tyr Gln Asn Ala Lys Arg Val Phe Tyr Thr 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 Met 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 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 (9)

1. A method for the preparation of fucosylated chondroitin, the method comprising the steps of:
mixing L-fucosyltransferase with substrate GDP-L-fucose and chondroitin oligosaccharide, and performing enzymatic reaction to obtain fucosylated chondroitin;
preferably, the nucleotide sequence of the L-fucosyltransferase Bf alpha 1,3FT is shown in SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification;
preferably, the nucleotide sequence of the L-fucosyltransferase Hp alpha 1,3FT S45F V30G is shown in SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 4, respectively.
2. The method of claim 1, further comprising the steps of: the single-site fucosylation modification is catalyzed by using chondroitin oligosaccharide and using L-fucosyltransferase Hp alpha 1,3FT S45F V30G at GlcA O-3 position of a non-reducing end structure D-GalNAc 1-4-beta-D-GlcA.
3. The method of claim 1, further comprising the steps of: fucosylation was modified using chondroitin oligosaccharide catalyzed by L-fucosyltransferase Bf α 1,3FT at all GlcA O-3 positions of D-GalNAc1-4- β -D-GlcA structure.
4. The method of claim 1, further comprising the steps of: when the non-reducing end of the fucosylated chondroitin oligosaccharide is not fucosylated and modified by two or more sugars, firstly, the backbone of fucosylated chondroitin is catalyzed to extend by chondroitin synthetase PmCS, and then, the fucosylation modification is further catalyzed at GlcA O-3 position of D-GalNAc 1-4-beta-D-GlcA by using L-fucosyltransferase Bf alpha 1,3FT or Hp alpha 1,3FT S45F V30G.
5. The method of claim 1, wherein the molar ratio of fucoidable GlcA to GDP-L-fucose in the chondroitin oligosaccharide is 0.5-2: 1-4.
6. The method of claim 1, wherein the fucosylated chondroitin has the structure:
Figure FDA0003724726210000011
Figure FDA0003724726210000021
Figure FDA0003724726210000031
7. the method of claim 1, wherein the chondroitin oligosaccharide is prepared by the method comprising the steps of: mixing chondroitin synthetase with a donor substrate and an acceptor substrate, and reacting to obtain chondroitin oligosaccharide;
preferably, said donor substrate is selected from: UDP-GalNAc, UDP-GlcA;
preferably, the acceptor substrate is selected from: GalNAc;
preferably, the molar ratio of the chondroitin oligosaccharide starting substrate to the sugar nucleotide is 0.5-2: 1-5;
preferably, the concentration of the chondroitin synthase is 0.1-2 mg/mL;
preferably, the method further comprises the step of separating;
preferably, the method further comprises the step of purification;
preferably, the chondroitin oligosaccharide has the following structure:
Figure FDA0003724726210000032
Figure FDA0003724726210000041
wherein n is 1 Is an integer selected from 1 to 10;
preferably, the chondroitin oligosaccharide is selected from the following structures:
Figure FDA0003724726210000042
8. the method of claim 7, wherein n is 1 Is an integer selected from 1 to 5, preferably, n is 1 Is an integer selected from 1 to 3.
9. A fucosyltransferase, wherein the nucleotide sequence of the fucosyltransferase is as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 4, respectively.
CN202210763300.XA 2022-06-30 2022-06-30 Fucosylated chondroitin and preparation method and application thereof Pending CN115058465A (en)

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US6878819B1 (en) * 1999-09-28 2005-04-12 Jari Natunen Fucosylated oligosaccharides and process for their preparation
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