Preparation method and application of immobilized enzyme

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a preparation method and application of immobilized enzyme. Compared with free enzyme, the immobilized enzyme prepared by selecting specific resin can be reused and has high stability; the immobilized enzyme is easy to separate from the reaction system, and can simplify the product purification process, improve the product yield and quality. Moreover, the present application develops a new strategy suitable for the large-scale synthesis of functional sugar chains such as sialylated sugar chains by immobilizing one kind of sugar nucleotide synthase and four kinds of sialyltransferase, improves the stability of related enzymes, realizes long-term preservation and reuse of the enzymes, and reduces the production cost of functional sugar chains.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing and applying an immobilized enzyme. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Sialic acid (Sia) is a type of noncarbon sugar containing a carbonyl group, widely found in various glycoconjugates (such as...). O - Polysaccharides, N -Non-reduced or branched terminal sites of sialic acid (glycans and glycosphingolipids). More than 50 structurally well-defined sialic acids and their natural derivatives have been identified, with the most common forms including N -acetylneuraminic acid N -Acetylneuramine Acid, Neu5Ac), N -Hydroxyacetylneuraminic acid (H2N) N α-Glycolylneuraminic acid (Neu5Gc) and deaminylneuraminic acid (2-keto-3-deoxy-nonulosonic acid, Kdn). Sialidized sugar chains are primarily linked to D-galactose (D-Galactose, Gal) residues via α2,3- or α2,6-glycosidic bonds, or to... N -Acetyl-D-galactosamine ( N It is widely present in various organisms in nature, either on α-Acetyl-D-galactosamine (GalNAc) residues or linked to sialic acid residues via α2,8 / α2,9-glycosidic bonds. It is often covalently linked to proteins or lipids and participates in regulating various physiological and pathological processes such as inflammatory responses, viral infections, and tumorigenesis.

[0004] Chemical synthesis is the primary method for obtaining sialylated glycans. While the raw materials for chemical synthesis are widely available, and the target product can be prepared through derivatization reactions, chemical synthesis struggles to achieve specific stereoselectivity in sialyl glycosylation. Furthermore, the total synthetic routes in chemical synthesis are often complex and cumbersome, resulting in low yields of the target product. The use of various reagents also has environmental impacts, leading to high overall costs. In contrast, enzymatic synthesis fully leverages the precise stereoselectivity and regioselectivity of glycosyltransferases. Microbial enzymes are also readily recombinant and expressible, exhibiting high catalytic efficiency and activity. However, current enzymatic synthesis processes mostly utilize free enzymes. Free enzymes suffer from poor stability, sensitivity to temperature and pH, and difficulties in separation and recovery, hindering their reusability and significantly limiting their application in industrial production. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing and applying an immobilized enzyme.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing an immobilized enzyme, comprising the following steps:

[0008] (1) Pretreatment of resin: Weigh the epoxy resin and place it in ammonium phosphate buffer solution. Stir at room temperature and discard the supernatant.

[0009] (2) Immobilization of free enzyme: The pretreated resin was placed in ammonium phosphate buffer, the free enzyme to be immobilized was added, the mixture was shaken for a set time, and the crude immobilized enzyme was obtained by filtration.

[0010] (3) Purification of immobilized crude enzyme: Add ammonium phosphate buffer to the immobilized crude enzyme, shake, and filter to obtain immobilized enzyme.

[0011] Further, in step (1), the epoxy resin is LX-1000HFA, and impurities on the epoxy resin LX-1000HFA are cleaned through pretreatment.

[0012] Further, in step (1), the mass-to-volume ratio of epoxy resin to ammonium phosphate buffer is 1:6-10 mg / μL.

[0013] Further, in step (1), the pH of the ammonium phosphate buffer is 6-8; the concentration of the ammonium phosphate buffer is 90-110 mM.

[0014] Furthermore, in step (1), the stirring time is 40-80 min.

[0015] Further, in step (2), the free enzyme is sialic acid glyconucleotide synthase (SUS). Neisseria meningitis CMP-sialic acid synthetase (NmCSS), α2,3-sialic acid transferase ( Pasteurella multocida 2,3-sialyltransferase (PmST1 M144D), α2,3-sialyltransferase ( Pasteurella multocida 2,3-sialyltransferase (PmST3), α2,6-sialyltransferase ( Photobacterium damsel α2,6-sialyltransferase (Pd2,6ST) or α2,8-sialyltransferase ( Campylobacter jejuni α2,8-sialyltransferase, CjCstII).

[0016] Further, in step (2), the mass ratio of the added free enzyme to the epoxy resin is 6-200:25000; preferably, the mass ratio of NmCSS to epoxy resin is 6-20:25000; the mass ratio of Pd2,6ST to epoxy resin is 100-200:25000; the mass ratio of PmST3 to epoxy resin is 60-120:25000; the mass ratio of PmST1 M144D to epoxy resin is 15-40:25000; and the mass ratio of CjCstII to epoxy resin is 60-120:25000.

[0017] Further, in step (2), the pH of the ammonium phosphate buffer is 6-8; the concentration of the ammonium phosphate buffer is 90-110 mM.

[0018] Furthermore, in step (2), the mixture is shaken at 18-22 ℃ for 22-24 hours.

[0019] Further, in step (3), the pH of the ammonium phosphate buffer is 6-8; the concentration of the ammonium phosphate buffer is 90-110 mM; and the amount of the ammonium phosphate buffer used is 2-3 times that used in step (1).

[0020] Furthermore, in step (3), the mixture is shaken for 4-6 hours at 28-32 ℃.

[0021] Secondly, the present invention provides an immobilized enzyme prepared by the method for preparing the immobilized enzyme described in the first aspect.

[0022] This invention uses epoxy resin as a carrier to immobilize a glyconucleotide synthase NmCSS and four sialyl transferases Pd2,6ST, PmST3, PmST1 M144D, and CjCstII. The glyconucleotide synthase NmCSS utilizes free sialic acid and cytidine triphosphate to convert sialic acid into the activated glyconucleotide donor cytidine monophosphate sialic acid (CMP-Neu5Ac). This sialic acid unit is then recognized by 2,6-sialyl transferases (Pd2,6ST), 2,3-sialyl transferases (PmST3), and 2,3-sialyl transferases (PmST1 M144D), respectively, and transferred to the corresponding acceptors' galactose and / or... N The sialic acid unit of acetylgalactosamine is transferred to the C6 hydroxyl group of galactose or the C3 hydroxyl group of galactose, or is recognized by 2,8-sialyltransferase (CjCstII), which transfers the sialic acid unit to the C8 hydroxyl group of the corresponding receptor, thereby generating the target product.

[0023] Thirdly, the present invention provides the application of the immobilized enzyme described in the second aspect in the sialylated glycan synthesis reaction.

[0024] Further, the sialylated sugar chain is selected from compounds 1 (Neu5Acα2-6Galβ1-4GlcβProN3), 2 (Neu5Acα2-3Galβ1-4GlcβProN3), 3 (Neu5Acα2-6GalNAcβProN3), 4 (Neu5Gcα2-6GalNAcβProN3), 5 (Kdnα2-6GalNAcβProN3), 6 (Neu5Acα2-6GalβProN3), 7 (Neu5Gcα2-6GalβProN3), 8 (Neu5Gcα2-6GalβProN3), and 9 (Neu5Acα2-3GalβProN3) with a 3-azidopropyl (βProN3) linker at the anomeric position. Compound 10 (Neu5Gcα2-3GalβproN3), compound 11 (Kdnα2-3GalβProN3), compound 12 (Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3), compound 13 (Neu5Gcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3), compound 14 (Neu5Acα2-8Neu5Acα2-3Galβ1-4GlcβProN3), and compounds 15 (Neu5Acα2-6Galβ1-4Glc) and 16 (Neu5Acα2-3Galβ1-4Glc) with hydroxyl groups at the anodic position.

[0025] The abbreviation Glc refers to D-glucose; the abbreviation GlcNAc indicates... N -acetyl-D-glucosamine ( N -Acetyl-D-Glucosamine).

[0026] The structures of sialylated glycan compounds 1-16 are shown below:

[0027]

[0028] Furthermore, the reaction donors include cytidine 5'-monophosphate- N - Acetylneuraminic acid (CMP-Neu5Ac), cytidine 5'-monophosphate- N -Hydroxyacetylneuraminic acid (CMP-Neu5Gc), Cytidine 5'-monophosphate-deaminoneuraminic acid (CMP-Kdn).

[0029] Furthermore, the acceptor compounds are selected from free compound 20 (Lactose), compound 21 (GalNAcβProN3), compound 22 (GalβProN3), compound 23 (Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3), compound 24 (Neu5Acα2-3Galβ1-4GlcβProN3), and compound 25 (LactoseβProN3) with a 3-azidopropyl (βProN3) linker arm at the anomeric position. The structures of acceptor compounds 20-25 are shown below:

[0030]

[0031] Furthermore, when the reaction acceptor is compound 20, the reaction donor is CMP-Neu5Ac;

[0032] When the acceptor is compound 21, the donor is CMP-Neu5Ac;

[0033] When the acceptor is compound 21, the donor is CMP-Neu5Gc;

[0034] When the acceptor is compound 21, the donor is CMP-Kdn;

[0035] When the acceptor is compound 22, the donor is CMP-Neu5Ac;

[0036] When the acceptor is compound 22, the donor is CMP-Neu5Gc;

[0037] When the acceptor is compound 22, the donor is CMP-Kdn;

[0038] When the acceptor is compound 23, the donor is CMP-Neu5Ac; when the acceptor is compound 23, the donor is CMP-Neu5Gc.

[0039] When the acceptor is compound 24, the donor is CMP-Neu5Ac;

[0040] When the acceptor is compound 25, the donor is CMP-Neu5Ac.

[0041] Fourthly, the present invention provides a method for synthesizing sialylated sugar chains, the method comprising using the immobilized enzyme described in the second aspect.

[0042] Further, the specific steps of the method are as follows: after dissolving the reaction acceptor, reaction donor, buffer solution and MgCl2, the immobilized enzyme described in the second aspect is added, and after reacting for a period of time, the sialylated sugar chain is obtained by filtration, concentration and purification.

[0043] This invention fully leverages the highly precise regioselectivity and stereoselectivity of enzymatic synthesis. By employing a glycan-based enzymatic modular assembly strategy, it starts from simple and inexpensive raw materials, avoiding the use of expensive and commercially unavailable nucleoside-activated glycosyl donors, thus efficiently constructing specific glycosidic bonds. Immobilization enhances enzyme stability, enabling reuse and significantly reducing costs for industrial applications. The immobilized enzyme is easily separated from the reaction system, reducing product separation and purification steps and greatly improving product yield and quality.

[0044] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0045] (1) The present invention provides a method for preparing immobilized enzymes. Compared with free enzymes, the immobilized enzymes prepared by the present invention using specific resins can be reused, have high stability, and reduce production costs. The immobilized enzymes are easy to separate from the reaction system, which can simplify the product purification process, improve product yield and quality, and the immobilized enzymes have a certain mechanical strength, which facilitates continuous and automated operation in industrial production.

[0046] (2) This invention develops a new strategy for the large-scale synthesis of functional sugar chains such as sialylated sugar chains by immobilizing a glyconucleotide generating enzyme and four sialyl transferases, thereby improving the stability of the relevant enzymes, enabling their long-term preservation and reuse, and reducing the production cost of functional sugar chains. Attached Figure Description

[0047] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0048] Figure 1 This is a TLC result of the immobilized enzyme Pd2,6ST catalytic activity in Example 1 of the present invention;

[0049] Figure 2 This is a stability diagram of the immobilized enzyme in Example 1 of the present invention;

[0050] Figure 3 This is a graph showing the reusability of the immobilized enzyme in Example 22 of the present invention. Detailed Implementation

[0051] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0053] Example 1

[0054] (1) Screening of epoxy resins

[0055] Pretreatment of resin: Weigh 25 mg of epoxy resin and place it in 200 μL of ammonium phosphate buffer (100 mM, pH 7.5). Stir the resin at room temperature for 1 hour and discard the supernatant.

[0056] Immobilization of free enzyme: 25 mg of pretreated resin was placed in ammonium phosphate buffer (100 mM, pH 7.5), and then the sialyl glycosyltransferase to be immobilized (Pd2,6ST) was added, with the total volume controlled at 500 μL. The mixture was shaken at 20 °C for 24 hours. After immobilization, the crude immobilized enzyme was obtained by filtration.

[0057] Purification of the immobilized enzyme crude product: Add 500 μL of ammonium phosphate buffer (100 mM, pH 7.5) to the immobilized enzyme crude product from the previous step, and shake at 30 °C for 6 hours; after the process, filter to obtain the immobilized enzyme.

[0058] The above-mentioned immobilization process was carried out using ten commonly used industrial epoxy resins: LXTE-600, LXTE-601, LXTE-602, LXTE-604, LXTE-605, LXTE-607, LXTE-608, LXTE-609, LXTE-707, and LX-1000HFA.

[0059] Using the above specific implementation scheme, thin-layer chromatography (TLC) was employed to track the reaction process and investigate the catalytic activity of enzymes immobilized with different types of epoxy resins in the synthesis of CMP-Neu5Ac, 6'-SLβProN3, 3'-SLβProN3, and GD3βProN3. Figure 1 This is a TLC result of the immobilized enzyme Pd2,6ST catalytic activity of this invention. Figure 1 In the figure, numbers 05, 06, 12, 13, 15, 21, 22, 29, 30, and 31 correspond to Pd2,6ST immobilized with epoxy resins LXTE-600, LXTE-601, LX-1000HFA, LXTE-602, LXTE-604, LXTE-605, LXTE-607, LXTE-608, LXTE-609, and LXTE-707, respectively. As can be seen from the figure, only Pd2,6ST immobilized with LX-1000HFA epoxy resin exhibits catalytic activity.

[0060] Using the same method described above, sialic acid glyconucleotide synthase (NmCSS) and sialic acid glycosyltransferases (PmST3, PmST1 M144D, CjCstII) were immobilized. The enzymes immobilized on LX-1000HFA epoxy resin all showed good catalytic activity and had a certain degree of universality.

[0061] (2) Storage conditions for immobilized enzymes

[0062] This invention investigated the storage conditions of immobilized enzymes, measuring enzyme activity retention over periods of one week, two weeks, one month, three months, six months, and one year, and using TLC to track the reaction progress. The results showed that sialyl glyconucleotide synthase (NmCSS), α2,6-sialyl transferase (Pd2,6ST), α2,3-sialyl transferase (PmST3), α2,3-sialyl transferase (PmST1 M144D), and α2,8-sialyl transferase (CjCstII) all maintained better activity under PBS solution and storage conditions at 4°C. Figure 2 This is a stability graph of the immobilized enzyme of this invention. Figure 2 It can be seen that:

[0063] Sialoglyconucleotide synthase (NmCSS) activity was approximately 82% and 69% of the initial activity after 1 month and 6 months of storage in PBS solution at 4°C, respectively.

[0064] After 1 month and 6 months of storage in PBS solution at 4°C, the activity of α2,6-sialyltransferase (Pd2,6ST) was approximately 89% and 75% of the initial activity, respectively.

[0065] After 1 month and 6 months of storage in PBS solution at 4°C, the activity of α2,3-sialyltransferase (PmST3) was approximately 85% and 71% of the initial activity, respectively.

[0066] The activity of α2,3-sialoyltransferase (PmST1 M144D) was approximately 86% and 72% of the initial activity after 1 month and 6 months of storage in PBS solution at 4°C, respectively.

[0067] After 1 month and 6 months of storage in PBS solution at 4°C, the activity of α2,8-sialyltransferase (CjCstII) was approximately 83% and 73% of the initial activity, respectively.

[0068] Example 2

[0069] Immobilization of sialyl glyconucleotide synthase (NmCSS) and sialyl glycosyltransferases (Pd2,6ST, PmST3, PmST1M144D, CjCstII)

[0070] (1) NmCSS fixation method:

[0071] Pretreatment of resin: Weigh 25 mg of epoxy resin LX-1000HFA and place it in 200 μL of ammonium phosphate buffer (100 mM, pH 7.5). Stir the resin at room temperature for 1 hour and discard the supernatant.

[0072] Immobilization of NmCSS: 25 mg of pretreated resin was placed in ammonium phosphate buffer (100 mM, pH 7.5), followed by the addition of 10 μg of NmCSS, bringing the total volume to 500 μL. The mixture was shaken at 20°C for 24 hours. After immobilization, the crude immobilized enzyme was obtained by filtration.

[0073] Purification of crude NmCSS immobilized enzyme: Add 500 μL of ammonium phosphate buffer (100 mM, pH 7.5) to the crude immobilized enzyme from the previous step, and shake at 30°C for 6 hours. After purification, filter the immobilized NmCSS.

[0074] (2) Pd2,6ST immobilization method:

[0075] Pretreatment of resin: Weigh 25 mg of epoxy resin LX-1000HFA and place it in 200 μL of ammonium phosphate buffer (100 mM, pH 7.5). Stir the resin at room temperature for 1 hour and discard the supernatant.

[0076] Immobilization of Pd2,6ST: 25 mg of pretreated resin was placed in ammonium phosphate buffer (100 mM, pH 7.5), followed by the addition of 150 μg of Pd2,6ST, bringing the total volume to 500 μL. The mixture was shaken at 20°C for 24 hours. After immobilization, the crude immobilized enzyme was obtained by filtration.

[0077] Purification of crude Pd2,6ST immobilized enzyme: Add 500 μL of ammonium phosphate buffer (100 mM, pH 7.5) to the crude immobilized enzyme from the previous step, and shake at 30°C for 6 hours. After purification, filter the immobilized Pd2,6ST.

[0078] (3) PmST3 immobilization method:

[0079] Pretreatment of resin: Weigh 25 mg of epoxy resin LX-1000HFA and place it in 200 μL of ammonium phosphate buffer (100 mM, pH 7.5). Stir the resin at room temperature for 1 hour and discard the supernatant.

[0080] PmST3 immobilization: 25 mg of pretreated resin was placed in ammonium phosphate buffer (100 mM, pH 7.5), followed by the addition of 100 μg of PmST3, bringing the total volume to 500 μL. The mixture was shaken at 20°C for 24 hours. After immobilization, the crude immobilized enzyme was obtained by filtration.

[0081] Purification of crude PmST3 immobilized enzyme: Add 500 μL of ammonium phosphate buffer (100 mM, pH 7.5) to the crude immobilized enzyme from the previous step, and shake at 30°C for 6 hours. After purification, filter the immobilized PmST3.

[0082] (4) PmST1 M144D immobilization method:

[0083] Pretreatment of resin: Weigh 25 mg of epoxy resin LX-1000HFA and place it in 200 μL of ammonium phosphate buffer (100 mM, pH 7.5). Stir the resin at room temperature for 1 hour and discard the supernatant.

[0084] Immobilization of PmST1 M144D: 25 mg of pretreated resin was placed in ammonium phosphate buffer (100 mM, pH 7.5), followed by the addition of 30 μg of PmST3, bringing the total volume to 500 μL. The mixture was shaken at 20°C for 24 hours. After immobilization, the crude immobilized enzyme was obtained by filtration.

[0085] Purification of PmST1 M144D immobilized crude enzyme: Add 500 μL of ammonium phosphate buffer (100 mM, pH 7.5) to the crude immobilized enzyme from the previous step, and shake at 30°C for 6 hours. After purification, filter the immobilized PmST1 M144D.

[0086] (5) CjcstII immobilization method:

[0087] Pretreatment of resin: Weigh 25 mg of epoxy resin LX-1000HFA and place it in 200 μL of ammonium phosphate buffer (100 mM, pH 7.5). Stir the resin at room temperature for 1 hour and discard the supernatant.

[0088] Immobilization of CjCstII: 25 mg of pretreated resin was placed in ammonium phosphate buffer (100 mM, pH 7.5), followed by the addition of 100 μg of CjCstII, bringing the total volume to 500 μL. The mixture was shaken at 20°C for 24 hours. After immobilization, the crude immobilized enzyme was obtained by filtration.

[0089] Purification of the crude CjCstII immobilized enzyme: Add 500 μL of ammonium phosphate buffer (100 mM, pH 7.5) to the crude immobilized enzyme from the previous step, and shake at 30°C for 6 hours. After purification, filter the immobilized CjCstII.

[0090] Example 3

[0091] Immobilized enzyme synthesizes cytidine 5'-monophosphate- N The synthesis method of β-acetylneuraminic acid (CMP-Neu5Ac) is as follows:

[0092] Will N Acetylneuraminic acid (Neu5Ac, 1.0 equivalent), cytosine triphosphate (CTP, 2.0 equivalent), MgCl2 (final concentration 20 mmol / L), and Tris-HCl buffer (final concentration 100 mmol / L) were thoroughly dissolved in water. The pH of the reaction solution was adjusted to 8.0. Immobilized glyconucleotide synthase (NmCSS) (NmCSS to epoxy resin mass ratio 8:25000) was then added to the reaction solution. The reaction time was 2 hours. After the reaction was completed, the cytosine 5'-monophosphate was obtained by separation and purification using an anion exchange column (DEAE) and gel size exclusion chromatography (Bio-gel P2). N - Acetylneuraminic acid (CMP-Neu5Ac) was used as a donor for the next immobilized enzyme reaction. The synthetic route is shown below.

[0093]

[0094] Example 4

[0095] Immobilized enzyme synthesizes cytidine 5'-monophosphate- N The synthesis method of β-hydroxyacetylneuraminic acid (CMP-Neu5Gc) is as follows:

[0096] Will N 5'-hydroxyacetylneuraminic acid (Neu5Gc, 1.0 equivalent), cytosine triphosphate (CTP, 2.0 equivalent), MgCl2 (final concentration 20 mmol / L), and Tris-HCl buffer (final concentration 100 mmol / L) were thoroughly dissolved in water. The pH of the reaction solution was adjusted to 8.0. Immobilized glyconucleotide synthase (NmCSS) (NmCSS to epoxy resin mass ratio 15:25000) was then added to the reaction solution. The reaction time was 10 hours. After the reaction was complete, the cytosine 5'-monophosphate was obtained by separation and purification using an anion exchange column (DEAE) and gel size exclusion chromatography (Bio-gel P2). N -Hydroxyacetylneuraminic acid (CMP-Neu5Gc) was used as a donor for the next immobilized enzyme reaction. The synthetic route is shown below.

[0097]

[0098] Example 5

[0099] The method for synthesizing cytidine 5'-monophosphate-deaminoneuraminic acid (CMP-Kdn) using immobilized enzymes is as follows:

[0100] After thoroughly dissolving deaminoneuraminic acid (Kdn, 1.0 equivalent), cytosine triphosphate (CTP, 2.0 equivalent), MgCl2 (final concentration 20 mmol / L), and Tris-HCl buffer (final concentration 100 mmol / L) in water, the pH of the reaction solution was adjusted to 8.0. Immobilized glyconucleotide synthase (NmCSS) (NmCSS to epoxy resin mass ratio 20:25000) was then added to the reaction solution, and the reaction time was 24 hours. After the reaction was complete, the solution was purified by DEAE anion exchange column and Bio-gel P2 size exclusion chromatography to obtain cytidine 5'-monophosphate-deaminoneuraminic acid (CMP-Kdn), which serves as the donor for the next immobilized enzyme reaction. The synthetic route is shown below.

[0101]

[0102] Example 6

[0103] The synthetic method for compound 1 (Neu5Acα2-6Galβ1-4Glc) using immobilized enzyme synthesis is as follows:

[0104] Lactose (50 mg), CMP-Neu5Ac (137 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 100:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 24 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain compound 1 (73.6 mg, 84%, α / β = 1:1.5). The NMR data are as follows: 1 H NMR (600 MHz, D2O) δ 5.18 (d, J =3.8 Hz, 1.00 H), 4.63 (d, J = 8.0 Hz, 1.69 H), 4.39 (d, J = 7.9 Hz, 2.84 H), 3.96– 3.73 (m, 25.60 H), 3.69 (d, J = 1.7 Hz, 1.53 H), 3.67 (d, J = 1.7 Hz, 1.17 H),3.64 – 3.47 (m, 25.04 H), 3.27 (t, J = 8.5 Hz, 1.57 H), 2.67 (dd, J = 12.4, 4.7Hz, 2.86 H), 1.99 (s, 7.91 H), 1.70 (t, J = 12.2 Hz, 2.83 H); 13C NMR (150 MHz, D2O) δ 174.76, 173.37, 103.10, 103.07, 100.13, 95.50, 91.67, 79.59, 79.47,74.52, 74.48, 73.55, 72.37, 72.20, 71.66, 71.49, 70.90, 70.64, 69.81, 68.37,68.23, 68.22, 63.45, 62.47, 60.10, 59.94, 51.64, 39.96, 21.92.

[0105] The synthetic route for compound 1 is shown below.

[0106]

[0107] Example 7:

[0108] The synthetic method for compound 2 (Neu5Acα2-3Galβ1-4Glc) using immobilized enzyme synthesis is as follows:

[0109] Lactose (50 mg), CMP-Neu5Ac (137 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST3 (PmST3 to epoxy resin mass ratio 100:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 24 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST3 was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 2 (82.6 mg, 94%, α / β = 1: 1.9). The NMR data are as follows: 1 H NMR (600 MHz, D2O) δ 5.15 (d, J = 3.7 Hz, 1.00 H), 4.59 (d, J = 8.0 Hz, 1.88 H), 4.46 (d, J = 7.9 Hz, 2.99 H), 4.07 – 4.02(m, 3.02 H), 3.91 – 3.47 (m, 54.83 H), 3.21 (t,J = 8.6 Hz, 1.70 H), 2.68 (dd, J = 12.4, 4.4 Hz, 3.07 H), 1.96 (s, 8.54 H), 1.73 (t, J = 12.1 Hz, 3.12 H); 13 C NMR (150 MHz, D2O) δ 174.80, 173.75, 102.42, 99.59, 95.60, 91.64, 78.02, 77.87,75.28, 74.99, 74.63, 74.15, 73.60, 72.68, 71.60, 71.21, 70.95, 69.91, 69.20,68.21, 67.88, 67.27, 62.36, 60.87, 59.84, 59.69, 51.48, 39.44, 21.86.

[0110] The synthetic route for compound 2 is shown below.

[0111]

[0112] Example 8:

[0113] The synthetic method for compound 3 (Neu5Acα2-6GalNAcβProN3) using immobilized enzyme synthesis is as follows:

[0114] GalNAcβProN3 (50 mg), CMP-Neu5Ac (141 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 150:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 28 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain compound 3 (87.1 mg, 89%). The NMR data are as follows: 1 H NMR (400 MHz, D2O) δ 4.40 (d, J= 8.4 Hz, 1H), 3.98 – 3.53 (m, 15 H), 3.35 (td, J = 6.6, 2.0 Hz, 2 H), 2.71 (dd, J = 12.5,4.6 Hz, 1 H), 2.02 (s, 3 H), 2.01 (s, 3 H), 1.81 (p, J = 6.4 Hz, 2 H), 1.67 (t, J = 12.1 Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 175.05, 174.69, 173.43, 101.73,100.43, 73.41, 72.63, 71.74, 70.78, 68.19, 67.74, 67.23, 63.44, 62.62, 52.35,51.84, 47.77, 40.17, 28.15, 22.21, 22.02.

[0115] The synthetic route for compound 3 is shown below.

[0116]

[0117] Example 9:

[0118] The synthetic method of compound 4 (Neu5Gcα2-6GalNAcβProN3) by immobilized enzyme synthesis is as follows:

[0119] GalNAcβProN3 (50 mg), CMP-Neu5Gc (135 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 150:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 32 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 4 (67.2 mg, 76%). NMR data are as follows: 1H NMR (400 MHz, D2O) δ 4.42 (d, J = 8.5 Hz, 1H), 4.11 (s, 2 H), 4.00 – 3.54 (m, 15 H), 3.42 – 3.31 (m, 2 H), 2.73 (dd, J =12.4, 4.6 Hz, 1 H), 2.03 (s, 3 H), 1.83 (p, J = 6.5 Hz, 2 H), 1.69 (t, J = 12.1Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 175.83, 174.71, 173.46, 101.74, 100.49,73.45, 72.38, 71.81, 70.82, 68.16, 67.96, 67.78, 67.26, 63.49, 62.62, 60.99,52.38, 51.56, 47.80, 40.23, 28.17, 22.23.

[0120] The synthetic route for compound 4 is shown below.

[0121]

[0122] Example 10:

[0123] The synthetic method for compound 5 (Kdnα2-6GalNAcβProN3) using immobilized enzyme synthesis is as follows:

[0124] GalNAcβProN3 (50 mg), CMP-Kdn (127 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 200:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 36 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain compound 5 (65.6 mg, 72%). NMR data are as follows:1 H NMR (400 MHz, D2O) δ 4.41 (d, J = 8.5 Hz, 1 H), 3.99 – 3.48 (m, 15 H), 3.36 (t, J = 6.6, 1.9 Hz, 2 H), 2.66 (dd, J = 12.5, 4.7Hz, 1H), 2.04 (s, 3H), 1.83 (p, J = 6.5 Hz, 2 H), 1.64 (t, J = 12.1 Hz, 1 H); 13 CNMR (100 MHz, D2O) δ 174.70, 173.62, 101.72, 100.43, 73.62, 73.43, 72.05,70.77, 70.14, 69.87, 67.84, 67.77, 67.23, 63.51, 62.68, 52.35, 47.76, 39.76,28.14, 22.21.

[0125] The synthetic route for compound 5 is shown below.

[0126]

[0127] Example 11:

[0128] The synthetic method for compound 6 (Neu5Acα2-6GalβProN3) using immobilized enzyme synthesis is as follows:

[0129] GalβProN3 (50 mg), CMP-Neu5Ac (163 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 100:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 20 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 6 (96.9 mg, 92%). The NMR data are as follows:1 H NMR (400 MHz, D2O) δ 4.36 (d, J = 7.9 Hz, 1 H), 4.03 – 3.39 (m, 17 H), 2.71 (dd, J = 12.6, 4.5 Hz, 1 H), 2.01 (s, 3 H), 1.89(p, J = 6.6 Hz, 2 H), 1.67 (t, J = 12.2 Hz, 1 H); 13 C NMR (100 MHz, D2O) δ175.04, 173.44, 102.88, 100.40, 73.38, 72.61, 72.55, 71.70, 70.64, 68.54,68.17, 67.44, 63.29, 62.58, 51.83, 47.85, 40.16, 28.25, 21.99.

[0130] The synthetic route for compound 6 is shown below.

[0131]

[0132] Example 12:

[0133] The synthetic method for compound 7 (Neu5Gcα2-6GalβProN3) using immobilized enzyme synthesis is as follows:

[0134] GalβProN3 (50 mg), CMP-Neu5Gc (156 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 150:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 34 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain compound 7 (78.3 mg, 83%). NMR data are as follows: 1 H NMR (400 MHz, D2O) δ 4.37 (d, J= 7.9 Hz, 1 H), 4.11 (s, 2 H), 4.02 – 3.41 (m, 17 H), 2.73 (dd, J = 12.4, 4.6 Hz, 1 H), 1.90(p, J = 6.6 Hz, 2 H), 1.69 (t, J = 12.1 Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 175.81,173.47, 102.89, 100.45, 73.40, 72.56, 72.34, 71.77, 70.66, 68.56, 68.11,67.93, 67.46, 63.32, 62.56, 60.95, 51.53, 47.87, 40.21, 28.27.

[0135] The synthetic route for compound 7 is shown below.

[0136]

[0137] Example 13:

[0138] The synthetic method of compound 8 (Kdnα2-6GalβProN3) by immobilized enzyme synthesis is as follows:

[0139] GalβProN3 (50 mg), CMP-Kdn (147 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 200:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 38 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain compound 8 (71.2 mg, 73%). NMR data are as follows: 1 H NMR (400 MHz, D2O) δ 4.36 (d, J = 7.9 Hz, 1 H), 4.03 –3.39 (m, 17 H), 2.66 (dd,J = 12.5, 4.6 Hz, 1 H), 1.89 (p, J = 6.6 Hz, 2 H), 1.63(t, J = 12.1 Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 173.63, 102.88, 100.43, 73.61,73.43, 72.57, 72.05, 70.66, 70.15, 69.88, 68.59, 67.84, 67.46, 63.37, 62.68,47.87, 39.78, 28.26.

[0140] The synthetic route for compound 8 is shown below.

[0141]

[0142] Example 14:

[0143] The synthetic method of compound 9 (Neu5Acα2,3GalβProN3) using immobilized enzymes is as follows:

[0144] GalβProN3 (50 mg), CMP-Neu5Ac (163 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST3 (PmST3 to epoxy resin mass ratio 80:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 40 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST3 was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 9 (95.8 mg, 91%). NMR data are as follows: 1 H NMR (400 MHz, D2O) δ 4.44 (d, J = 7.9 Hz, 1 H), 4.07 (d, J =9.8 Hz, 1 H), 4.01 – 3.49 (m, 14 H), 3.43 (t, J = 6.6 Hz, 2 H), 2.73 (dd,J =12.4, 4.1 Hz, 1 H), 2.00 (s, 3 H), 1.89 (p, J = 6.4 Hz, 2 H), 1.77 (t, J = 12.1Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 174.99, 173.87, 102.54, 99.80, 75.78, 74.87,72.82, 71.72, 69.11, 68.33, 68.03, 67.49, 67.15, 62.52, 60.91, 51.66, 47.89, 39.60, 28.24, 22.02.

[0145] The synthetic route for compound 9 is shown below.

[0146]

[0147] Example 15:

[0148] The synthetic method for compound 10 (Neu5Gcα2,3GalβproN3) using immobilized enzymes is as follows:

[0149] GalβProN3 (50 mg), CMP-Neu5Gc (156 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST3 (PmST3 to epoxy resin mass ratio 100:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 43 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST3 was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 10 (74.5 mg, 79%). The NMR data are as follows: 1 H NMR (400 MHz, D2O) δ 4.46 (d, J = 7.9 Hz, 1 H), 4.12 –3.50 (m, 17 H), 3.45 (t, J = 6.7 Hz, 2 H), 2.76 (dd, J= 12.4, 4.7 Hz, 1 H), 1.90(p, J = 6.6 Hz, 2 H), 1.80 (t, J = 12.1 Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 175.76,173.89, 102.53, 99.81, 75.78, 74.88, 72.53, 71.78, 69.11, 68.08, 67.95,67.46, 67.14, 62.48, 60.93, 51.34, 47.88, 39.67, 28.23.

[0150] The synthetic route for compound 10 is shown below.

[0151]

[0152] Example 16:

[0153] Synthesis of compound 11 (Kdnα2,3GalβproN3) by immobilized enzyme method:

[0154] GalβProN3 (50 mg), CMP-Kdn (147 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST3 (PmST3 to epoxy resin mass ratio 120:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 48 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST3 was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 11 (72.2 mg, 74%). NMR data are as follows: 1 H NMR (400 MHz, D2O) δ 4.45 (d, J = 7.9 Hz, 1 H), 4.06(dd, J = 9.8, 3.2 Hz, 1 H), 4.02 – 3.49 (m, 14 H), 3.45 (t, J = 6.7 Hz, 2 H), 2.70 (dd, J= 12.5, 4.4 Hz, 1 H), 1.90 (p, J = 6.6 Hz, 2 H), 1.74 (t, J = 12.0 Hz, 1 H); 13 C NMR (100 MHz, D2O) δ 174.02, 102.55, 99.78, 75.76, 74.89, 73.85,72.02, 70.23, 69.71, 69.11, 67.66, 67.41, 67.14, 62.59, 60.92, 47.89, 39.28,28.23.

[0155] The synthetic route for compound 11 is shown below.

[0156]

[0157] Example 17:

[0158] The synthetic method of compound 12 (Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3) using immobilized enzyme method is as follows:

[0159] Compound 23 (50 mg), CMP-Neu5Ac (60.6 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST1 M144D (PmST1 M144D to epoxy resin mass ratio 15:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 36 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST1 M144D was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 12 (56.6 mg, 84%). NMR data are as follows: (88 mg, 94%). 1 H NMR (600 MHz, D2O) δ 4.70(d, J = 8.4 Hz, 1H), 4.70 (d, J = 7.8 Hz, 1H), 4.56 (d, J= 7.8 Hz, 1H), 4.49 (d, J =7.8 Hz, 1H), 4.47(d, J = 7.8 Hz, 1H), 4.44 (d, J = 7.8 Hz, 1H), 4.16 (t, J = 3.0Hz, 2H), 4.12 (dd, J = 3.0,10.2 Hz,1H), 4.02-3.56 (m, 42H), 3.46 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 8.4 Hz, 1H), 2.76 (dd, J = 4.2,12 Hz,1H), 2.04 (s, 9H), 1.92(p, J = 6.6 Hz, 2H), 1.80 (t, J = 12 Hz, 1H); 13 C NMR (150 MHz, D2O) 174.99,174.88, 174.87, 173.85, 102.92, 102.85, 102.78, 102.73, 102.52, 102.09,99.78, 82.05,82.01, 78.32, 78.13, 77.95, 75.46, 75.15, 74.86, 74.85, 74.74,74.52, 74.33, 72.86, 72.76, 72.14, 72.11, 71.74, 69.94, 69.35, 68.31, 68.30, 68.28, 68.05, 67.34, 62.55, 61.02, 60.95, 60.93, 60.03, 59.80, 55.16, 55.11, 51.66, 47.85, 28.21, 22.16, 22.02.

[0160] The synthetic route for compound 12 is shown below:

[0161]

[0162] Example 18

[0163] The synthetic method of compound 13 (Neu5Gcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3) using immobilized enzyme method is as follows:

[0164] Compound 23 (50 mg), CMP-Neu5Gc (58.3 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST1 M144D (PmST1 M144D to epoxy resin mass ratio 40:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 36 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST1 M144D was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 13 (56.6 mg, 82%). NMR data are as follows: 1 H NMR (600 MHz, D2O) δ 4.70 (d, J = 8.4 Hz, 2H), 4.56 (d, J = 8.4 Hz, 1H), 4.49 (d, J = 7.8 Hz, 1H), 4.47 (d, J = 7.8 Hz, 1H), 4.44(d, J = 7.8 Hz, 1H), 4.16 (t, J = 3.0 Hz, 2H), 4.13 (dd, J = 3.0,6.0 Hz, 1H),4.12 (s, 2H), 4.02-3.57(m, 42H), 3.46 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 8.4 Hz, 1H), 2.78 (dd, J = 4.2,12 Hz, 1H), 2.03 (s,6H), 1.91 (p, J = 6.6 Hz, 2H), 1.82(t, J = 12 Hz, 1H); 13C NMR (150 MHz, D2O) δ 175.76, 174.87,173.88, 102.92,102.86, 102.79, 102.73, 102.53, 102.09, 99.81, 82.06, 82.02, 78.32, 78.14,77.96,75.46, 75.16, 74.87, 74.85, 74.75, 74.53, 74.34, 72.77, 72.59, 72.14,72.11, 71.81, 69.94, 69.37,68.31, 68.29, 68.06, 67.99, 67.45, 67.34, 62.53, 61.03, 60.96, 60.93, 60.03, 59.82, 55.16, 55.12, 51.37, 47.85, 28.22, 22.17.

[0165] The synthetic route for compound 13 is shown below.

[0166]

[0167] Example 19

[0168] The synthetic method of compound 14 (Neu5Acα2-8Neu5Acα2-3Galβ1-4GlcβProN3) by immobilized enzyme method is as follows:

[0169] Compound 24 (50 mg), CMP-Neu5Ac (57.2 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized CjCstII (CjCstII to epoxy resin mass ratio 100:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 38 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized CjCstII was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 14 (53.5 mg, 76%). NMR data are as follows: 1 H NMR (600 MHz, D2O) δ 4.53 (d, J = 8.0 Hz, 1H), 4.49 (d,J = 7.9 Hz, 1H), 4.21 – 3.53 (m, 27H), 3.47 (t, J = 6.7 Hz, 2H), 3.32(t, J = 8.6 Hz, 1H), 2.79 (dd, J = 12.3, 4.8 Hz, 1H), 2.69 (dd, J = 12.4, 4.5 Hz,1H), 2.07 (s, 3H), 2.04(s, 3H), 1.92 (p, J = 6.5 Hz, 2H), 1.75 (t, J = 12.1 Hz, 2H); 13 C NMR (150 MHz, D2O) δ 176.18, 174.59, 103.44, 102.88, 101.30, 101.07,78.70, 76.01, 75.74, 75.37, 74.84, 74.55, 73.40, 73.21, 72.30, 69.89, 69.81,68.99, 68.68, 68.44, 68.00, 67.89, 63.09, 62.06, 61.57, 60.51, 52.71, 52.15,48.30, 40.83, 40.05, 28.52, 22.59, 22.31.

[0170] The synthetic route for compound 14 is shown below.

[0171]

[0172] Example 20

[0173] The synthetic method of compound 15 (Neu5Acα2-6Galβ1-4GlcβProN3) by immobilized enzyme synthesis is as follows:

[0174] LactoseβProN3 (50 mg), CMP-Neu5Ac (125 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized Pd2,6ST (Pd2,6ST to epoxy resin mass ratio 100:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 24 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized Pd2,6ST was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 size exclusion chromatography and DEAE anion exchange column to obtain compound 15 (74 mg, 88%). NMR data are as follows: 1 H NMR (600 MHz, D2O) δ 4.51 (d, J = 8.0 Hz, 1H), 4.45 (d, J = 7.9 Hz, 1 H), 4.06 – 3.61 (m, 18 H), 3.59 (dd, J = 9.1, 1.5Hz, 1 H), 3.55 (dd, J = 9.9, 7.9 Hz, 1 H), 3.49 (t, J = 6.7 Hz, 2 H), 3.36(t, J = 8.6 Hz, 1 H), 2.73 (dd, J = 12.4, 4.7 Hz, 1 H), 2.05 (s, 3 H), 1.94(p, J = 6.7 Hz, 2 H), 1.76 (t, J = 12.2 Hz, 1 H); 13 C NMR (151 MHz, D2O) δ174.92, 173.48, 103.20, 102.01, 100.30, 79.59, 74.64, 74.60, 73.68, 72.73,72.52, 72.36, 71.79, 70.79, 68.52, 68.37, 68.35, 67.33, 63.55, 62.64, 60.26,51.79, 47.89, 40.09, 28.24, 22.09.

[0175] The synthetic route for compound 15 is shown below.

[0176]

[0177] Example 21

[0178] The synthetic method of compound 16 (Neu5Acα2-3Galβ1-4GlcβProN3) by immobilized enzyme is as follows:

[0179] LactoseβProN3 (50 mg), CMP-Neu5Ac (125 mg), Tris-HCl buffer (final concentration 100 mmol / L, pH 7.5), and MgCl2 (final concentration 20 mmol / L) were dissolved in a 50 mL centrifuge tube. Immobilized PmST3 (PmST3 to epoxy resin mass ratio 60:25000) was added, and double-distilled water was added to a total volume of 3 mL. The reaction system was then incubated in a shaker at 37 °C and 140 r / min for 24 hours. Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5) was performed. in / out After the reaction was completed, the immobilized PmST3 was separated by filtration to terminate the reaction. The supernatant of the reaction system was then concentrated by rotary evaporation and purified by Bio-gel P2 gel size exclusion chromatography and DEAE anion exchange column to obtain compound 16 (82.6 mg, 90%). NMR data are as follows: 1 H NMR (600 MHz, D2O) δ 4.51 (d, J = 7.9 Hz, 1 H), 4.47 (d, J = 8.0 Hz, 1 H), 4.09 (dd, J = 9.9, 2.7 Hz, 1 H), 4.00 – 3.95 (m, 2H), 3.94 (d, J = 2.6 Hz, 1 H), 3.90 – 3.53 (m, 16 H), 3.44 (t, J = 6.7 Hz, 2H), 3.29 (t, J = 8.3 Hz, 1 H), 2.74 (dd, J = 12.4, 4.5 Hz, 1 H), 2.01 (s, 3H), 1.89 (p, J = 6.5 Hz, 2 H), 1.78 (t, J = 12.1 Hz, 1 H); 13C NMR (151 MHz,D2O) δ 174.68, 173.57, 102.33, 101.82, 99.48, 77.92, 75.16, 74.84, 74.45,74.02, 72.56, 72.50, 71.46, 69.05, 68.02, 67.78, 67.16, 67.05, 62.27, 60.72, 59.09, 51.38, 47.56, 39.31, 27.94, 21.77.

[0180] The synthetic route for compound 16 is shown below.

[0181]

[0182] Example 22

[0183] Immobilized enzymes exhibit enhanced stability and improved reusability. This invention investigates their reusability using TLC to track the reactions in the specific embodiments described above. After the reaction is complete, the immobilized enzyme is separated, washed with water to remove residual reactants from its surface, and then fed into the same new reaction. Figure 3 This is a graph showing the reusability of the immobilized enzyme in an embodiment of the present invention. Figure 3 It can be seen from this:

[0184] Using Example 3 as a reaction template, repeated experiments were conducted. After 5 and 10 repeated uses, the activity of sialyl glyconucleotide synthase (NmCSS) was approximately 72% and 36% of the initial activity, respectively.

[0185] Using Example 20 as a reaction template, the experiment was repeated. After 5 and 10 repeated uses, the activity of α2,6-sialyltransferase (Pd2,6ST) was approximately 80% and 62% of the initial activity, respectively.

[0186] Using Example 21 as a reaction template, the experiment was repeated. After 5 and 10 repeated uses, the activity of α2,3-sialyltransferase (PmST3) was approximately 75% and 49% of the initial activity, respectively.

[0187] Using Example 17 as a reaction template, the experiment was repeated. After 5 and 10 repeated uses, the activity of α2,3-sialyltransferase (PmST1 M144D) was approximately 76% and 50% of the initial activity, respectively.

[0188] Using Example 19 as a reaction template, the experiment was repeated. After 5 and 10 repeated uses, the activity of α2,8-sialyltransferase (CjCstII) was approximately 79% and 55% of the initial activity, respectively.

[0189] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an immobilized enzyme, characterized in that, Includes the following steps: (1) Pretreatment of resin: Weigh the epoxy resin and place it in ammonium phosphate buffer solution, stir at room temperature, and discard the supernatant; (2) Immobilization of free enzyme: The pretreated resin is placed in ammonium phosphate buffer, the free enzyme to be immobilized is added, the mixture is shaken for a set time, and then filtered to obtain crude immobilized enzyme. (3) Purification of immobilized crude enzyme: Add ammonium phosphate buffer to the immobilized crude enzyme, shake, and filter to obtain immobilized enzyme; The epoxy resin is LX-1000HFA; The free enzyme is sialyl glyconucleotide synthase NmCSS, α2,3-sialyl transferase PmST1 M144D, α2,3-sialyl transferase PmST3, α2,6-sialyl transferase Pd2,6ST or α2,8-sialyl transferase CjCstII. The mass ratio of the added free enzyme to the epoxy resin is 6-200:25000; In step (2), the mixture is shaken at 18-22℃ for 22-24 hours; In step (3), the mixture is shaken at 28-32℃ for 4-6 hours; the amount of ammonium phosphate buffer used is 2-3 times that used in step (1).

2. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of epoxy resin to ammonium phosphate buffer is 1:6-10 mg / μL; or, the pH of the ammonium phosphate buffer is 6-8; the concentration of the ammonium phosphate buffer is 90-110 mM; or, the stirring time is 40-80 min.

3. The preparation method according to claim 1, characterized in that, The mass ratio of NmCSS to epoxy resin is 6-20:25000; or, the mass ratio of Pd2,6ST to epoxy resin is 100-200:25000; or, the mass ratio of PmST3 to epoxy resin is 60-120:25000; or, the mass ratio of PmST1 M144D to epoxy resin is 15-40:25000; or, the mass ratio of CjCstII to epoxy resin is 60-120:25000.

4. The immobilized enzyme prepared by the preparation method according to any one of claims 1-3.

5. The application of the immobilized enzyme as described in claim 4 in the sialylated glycan synthesis reaction.

6. The application as described in claim 5, characterized in that, The sialylated sugar chains are selected from compounds 1 Neu5Acα2-6Galβ1-4GlcβProN3, 2 Neu5Acα2-3Galβ1-4GlcβProN3, 3 Neu5Acα2-6GalNAcβProN3, 4 Neu5Gcα2-6GalNAcβProN3, 5 Kdnα2-6GalNAcβProN3, 6 Neu5Acα2-6GalβProN3, 7 Neu5Gcα2-6GalβProN3, 8 Neu5Gcα2-6GalβProN3, 9 Neu5Acα2-3GalβProN3, 10 Neu5Gcα2-3GalβproN3, 11 Kdnα2-3GalβProN3, and 12, which have a 3-azidopropylβProN3 linker at the anomeric position. Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3, compound 13 Neu5Gcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3, compound 14 Neu5Acα2-8Neu5Acα2-3Galβ1-4GlcβProN3, and compound 15 Neu5Acα2-6Galβ1-4Glc and compound 16 Neu5Acα2-3Galβ1-4Glc with hydroxyl groups at the anodic position; The structures of sialylated glycan compounds 1-16 are shown below: 。 7. The application as described in claim 6, characterized in that, The reaction acceptors were selected from free compound 20 Lactose, compound 21 GalNAcβProN3, compound 22 GalβProN3, compound 23 Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcβProN3, compound 24 Neu5Acα2-3Galβ1-4GlcβProN3, and compound 25 LactoseβProN3 with a 3-azidopropylβProN3 linker at the anotope. The structure of the receptor compound 20-25 is shown below: ； When the acceptor is compound 20, the donor is CMP-Neu5Ac; When the acceptor is compound 21, the reaction donor is CMP-Neu5Ac; or, when the acceptor is compound 21, the reaction donor is CMP-Neu5Gc; or, when the acceptor is compound 21, the reaction donor is CMP-Kdn. When the acceptor is compound 22, the reaction donor is CMP-Neu5Ac; or, when the acceptor is compound 22, the reaction donor is CMP-Neu5Gc; or, when the acceptor is compound 22, the reaction donor is CMP-Kdn. When the acceptor is compound 23, the donor is CMP-Neu5Ac; or, when the acceptor is compound 23, the donor is CMP-Neu5Gc. When the acceptor is compound 24, the donor is CMP-Neu5Ac; When the acceptor is compound 25, the donor is CMP-Neu5Ac.

8. A method for synthesizing sialylated sugar chains, characterized in that, The method includes using the immobilized enzyme as described in claim 4.

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