Chemical enzyme synthesis method of blood group trisaccharide

Abstract

The invention provides a chemical enzymatic synthesis method of blood group trisaccharide, which comprises the following steps: dissolving a substrate, GDP-Fuc and magnesium chloride in water, adjusting the pH value of the solution, adding enzyme alpha1 and 2-FucT, and stirring for reaction to obtain a first product reaction solution; adding an enzyme-removing solvent, standing, centrifugally filtering, taking supernatant, and evaporating to remove the enzyme-removing solvent, so as to obtain a first product aqueous solution; adding glycosylate, dissolving, adjusting the pH value, and adding glycosyltransferase to obtain a second product reaction solution; removing the glycosyl transferase by using an enzyme removal solvent, standing, centrifugally filtering, taking supernatant, and evaporating to remove the enzyme removal solvent, so as to obtain a second product aqueous solution; separating and purifying the second product aqueous solution, and freeze-drying to obtain a solid product; dissolving the solid product, adding Pd / C, introducing hydrogen, stirring, filtering to remove Pd / C, concentrating and freeze-drying to obtain the hemotriose. The method provides a new technical thought of synthesizing the blood group trisaccharide by a chemical enzyme method, is beneficial to popularization and application, and solves the problems of low yield and high price.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a chemical enzymatic synthesis method of blood type trisaccharide. Background Art

[0002] Blood group trisaccharide A and blood group trisaccharide B are commonly used biochemical reagents, both of which are used to express a type of molecule on the surface of red blood cells (RBCs) that can stimulate the body's natural or artificial immunity to produce corresponding antibodies. At present, in the blood group system, the clinical value and research significance of the ABO blood group system antigens and the Rh blood group system antigens are the most extensive. However, the market price of these substances is high and the suppliers are scarce, which has become a major bottleneck in research and application.

[0003] Although the research value of blood group trisaccharides is significant, its chemoenzymatic synthesis technology faces challenges. At present, there is no report on the chemoenzymatic synthesis of blood group trisaccharides A and B. Although there are studies that disclose the synthesis technology of blood group trisaccharides proNH2, the title "Diversity-oriented Enzymatic Modular Assembly of ABO Histo-blood Group Antigens" [Acs Catalysis, 2016:8140-8144.] discloses the synthesis and application of blood group trisaccharides proNH2, however, the final molecules synthesized are all linker-modified, non-oligosaccharide molecules, and cannot meet the needs of directly preparing blood group trisaccharides A and B.

[0004] Therefore, further research on blood type trisaccharides and breakthroughs in chemical enzymatic synthesis methods have become important issues that need to be urgently addressed in this field. The present invention is proposed in this context, aiming to achieve efficient and direct preparation of blood type trisaccharides A and B through an innovative chemical enzymatic synthesis method, providing strong support for blood type-related research and applications. Summary of the invention

[0005] Based on the above description, the present invention provides a chemical enzymatic synthesis method of blood type trisaccharide to achieve efficient and direct preparation of blood type trisaccharide A and B.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: The present invention provides a chemical enzymatic synthesis method of blood type trisaccharide, comprising the following steps: S1: dissolving the substrate, GDP-Fuc, and magnesium chloride in water, adjusting the pH of the solution, adding the enzyme α1,2-FucT, and stirring the reaction to obtain the first product reaction solution; S2, removing the enzyme α1,2-FucT in the first product reaction solution by using an enzyme removal solvent, leaving it for a while, centrifuging and filtering, taking the supernatant, and evaporating and removing the enzyme removal solvent to obtain a first product aqueous solution; S3, adding glycosylation to the aqueous solution of the first product, adjusting the pH of the solution after complete dissolution, and then adding glycosyltransferase, stirring and reacting to obtain a second product reaction solution; S4, removing the glycosyltransferase in the second product reaction solution by using an enzyme removal solvent, leaving it for a while, centrifuging and filtering, taking the supernatant, and evaporating and removing the enzyme removal solvent to obtain a second product aqueous solution; S5, separating and purifying the aqueous solution of the second product, and freeze-drying to obtain a solid product; S6. After dissolving the solid product, add Pd / C, introduce hydrogen, stir sufficiently, filter to remove Pd / C, concentrate and freeze-dry to obtain solid powdered blood type trisaccharide.

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, in step S3, the glycosylation product is a UDP-Gal solid or a UDP-GalNAc solid.

[0009] Furthermore, in step S3, when the glycosylated product is a UDP-Gal solid, the glycosyltransferase is an enzyme GTB; the molar ratio of the UDP-Gal solid to the first product in the first product aqueous solution is 2:1; The pH of the solution was adjusted to 7.0-8.0, and the stirring reaction temperature was 37±1°C.

[0010] Further, in step S3, when the glycosylated substance is a UDP-GalNAc solid, the glycosyltransferase is the enzyme BGTA; The molar ratio of UDP-GalNAc solid to the first product in the first product aqueous solution is 1:0.8; The pH of the solution was adjusted to 7.0-8.0, and the stirring reaction temperature was 37±1°C.

[0011] Further, in step S1, the substrate is a β-benzylgalactose substrate; The molar ratio of the substrate to the GDP-Fuc is 1:1; after magnesium chloride is added, the final concentration of magnesium chloride in the solution is 5 mM; Adjust the solution pH to 7.0-8.0 and stir the reaction at a temperature of 37±1°C.

[0012] Furthermore, in steps S2 and S4, the enzyme removal solvent is anhydrous ethanol, and the amounts of the enzyme removal solvent added twice are the same as the volumes of the first product reaction solution and the second product reaction solution, respectively.

[0013] Furthermore, in steps S2 and S4, the placing treatment after adding the enzyme removal solvent specifically includes: placing at 4° C. for 3-5 hours.

[0014] Furthermore, step S5 specifically includes: Step S501: Load the second product aqueous solution onto the pretreated C 18 The chromatography column is eluted with an elution solvent to obtain a second product solution; Step S502, subjecting the second product solution to rotary evaporation, concentration and freeze-drying to obtain the solid product.

[0015] Furthermore, in step S501, the elution solvent is a mixed solution of anhydrous methanol and water, wherein the volume ratio of anhydrous methanol to water is 1:4.

[0016] Furthermore, in step S6, the solvent for dissolving the solid product is anhydrous methanol.

[0017] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: The chemical enzymatic synthesis method of blood type trisaccharide provided by the present invention has the following beneficial effects compared with the existing methods: 1. The chemical enzymatic synthesis method of blood type trisaccharide solves the process of synthesis and amplification of blood type trisaccharide, provides a new technical idea of ​​chemical enzymatic synthesis method, which can be applied in the synthesis of similar oligosaccharide compounds, is conducive to promotion and application, and solves the problem of low yield and high price.

[0018] 2. The chemoenzymatic synthesis method of blood type trisaccharide can simply and directly obtain blood type trisaccharide to obtain the key raw material of carbohydrate-modified compounds, providing new ideas for sugar-protein / antibody research, conjugated drug development, in vitro diagnosis, medical device consumables and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 A schematic diagram of a process for the chemical enzymatic synthesis of blood type trisaccharide provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the test results of blood type trisaccharide A of the sample prepared in Example 1 of the present invention; Figure 3This is the mass spectrum of the blood type trisaccharide A sample prepared in Example 1 of the present invention; Figure 4 This is the hydrogen spectrum of the sample blood type trisaccharide A prepared in Example 1 of the present invention; Figure 5 This is the mass spectrum of the reaction result from substrate 1 to intermediate product 2 during the preparation process of Example 1 of the present invention; Figure 6 This is a TLC intuitive diagram of the reaction from substrate 1 to intermediate product 2 during the preparation process of Example 1 of the present invention; Figure 7 This is a TLC intuitive diagram of the reaction from intermediate product 2 to intermediate product 3 in the preparation process of Example 1 of the present invention; Figure 8 This is a mass spectrum of the purified intermediate product 2 obtained in the preparation process of Example 1 of the present invention; Fig. 9 This is a mass spectrum of the purified intermediate product 3 obtained in the preparation process of Example 1 of the present invention; Fig.10 This is a schematic diagram of the test results of the blood type trisaccharide B sample prepared in Example 2 of the present invention; Fig.11 This is the mass spectrum of the sample blood type trisaccharide B prepared in Example 2 of the present invention; Fig.12 This is the hydrogen spectrum of the sample blood type trisaccharide B prepared in Example 2 of the present invention; Fig.13 This is the mass spectrum of the reaction result from substrate 1 to intermediate product 2 in the preparation process of Example 2 of the present invention; Fig.14 This is the mass spectrum of the reaction result from intermediate product 2 to intermediate product 3 in the preparation process of Example 2 of the present invention; Fig.15 This is a TLC intuitive diagram of the reaction from substrate 1 to intermediate product 2 during the preparation process of Example 2 of the present invention; Fig.16 This is a TLC intuitive diagram of the reaction from intermediate product 2 to intermediate product 3 during the preparation process of Example 2 of the present invention; Fig.17 This is a mass spectrum of the purified intermediate product 2 obtained in the preparation process of Example 2 of the present invention; Fig.18 This is the mass spectrum of the intermediate product 3 obtained in the preparation process of Example 2 of the present invention after purification. DETAILED DESCRIPTION

[0021] The specific implementation of the present invention is described in detail below. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the protection scope of the present invention.

[0022] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0023] Unless defined otherwise, 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 belongs.

[0024] In the present invention, conventional reagents used in the experiments are all commercially available, and core raw materials (substrates, sugar nucleotides, enzymes, etc.) are all self-produced.

[0025] Combined with Figure 1 The chemical enzymatic synthesis method of blood type trisaccharide provided by the present invention comprises the following steps: S1: Dissolve the substrate, GDP-Fuc, and magnesium chloride in water, adjust the pH of the solution, add the enzyme α1,2-FucT, stir the reaction, and obtain the first product reaction solution.

[0026] Specifically, the substrate is Gal-R, wherein the R group is benzyl, specifically β-benzylgalactose substrate - D-Gal-β-Bz. The first product is H2-R.

[0027] The molar ratio of substrate to GDP-Fuc was 1:1; after magnesium chloride was added, the final concentration of magnesium chloride in the solution was 5 mM; In this step, the pH of the solution was adjusted to 7.0-8.0, and the stirring reaction temperature was 37±1°C.

[0028] S2. Remove the enzyme α1,2-FucT in the first product reaction solution by using an enzyme removal solvent, and place it at 4° C. for 3-5 hours; centrifuge and filter, take the supernatant, and evaporate to remove the enzyme removal solvent to obtain a first product aqueous solution.

[0029] The enzyme removal solvent is anhydrous ethanol, and the amount of the enzyme removal solvent added is the same as the volume of the first product reaction solution.

[0030] S3, add glycosylation to the aqueous solution of the first product, adjust the pH of the solution after complete dissolution, add glycosyltransferase, stir and react, and obtain a second product reaction solution. The second product is A3 / B3-R.

[0031] Specifically, the pH of the solution was adjusted to 7.0-8.0, and the stirring reaction temperature was 37±1°C.

[0032] Wherein, the glycosylation product is UDP-Gal solid or UDP-GalNAc solid.

[0033] When the glycosylated product is a UDP-Gal solid, the glycosyltransferase is the enzyme GTB; the molar ratio of the UDP-Gal solid to the first product in the first product aqueous solution is 2:1; When the glycosylated product is UDP-GalNAc solid, the glycosyltransferase is enzyme BGTA; the molar ratio of UDP-GalNAc solid to the first product in the first product aqueous solution is 1:0.8.

[0034] S4. Remove the glycosyltransferase in the second product reaction solution by using an enzyme removal solvent, and place it at 4° C. for 3-5 hours; centrifuge and filter, take the supernatant, and evaporate to remove the enzyme removal solvent to obtain a second product aqueous solution.

[0035] The enzyme removal solvent is anhydrous ethanol, and the amount of the enzyme removal solvent added is the same as the volume of the second product reaction solution.

[0036] S5, separating and purifying the second product aqueous solution, and obtaining a solid product after freeze-drying.

[0037] This step specifically includes: Step S501: Load the second product aqueous solution onto the pretreated C 18 The chromatography column is eluted with an elution solvent to obtain a second product solution; Step S502: rotary evaporate the second product solution, concentrate it, and then freeze-dry it to obtain a solid product.

[0038] S6. After the solid product is dissolved, Pd / C is added, and hydrogen is introduced, the mixture is stirred sufficiently, the Pd / C is removed by filtration, and the mixture is concentrated and freeze-dried to obtain solid powdered blood type trisaccharide A / B.

[0039] Wherein, the solvent for dissolving the solid product is anhydrous methanol.

[0040] Correspondingly, when the glycosylated substance is a UDP-Gal solid and the glycosyltransferase is the enzyme GTB, the above-mentioned synthesis idea is:

[0041] When the glycosylated substance is a UDP-GalNAc solid and the glycosyltransferase is the enzyme BGTA, the above-mentioned synthesis idea is:

[0042] The following is combined with Figures 2 to 18 , further provide multiple embodiments for introduction: Example 1 The chemoenzymatic synthesis method of blood group trisaccharide A provided in this embodiment, the chemoenzymatic synthesis idea in this embodiment is as follows:

[0043] The specific steps include: 1. Dissolve 16g of substrate 1 (β-benzylgalactose substrate - D-Gal-β-Bz), 37g of GDP-Fuc, and 4.76g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), add enzyme α1,2-FucT, and react at 37±1℃. Use TLC to detect the reaction end point to obtain product 2 reaction solution; 2. Add an equal amount of anhydrous ethanol to the reaction solution of product 2 to precipitate the enzyme, place at 4°C for 4 hours, and then centrifuge and filter to obtain the supernatant to obtain the ethanol aqueous solution of product 2; 3. The ethanol solution of product 2 was subjected to rotary evaporation at 40°C to remove ethanol, to obtain an aqueous solution of product 2. The HPLC quantification showed that the amount of product 2 in the aqueous solution of product 2 was 22 g; 4. Add 41 g of UDP-GalNAc solid to the aqueous solution of product 2 (product 2 is 22 g). After complete dissolution, adjust the pH to 7.5 (±0.5), add enzyme BGTA, and react at 37±1°C. Use TLC to detect the reaction end point to obtain a product 3 reaction solution; 5. Add an equal amount of anhydrous ethanol to the reaction solution of product 3 to precipitate the enzyme, place at 4°C for 4 hours, and then centrifuge and filter to obtain the supernatant to obtain the ethanol solution of product 3; 6. The product 3 ethanol aqueous solution is subjected to rotary evaporation at 40°C to remove ethanol to obtain a product 3 aqueous solution; 7. The product 3 aqueous solution obtained in the previous step is loaded onto the pretreated C 18 The product was eluted using a mixed solution of anhydrous methanol and water (the volume ratio of anhydrous methanol to water was 1:4) on a chromatography column to obtain a methanol-water solution of product 3; 8. The methanol-water solution of product 3 was concentrated by rotary evaporation and freeze-dried to obtain 26 g of solid product 3; 9. The solid product 3 was dissolved in anhydrous methanol, and then Pd / C was added, and hydrogen was introduced, and stirred overnight; after TLC detection, the Pd / C was filtered out to obtain a methanol solution of the product A3; 10. The methanol solution of product A3 was concentrated by rotary evaporation, and water was added to dissolve it, followed by freeze-drying to obtain 19 g of solid powder of blood type trisaccharide A.

[0044] The purity of blood type trisaccharide A in the tested sample was >95%; the overall yield was 61%. Figure 2 Its mass spectrum and hydrogen spectrum are shown as follows: Figure 3 and Figure 4 shown.

[0045] Among them, the relevant verification of intermediate products 2 and 3 in this embodiment is as follows Figures 5 to 9 As shown, Figure 5 Shown is the mass spectrum (MS) of intermediate 2; Figure 6 and Figure 7 Shown is a TLC intuitive diagram of the reaction of intermediate product 2 and intermediate product 3; Figure 8 and Fig. 9 Shown are the mass spectra of the purified intermediate product 2 and intermediate product 3. According to the above figures, it can be seen that in this embodiment, both the intermediate product and the final product are verified.

[0046] Example 2 The chemoenzymatic synthesis method of blood group trisaccharide B provided in this embodiment, the chemoenzymatic synthesis idea in this embodiment is as follows:

[0047] The specific steps include: 1. Dissolve 16g of substrate 1 (β-benzylgalactose substrate - D-Gal-β-Bz), 37g of GDP-Fuc, and 4.76g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), add enzyme α1,2-FucT, and react at 37±1℃. Use TLC to detect the reaction end point to obtain product 2 reaction solution; 2. Add an equal amount of anhydrous ethanol to the reaction solution of product 2 to precipitate the enzyme, place at 4°C for 5 hours, and centrifuge to obtain the supernatant to obtain the ethanol solution of product 2; 3. The ethanol solution of product 2 was subjected to rotary evaporation at 40°C to remove ethanol, to obtain an aqueous solution of product 2. The HPLC quantification showed that the amount of product 2 in the aqueous solution of product 2 was 22 g; 4. Add 41 g of UDP-Gal solid to the aqueous solution of product 2 (product 2 is 22 g). After complete dissolution, adjust the pH to 7.5 (±0.5), add enzyme GTB, and react at 37±1°C. Use TLC to detect the reaction end point to obtain a product 3 reaction solution; 5. Add an equal amount of anhydrous ethanol to the reaction solution of product 3 to precipitate the enzyme, place at 4°C for 4 hours, and then centrifuge and filter to obtain the supernatant to obtain the ethanol solution of product 3; 6. The product 3 ethanol aqueous solution is subjected to rotary evaporation at 40°C to remove ethanol to obtain a product 3 aqueous solution; 7. The product 3 aqueous solution obtained in the previous step is loaded onto the pretreated C 18 The product was eluted using a mixed solution of anhydrous methanol and water (the volume ratio of anhydrous methanol to water was 1:4) on a chromatography column to obtain a methanol-water solution of product 3; 8. The methanol-water solution of product 3 was concentrated by rotary evaporation and freeze-dried to obtain 12 g of solid product 3; 9. After dissolving the product 3 in anhydrous methanol, add Pd / C, introduce hydrogen, and stir overnight; after TLC detection, filter and remove Pd / C after the reaction is complete to obtain a methanol solution of the product B3; 10. The methanol solution of product B3 was concentrated by rotary evaporation, and water was added to dissolve it, followed by freeze-drying to obtain 9.8 g of solid powder of blood type trisaccharide B.

[0048] The purity of blood type trisaccharide B in the tested sample was >95%; the overall yield was 33%. Fig.10 Its mass spectrum and hydrogen spectrum are shown as follows: Fig.11 and Fig.12 shown.

[0049] Among them, the relevant verification of intermediate products 2 and 3 in this embodiment is as follows Figures 13 to 18 As shown, Fig.13 and Fig.14 Shown are the mass spectra (MS) of intermediate 2 and intermediate 3; Fig.15 and Fig.16 Shown is a TLC intuitive diagram of the reaction of intermediate product 2 and intermediate product 3; Fig.17 and Fig.18 Shown are the mass spectra of the purified intermediate product 2 and intermediate product 3. According to the above figures, it can be seen that in this embodiment, both the intermediate product and the final product are verified.

[0050] Comparative Example 1 Based on Example 1, the difference of this comparative example is that 1 g of substrate 4 (R group is H), 1.7 g of GDP-Fuc, and 0.4 g of magnesium chloride are directly dissolved in water, the pH is adjusted to 7.5 (±0.5), and then the enzyme α1,2-FucT is added, and the reaction is carried out at 37±1°C, and the reaction is detected by TLC.

[0051] The corresponding ideal synthesis idea is:

[0052] TLC detection revealed no intermediate 5, and MS verification confirmed that no intermediate was generated, and the reaction was terminated.

[0053] Comparative Example 2 On the basis of Example 1, the difference of this comparative example is: The corresponding ideal synthesis idea is:

[0054] Dissolve 1 g of substrate 6 (R group is methyl), 3.7 g of GDP-Fuc, and 0.4 g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), add enzyme α1,2-FucT, and react at 37±1°C. Detect the reaction end point by TLC to obtain a reaction solution of product 7; Add an equal amount of anhydrous ethanol to the reaction solution of product 7 to precipitate the enzyme, place at 4°C for 4 hours, and then centrifuge and filter to obtain the supernatant to obtain the ethanol aqueous solution of product 7; The ethanol solution of product 7 was subjected to rotary evaporation at 40°C to remove ethanol, thereby obtaining an aqueous solution of product 7. The HPLC quantification showed that the amount of product 7 in the aqueous solution of product 7 was 1.4 g. 3.3 g of UDP-GalNAc solid was added to the aqueous solution of product 7 (1.4 g of product 7), and after complete dissolution, the pH was adjusted to 7.5 (±0.5), and then the enzyme BGTA was added, and the reaction was carried out at 37±1°C. The reaction end point was detected by TLC to obtain a reaction solution of product 8; An equal amount of anhydrous ethanol was added to the reaction solution of product 8 to precipitate the enzyme, and after standing at 4°C for 4 hours, the supernatant was centrifuged and filtered to obtain an ethanol aqueous solution of product 8; The product 8 ethanol aqueous solution was subjected to rotary evaporation at 40°C to remove ethanol, thereby obtaining a product 8 aqueous solution; The aqueous solution of product 8 obtained in the previous step was loaded onto the pretreated C 18 The product was eluted using a mixed solution of anhydrous methanol and water (the volume ratio of anhydrous methanol to water was 1:4) on a chromatography column to obtain a methanol-water solution of product 8; The methanol-water solution of product 8 was concentrated by rotary evaporation and then freeze-dried to obtain 1.1 g of solid product 8; The product 8 was dissolved in anhydrous methanol, Pd / C was added, hydrogen was introduced, and the mixture was stirred overnight; Finally, TLC and MS combined detection revealed that no product A3 was generated, and the reaction was terminated by filtration.

[0055] Comparative Example 3 On the basis of Example 2, the difference of this comparative example is: Directly dissolve 1 g of substrate 4 (R group is H), 1.7 g of GDP-Fuc, and 0.4 g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), add enzyme α1,2-FucT, and react at 37±1°C. Use TLC to detect the reaction.

[0056] The corresponding ideal synthesis idea is:

[0057] TLC detection revealed no intermediate product, and MS verification confirmed that no intermediate product was generated, and the reaction was terminated.

[0058] Comparative Example 4 On the basis of Example 2, the difference of this comparative example is: The corresponding ideal synthesis idea is:

[0059] Dissolve 1g of substrate 6 (R group is methyl), 3.7g of GDP-Fuc, and 0.4g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), add enzyme α1,2-FucT, and react at 37±1℃. Detect the reaction end point by TLC to obtain the reaction solution of product 7; Add an equal amount of anhydrous ethanol to the reaction solution of product 7 to precipitate the enzyme, place at 4°C for 4 hours, and then centrifuge and filter to obtain the supernatant to obtain the ethanol aqueous solution of product 7; The ethanol solution of product 7 was subjected to rotary evaporation at 40°C to remove ethanol, thereby obtaining an aqueous solution of product 7. The HPLC quantification showed that the amount of product 7 in the aqueous solution of product 7 was 1.4 g. 4 g of UDP-Gal solid was added to the aqueous solution of product 7 (1.4 g of product 7), and after complete dissolution, the pH was adjusted to 7.5 (±0.5), and then enzyme GTB was added, and the reaction was carried out at 37±1°C. The reaction end point was detected by TLC to obtain a reaction solution of product 8; An equal amount of anhydrous ethanol was added to the reaction solution of product 8 to precipitate the enzyme, and after standing at 4°C for 4 hours, the supernatant was centrifuged and filtered to obtain an ethanol aqueous solution of product 8; The product 8 ethanol aqueous solution was subjected to rotary evaporation at 40°C to remove ethanol, thereby obtaining a product 8 aqueous solution; The aqueous solution of product 8 obtained in the previous step was loaded onto a pretreated C18 chromatography column, and the product was eluted with a mixed solution of anhydrous methanol and water (the volume ratio of anhydrous methanol to water was 1:4) to obtain a methanol aqueous solution of product 8; The methanol-water solution of product 8 was concentrated by rotary evaporation and then freeze-dried to obtain 720 mg of solid product 8; The product 8 was dissolved in anhydrous methanol, Pd / C was added, hydrogen was introduced, and the mixture was stirred overnight; Finally, TLC and MS combined detection revealed that no product B3 was generated, and the reaction was terminated by filtration.

[0060] Combining the above embodiments and comparative examples, it can be seen that only when the R group in the substrate, i.e., the linker, is a benzyl group, can the oligosaccharide blood type trisaccharide A / B be directly synthesized; when the R group is H or methyl, the oligosaccharide blood type trisaccharide cannot be directly synthesized.

[0061] In summary, the chemoenzymatic synthesis method of blood type trisaccharide provided in the embodiment of the present invention solves the process of synthesis and amplification of blood type trisaccharide, provides a new technical idea of ​​chemoenzymatic synthesis method, which can be applied in the synthesis of similar oligosaccharide compounds, is conducive to promotion and application, and solves the problem of low yield and high price; the chemoenzymatic synthesis method of blood type trisaccharide can simply and directly obtain blood type trisaccharide to obtain the key raw material of carbohydrate-modified compounds, and provides new ideas for many fields such as sugar-protein / antibody research, conjugated drug development, in vitro diagnosis, medical device consumables, etc.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chemical enzymatic synthesis method of blood type trisaccharide, characterized in that: The steps include: S1: dissolving the substrate, GDP-Fuc, and magnesium chloride in water, adjusting the pH of the solution, adding the enzyme α1,2-FucT, and stirring the reaction to obtain the first product reaction solution; S2, removing the enzyme α1,2-FucT in the first product reaction solution by using an enzyme removal solvent, leaving it for a while, centrifuging and filtering, taking the supernatant, and evaporating and removing the enzyme removal solvent to obtain a first product aqueous solution; S3, adding glycosylation to the aqueous solution of the first product, adjusting the pH of the solution after complete dissolution, and then adding glycosyltransferase, stirring and reacting to obtain a second product reaction solution; S4, removing the glycosyltransferase in the second product reaction solution by using an enzyme removal solvent, leaving it for a while, centrifuging and filtering, taking the supernatant, and evaporating and removing the enzyme removal solvent to obtain a second product aqueous solution; S5, separating and purifying the aqueous solution of the second product, and freeze-drying to obtain a solid product; S6. After dissolving the solid product, add Pd / C, introduce hydrogen, stir sufficiently, filter to remove Pd / C, concentrate and freeze-dry to obtain solid powdered blood type trisaccharide.

2. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: In step S3, the glycosylation product is UDP-Gal solid or UDP-GalNAc solid.

3. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 2, characterized in that: In step S3, when the glycosylated product is a UDP-Gal solid, the glycosyltransferase is the enzyme GTB; the molar ratio of the UDP-Gal solid to the first product in the first product aqueous solution is 2:1; The pH of the solution was adjusted to 7.0-8.0, and the stirring reaction temperature was 37±1°C.

4. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 2, characterized in that: In step S3, when the glycosylated product is UDP-GalNAc solid, the glycosyltransferase is enzyme BGTA; the molar ratio of UDP-GalNAc solid to the first product in the first product aqueous solution is 1:0.8; The pH of the solution was adjusted to 7.0-8.0, and the stirring reaction temperature was 37±1°C.

5. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: In step S1, the substrate is β-benzylgalactose substrate; The molar ratio of the substrate to the GDP-Fuc is 1:1; after magnesium chloride is added, the final concentration of magnesium chloride in the solution is 5 mM; Adjust the solution pH to 7.0-8.0 and stir the reaction at a temperature of 37±1°C.

6. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: In steps S2 and S4, the enzyme removal solvent is anhydrous ethanol, and the amount of the enzyme removal solvent added twice is the same as the volume of the first product reaction solution and the second product reaction solution, respectively.

7. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: In steps S2 and S4, the placing treatment after adding the enzyme removal solvent specifically includes: placing at 4° C. for 3-5 hours.

8. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: Step S5 specifically includes: Step S501: Load the second product aqueous solution onto the pretreated C 18 The chromatography column is eluted with an elution solvent to obtain a second product solution; Step S502, subjecting the second product solution to rotary evaporation, concentration and freeze-drying to obtain the solid product.

9. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: In step S501, the elution solvent is a mixed solution of anhydrous methanol and water, wherein the volume ratio of anhydrous methanol to water is 1:

4.

10. The chemical enzymatic synthesis method of blood type trisaccharide according to claim 1, characterized in that: In step S6, the solvent for dissolving the solid product is anhydrous methanol.

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