A chemoenzymatic synthesis method of a blood group trisaccharide

Abstract

The present invention provides a chemoenzymatic synthesis method for blood group trisaccharide, which includes: dissolving a substrate, GDP-Fuc, and magnesium chloride in water, adjusting the pH of the solution, adding the enzyme α1,2-FucT, and stirring for reaction to obtain a first product reaction solution; adding a de-enzyme solvent, standing, centrifuging and filtering, taking the supernatant, and evaporating to remove the de-enzyme solvent to obtain an aqueous solution of the first product; adding a glycosylated product, adjusting the pH after dissolution, and adding a glycosyltransferase to obtain a second product reaction solution; using a de-enzyme solvent to remove the glycosyltransferase, standing, centrifuging and filtering, taking the supernatant, and evaporating to remove the de-enzyme solvent to obtain an aqueous solution of the second product; separating and purifying the aqueous solution of the second product, and freeze-drying to obtain a solid product; dissolving the solid product, adding Pd / C, introducing hydrogen gas, stirring and filtering to remove Pd / C, concentrating and freeze-drying to obtain the blood group trisaccharide. This method provides a new technical idea for the chemoenzymatic synthesis of blood group trisaccharide, which is conducive 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:

[0007] The present invention provides a chemical enzymatic synthesis method of blood type trisaccharide, comprising the following steps:

[0008] 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;

[0009] S2. Remove the enzyme α1,2-FucT in the first product reaction solution with an enzyme removal solvent. After standing, centrifuge and filter, take the supernatant, and evaporate to remove the enzyme removal solvent to obtain an aqueous solution of the first product;

[0010] S3. Add a glycosylating agent to the aqueous solution of the first product. After complete dissolution, adjust the pH of the solution, and then add a glycosyltransferase, and stir and react to obtain a second product reaction solution;

[0011] S4. Remove the glycosyltransferase in the second product reaction solution with an enzyme removal solvent. After standing, centrifuge and filter, take the supernatant, and evaporate to remove the enzyme removal solvent to obtain an aqueous solution of the second product;

[0012] S5. Separate and purify the aqueous solution of the second product, and freeze-dry to obtain a solid product;

[0013] S6. After dissolving the solid product, add Pd / C, and introduce hydrogen, stir well, filter to remove Pd / C, concentrate and freeze-dry to obtain a solid powder of blood group trisaccharide.

[0014] Based on the above technical solutions, the present invention can be further improved as follows.

[0015] Further, in step S3, the glycosylating agent is UDP-Gal solid or UDP-GalNAc solid.

[0016] Further, in step S3, when the glycosylating agent is UDP-Gal solid, the glycosyltransferase is enzyme GTB; the molar ratio of UDP-Gal solid to the first product in the aqueous solution of the first product is 2:1;

[0017] Among them, adjust the pH of the solution to 7.0 - 8.0, and the stirring reaction temperature is 37 ± 1°C.

[0018] Further, in step S3, when the glycosylating agent is UDP-GalNAc solid, the glycosyltransferase is enzyme BGTA;

[0019] The molar ratio of UDP-GalNAc solid to the first product in the aqueous solution of the first product is 1:0.8;

[0020] Among them, adjust the pH of the solution to 7.0 - 8.0, and the stirring reaction temperature is 37 ± 1°C.

[0021] Further, in step S1, the substrate is a β-benzyl galactose substrate;

[0022] The molar ratio of the substrate to GDP-Fuc is 1:1; after adding magnesium chloride, the final concentration of magnesium chloride in the solution is 5 mM;

[0023] Adjust the pH of the solution to 7.0 - 8.0, and the stirring reaction temperature is 37 ± 1°C.

[0024] Furthermore, in steps S2 and S4, the enzyme-removing solvent is anhydrous ethanol, and the addition amounts of the two enzyme-removing solvents are respectively the same as the volumes of the first product reaction solution and the second product reaction solution.

[0025] Furthermore, in steps S2 and S4, the placement treatment after adding the enzyme-removing solvent specifically includes: placing at 4°C for 3 - 5 h.

[0026] Furthermore, step S5 specifically includes:

[0027] Step S501: Load the aqueous solution of the second product onto a pre-treated C 18 chromatography column, and elute with an elution solvent to obtain a second product solution;

[0028] Step S502: Rotate and evaporate the second product solution, and lyophilize after concentration to obtain the solid product.

[0029] 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.

[0030] Furthermore, in step S6, the solvent for dissolving the solid product is anhydrous methanol.

[0031] Compared with the prior art, the technical solution of the present application has the following beneficial technical effects:

[0032] The chemoenzymatic synthesis method of blood group trisaccharide provided by the present invention has the following beneficial effects compared with the existing methods:

[0033] 1. The chemoenzymatic synthesis method of blood group trisaccharide solves the process of synthesizing and scaling up blood group trisaccharide, provides a new technical idea of chemoenzymatic synthesis method, can be applied in the synthesis of similar oligosaccharide compounds, is conducive to popularization and application, and solves the problems of low yield and high price.

[0034] 2. The chemoenzymatic synthesis method of blood group trisaccharide can simply and directly obtain blood group trisaccharide to obtain the key raw material for sugar-modified compounds, and provides new ideas for multiple fields such as glycoprotein / antibody research, conjugate drug development, in vitro diagnosis, and medical device consumables. Description of the Drawings

[0035] To more clearly illustrate the technical solution of the present invention, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0036] Figure 1 Schematic flow chart of the chemoenzymatic synthesis method of blood group trisaccharide provided by the embodiment of the present invention;

[0037] Figure 2 Schematic diagram of the detection result of the sample blood group trisaccharide A prepared in Example 1 of the present invention;

[0038] Figure 3 Mass spectrum of the sample blood group trisaccharide A prepared in Example 1 of the present invention;

[0039] Figure 4 1H NMR spectrum of the sample blood group trisaccharide A prepared in Example 1 of the present invention;

[0040] Figure 5 Mass spectrum of the reaction result from substrate 1 to intermediate 2 during the preparation process of Example 1 of the present invention;

[0041] Figure 6 TLC visualization of the reaction from substrate 1 to intermediate 2 during the preparation process of Example 1 of the present invention;

[0042] Figure 7 TLC visualization of the reaction from intermediate 2 to intermediate 3 during the preparation process of Example 1 of the present invention;

[0043] Figure 8 Purified mass spectrum of intermediate 2 obtained during the preparation process of Example 1 of the present invention;

[0044] Figure 9 Purified mass spectrum of intermediate 3 obtained during the preparation process of Example 1 of the present invention;

[0045] Figure 10 Schematic diagram of the detection result of the sample blood group trisaccharide B prepared in Example 2 of the present invention;

[0046] Figure 11 Mass spectrum of the sample blood group trisaccharide B prepared in Example 2 of the present invention;

[0047] Figure 12 1H NMR spectrum of the sample blood group trisaccharide B prepared in Example 2 of the present invention;

[0048] Figure 13This is the mass spectrometry map of the reaction result from substrate 1 to intermediate product 2 during the preparation process of Example 2 of the present invention;

[0049] Figure 14 This is the mass spectrometry map of the reaction result from intermediate product 2 to intermediate product 3 during the preparation process of Example 2 of the present invention;

[0050] Figure 15 This is the TLC visualization diagram of the reaction from substrate 1 to intermediate product 2 during the preparation process of Example 2 of the present invention;

[0051] Figure 16 This is the TLC visualization diagram of the reaction from intermediate product 2 to intermediate product 3 during the preparation process of Example 2 of the present invention;

[0052] Figure 17 This is the purified mass spectrometry map of intermediate product 2 obtained during the preparation process of Example 2 of the present invention;

[0053] Figure 18 This is the purified mass spectrometry map of intermediate product 3 obtained during the preparation process of Example 2 of the present invention. Detailed Embodiments

[0054] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the protection scope of the present invention.

[0055] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs.

[0057] In the present invention, the conventional reagents used in the experiments are all commercially available, and the core raw materials (substrates, sugar nucleotides, enzymes, etc.) are all self-produced.

[0058] Combined with the attached Figure 1 , the chemoenzymatic synthesis method of the blood group trisaccharide provided by the present invention includes the following steps:

[0059] S1: Dissolve the substrate, GDP-Fuc, and magnesium chloride in water, adjust the pH of the solution, and then add the enzyme α1,2-FucT, and stir and react to obtain the first product reaction solution.

[0060] Specifically, the substrate is Gal-R, where the R group is benzyl, specifically the β-benzyl galactose substrate - D-Gal-β-Bz. The first product is H2-R.

[0061] The molar ratio of the substrate to GDP-Fuc is 1:1; after adding magnesium chloride, the final concentration of magnesium chloride in the solution is 5 mM;

[0062] In this step, the pH of the solution is adjusted to 7.0 - 8.0, and the stirring reaction temperature is 37 ± 1°C.

[0063] S2. Use an enzyme-removing solvent to remove the enzyme α1,2-FucT from the first product reaction solution. After standing, specifically, stand at 4°C for 3 - 5 h; centrifuge and filter, take the supernatant, and evaporate to remove the enzyme-removing solvent to obtain an aqueous solution of the first product.

[0064] Among them, the enzyme-removing solvent is anhydrous ethanol, and the added amount of the enzyme-removing solvent is the same as the volume of the first product reaction solution respectively.

[0065] S3. Add a glycosylating agent to the aqueous solution of the first product. After complete dissolution, adjust the pH of the solution, and then add a glycosyltransferase and stir to react to obtain a second product reaction solution. The second product is A3 / B3-R.

[0066] Specifically, the pH of the solution is adjusted to 7.0 - 8.0, and the stirring reaction temperature is 37 ± 1°C.

[0067] Among them, the glycosylating agent is UDP-Gal solid or UDP-GalNAc solid.

[0068] When the glycosylating agent is UDP-Gal solid, the glycosyltransferase is enzyme GTB; the molar ratio of UDP-Gal solid to the first product in the aqueous solution of the first product is 2:1;

[0069] When the glycosylating agent is UDP-GalNAc solid, the glycosyltransferase is enzyme BGTA; the molar ratio of UDP-GalNAc solid to the first product in the aqueous solution of the first product is 1:0.8.

[0070] S4. Use an enzyme-removing solvent to remove the glycosyltransferase from the second product reaction solution. After standing, specifically, stand at 4°C for 3 - 5 h; centrifuge and filter, take the supernatant, and evaporate to remove the enzyme-removing solvent to obtain an aqueous solution of the second product.

[0071] Among them, the enzyme-removing solvent is anhydrous ethanol, and the added amount of the enzyme-removing solvent is the same as the volume of the second product reaction solution respectively.

[0072] S5. Separate and purify the aqueous solution of the second product, and obtain a solid product after freeze-drying.

[0073] This step specifically includes:

[0074] Step S501: Load the aqueous solution of the second product onto the pre-treated C 18 chromatographic column, and elute with an elution solvent to obtain a solution of the second product;

[0075] Step S502: Rotavaporize the solution of the second product, concentrate it, and then lyophilize to obtain a solid product.

[0076] S6: After dissolving the solid product, add Pd / C, introduce hydrogen, stir thoroughly, filter to remove Pd / C, concentrate, and lyophilize to obtain the solid powder of blood group trisaccharide A / B.

[0077] Among them, the solvent for dissolving the solid product is anhydrous methanol.

[0078] Correspondingly, when the glycosyl donor is UDP-Gal solid and the glycosyltransferase is enzyme GTB, the above synthetic idea is as follows:

[0079]

[0080] When the glycosyl donor is UDP-GalNAc solid and the glycosyltransferase is enzyme BGTA, the above synthetic idea is as follows:

[0081]

[0082] The following further provides multiple examples in combination with the attached Figures 2 to 18 , for introduction: Example 1

[0083] The chemoenzymatic synthesis method of blood group trisaccharide A provided in this example, the synthetic idea of the chemoenzymatic method in this example is as follows:

[0084]

[0085] The specific steps include:

[0086] 1. Dissolve 16 g of substrate 1 (β-benzyl galactose substrate - D-Gal-β-Bz), 37 g of GDP-Fuc, and 4.76 g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), then add enzyme α1,2-FucT, place it under the condition of 37 ± 1 °C for reaction, and use TLC to detect the end point of the reaction to obtain the reaction solution of product 2;

[0087] 2. Add an equal amount of absolute ethanol to the reaction solution of product 2 to precipitate the enzyme, place it at 4 °C for 4 h, then centrifuge and filter to take the supernatant to obtain the ethanol aqueous solution of product 2;

[0088] 3. Rotavaporize the ethanol aqueous solution of product 2 at 40 °C to remove ethanol, obtain the aqueous solution of product 2, and quantitatively analyze by HPLC. The amount of product 2 in the aqueous solution of product 2 is 22 g;

[0089] 4. Add 41 g of solid UDP-GalNAc to the aqueous solution of product 2 (22 g of product 2). After complete dissolution, adjust the pH to 7.5 (±0.5), then add the enzyme BGTA, and place it under the condition of 37 ± 1 °C for reaction. Use TLC to detect the end point of the reaction to obtain the reaction solution of product 3;

[0090] 5. Add an equal amount of absolute ethanol to the reaction solution of product 3 to precipitate and remove the enzyme. After standing at 4 °C for 4 h, centrifuge and filter to take the supernatant to obtain the ethanol aqueous solution of product 3;

[0091] 6. Rotate and evaporate the ethanol aqueous solution of product 3 at 40 °C to remove ethanol to obtain the aqueous solution of product 3;

[0092] 7. Load the aqueous solution of product 3 obtained in the previous step onto the pre-treated C 18 chromatography column, and elute the product with a mixed solution of absolute methanol and water (the volume ratio of absolute methanol to water is 1:4) to obtain the methanol aqueous solution of product 3;

[0093] 8. After rotating and evaporating and concentrating the methanol aqueous solution of product 3, freeze-dry it to obtain 26 g of solid product 3;

[0094] 9. Dissolve the solid product 3 in absolute methanol, add Pd / C, and introduce hydrogen gas, and stir overnight; after TLC detects that the reaction is complete, filter to remove Pd / C to obtain the methanol solution of product A3;

[0095] 10. Rotate and evaporate and concentrate the methanol solution of product A3, add water to dissolve it, and then freeze-dry it to obtain 19 g of solid powder of blood group trisaccharide A.

[0096] After detection, the purity of blood group trisaccharide A in the sample > 95%; the overall yield is 61%, as Figure 2 shown. Its mass spectrum and hydrogen spectrum are respectively as Figure 3 and Figure 4 shown.

[0097] Among them, the relevant verification of intermediate products 2 and 3 in this example is as Figures 5 to 9 shown, where Figure 5 shown is the mass spectrum (MS) of intermediate product 2; Figure 6 and Figure 7 shown are the reaction TLC visualization diagrams of intermediate product 2 and intermediate product 3; Figure 8 and Figure 9 shown are the purified mass spectra of intermediate product 2 and intermediate product 3. According to the above diagrams, it can be seen that in this example, both intermediate products and the final product are verified.

[0098] Example 2

[0099] The chemoenzymatic synthesis method of blood group trisaccharide B provided in this example has the following synthetic idea for the chemoenzymatic method in this example:

[0100]

[0101] The specific steps are as follows:

[0102] 1. Dissolve 16 g of substrate 1 (β-benzyl galactose substrate - D-Gal-β-Bz), 37 g of GDP-Fuc, and 4.76 g of magnesium chloride in water, adjust the pH to 7.5 (±0.5), then add the enzyme α1,2-FucT, and place it under the condition of 37 ± 1 °C for reaction. Use TLC to detect the end point of the reaction to obtain the reaction solution of product 2;

[0103] 2. Add an equal amount of absolute ethanol to the reaction solution of product 2 to precipitate and remove the enzyme. After placing it at 4 °C for 5 h, centrifuge and filter to take the supernatant to obtain the ethanol aqueous solution of product 2;

[0104] 3. Rotavaporize the ethanol aqueous solution of product 2 at 40 °C to remove ethanol, obtain the aqueous solution of product 2, and quantify it by HPLC. The product 2 in the aqueous solution of product 2 is 22 g;

[0105] 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), then add the enzyme GTB, and place it under the condition of 37 ± 1 °C for reaction. Use TLC to detect the end point of the reaction to obtain the reaction solution of product 3;

[0106] 5. Add an equal amount of absolute ethanol to the reaction solution of product 3 to precipitate and remove the enzyme. After placing it at 4 °C for 4 h, centrifuge and filter to take the supernatant to obtain the ethanol aqueous solution of product 3;

[0107] 6. Rotavaporize the ethanol aqueous solution of product 3 at 40 °C to remove ethanol to obtain the aqueous solution of product 3;

[0108] 7. Load the aqueous solution of product 3 obtained in the previous step onto a pre-treated C 18 chromatographic column, and elute the product with a mixed solution of absolute methanol and water (the volume ratio of absolute methanol to water is 1:4) to obtain the methanol aqueous solution of product 3;

[0109] 8. After rotary evaporation and concentration of the methanol aqueous solution of product 3, lyophilize it to obtain 12 g of solid product 3;

[0110] 9. Dissolve product 3 in absolute methanol, add Pd / C, and introduce hydrogen gas, and stir overnight; after detecting the complete reaction by TLC, filter to remove Pd / C to obtain the methanol solution of product B3;

[0111] 10. After the methanol solution of product B3 was concentrated by rotary evaporation, water was added for dissolution, and then freeze-dried to obtain 9.8 g of solid powder of blood group trisaccharide B.

[0112] The purity of blood group trisaccharide B in the detected sample was >95%; the overall yield was 33%, as Figure 10 shown. Its mass spectrum and hydrogen spectrum are shown in Figure 11 and Figure 12 respectively.

[0113] Among them, the relevant verification of intermediate products 2 and 3 in this example is shown in Figures 13 to 18 where Figure 13 and Figure 14 are the mass spectrometry (MS) spectra of intermediate product 2 and intermediate product 3; Figure 15 and Figure 16 are the reaction TLC visualization diagrams of intermediate product 2 and intermediate product 3; Figure 17 and Figure 18 are the mass spectra after purification of intermediate product 2 and intermediate product 3. According to the above diagrams, it can be seen that in this example, both the intermediate product and the final product were verified.

[0114] Comparative Example 1

[0115] Based on Example 1, the difference in 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 were directly dissolved in water, the pH was adjusted to 7.5 (±0.5), and then enzyme α1,2-FucT was added, and the reaction was carried out at 37 ± 1 °C, and the reaction was detected by TLC.

[0116] The corresponding ideal synthesis idea is:

[0117]

[0118] No intermediate product 5 was found by TLC detection, and it was verified by MS that no intermediate product was formed, and the reaction was terminated.

[0119] Comparative Example 2

[0120] Based on Example 1, the difference in this comparative example is that:

[0121] The corresponding ideal synthesis idea is:

[0122]

[0123] 1 g of substrate 6 (R group is methyl), 3.7 g of GDP-Fuc, and 0.4 g of magnesium chloride were dissolved in water, the pH was adjusted to 7.5 (±0.5), and then enzyme α1,2-FucT was added, and the reaction was carried out at 37 ± 1 °C, and the reaction end point was detected by TLC to obtain the reaction solution of product 7.

[0124] An equal amount of absolute ethanol was added to the reaction solution of product 7 to precipitate and remove the enzyme. After standing at 4 °C for 4 h, the supernatant was obtained by centrifugation and filtration to obtain an ethanol aqueous solution of product 7.

[0125] The ethanol aqueous solution of product 7 was rotary evaporated at 40 °C to remove ethanol, obtaining an aqueous solution of product 7. Quantified by HPLC, in the aqueous solution of product 7, product 7 was 1.4 g.

[0126] 3.3 g of solid UDP-GalNAc was added to the aqueous solution of product 7 (product 7 was 1.4 g). After complete dissolution, the pH was adjusted to 7.5 (±0.5), and then enzyme BGTA was added. The reaction was carried out at 37 ± 1 °C, and the reaction end point was detected by TLC to obtain the reaction solution of product 8.

[0127] An equal amount of absolute ethanol was added to the reaction solution of product 8 to precipitate and remove the enzyme. After standing at 4 °C for 4 h, the supernatant was obtained by centrifugation and filtration to obtain an ethanol aqueous solution of product 8.

[0128] The ethanol aqueous solution of product 8 was rotary evaporated at 40 °C to remove ethanol, obtaining an aqueous solution of product 8.

[0129] The aqueous solution of product 8 obtained in the previous step was loaded onto a pre-treated C 18 chromatography column, and the product was eluted with a mixed solution of absolute methanol and water (the volume ratio of absolute methanol to water was 1:4) to obtain a methanol aqueous solution of product 8.

[0130] The methanol aqueous solution of product 8 was rotary evaporated and concentrated and then freeze-dried to obtain 1.1 g of solid product 8.

[0131] Product 8 was dissolved in absolute methanol, Pd / C was added, and hydrogen was introduced, followed by stirring overnight.

[0132] Finally, it was found by combined detection of TLC and MS that no product A3 was generated, and the reaction was terminated by filtration.

[0133] Comparative Example 3

[0134] Based on Example 2, the difference in this comparative example is that:

[0135] 1 g of substrate 4 (R group is H), 1.7 g of GDP-Fuc, and 0.4 g of magnesium chloride were directly dissolved in water, the pH was adjusted to 7.5 (±0.5), and then enzyme α1,2-FucT was added. The reaction was carried out at 37 ± 1 °C, and the reaction was detected by TLC.

[0136] The corresponding ideal synthesis idea is:

[0137]

[0138] TLC detection found no formation of intermediate products, and MS verification confirmed the absence of intermediate products, so the reaction was terminated.

[0139] Comparative Example 4

[0140] Based on Example 2, the difference in this comparative example is that:

[0141] The corresponding ideal synthesis idea is:

[0142]

[0143] 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), then add the enzyme α1,2-FucT, and place it at 37 ± 1 °C for reaction. Use TLC to detect the end point of the reaction to obtain the reaction solution of product 7;

[0144] Add an equal amount of absolute ethanol to the reaction solution of product 7 to precipitate and remove the enzyme. After standing at 4 °C for 4 h, centrifuge and filter to take the supernatant to obtain the ethanol aqueous solution of product 7;

[0145] The ethanol aqueous solution of product 7 is rotary evaporated at 40 °C to remove ethanol to obtain the aqueous solution of product 7. Quantified by HPLC, in the aqueous solution of product 7, product 7 is 1.4 g;

[0146] Add 4 g of UDP-Gal solid to the aqueous solution of product 7 (product 7 is 1.4 g). After complete dissolution, adjust the pH to 7.5 (±0.5), then add the enzyme GTB, and place it at 37 ± 1 °C for reaction. Use TLC to detect the end point of the reaction to obtain the reaction solution of product 8;

[0147] Add an equal amount of absolute ethanol to the reaction solution of product 8 to precipitate and remove the enzyme. After standing at 4 °C for 4 h, centrifuge and filter to take the supernatant to obtain the ethanol aqueous solution of product 8;

[0148] The ethanol aqueous solution of product 8 is rotary evaporated at 40 °C to remove ethanol to obtain the aqueous solution of product 8;

[0149] The aqueous solution of product 8 obtained in the previous step is loaded onto a pretreated C18 chromatography column, and the product is eluted with a mixed solution of absolute methanol and water (the volume ratio of absolute methanol to water is 1:4) to obtain the methanol aqueous solution of product 8;

[0150] The methanol aqueous solution of product 8 is rotary evaporated and concentrated and then freeze-dried to obtain 720 mg of solid product 8;

[0151] Dissolve product 8 in absolute methanol, add Pd / C, and introduce hydrogen gas, and stir overnight;

[0152] Finally, combined detection by TLC and MS found no formation of product B3, and the reaction was terminated by filtration.

[0153] From the above examples and comparative examples, it can be seen that only when the R group in the substrate, that is, the linker is benzyl, can the oligosaccharide blood group trisaccharide A / B be directly synthesized; when the R group is H and methyl, the oligosaccharide blood group trisaccharide cannot be directly synthesized.

[0154] In summary, the chemoenzymatic synthesis method of blood group trisaccharide provided by the embodiments of the present invention solves the process of synthesizing and amplifying blood group trisaccharide, provides a new technical idea of chemoenzymatic synthesis method, can be applied in the synthesis of similar oligosaccharide compounds, is conducive to popularization and application, and solves the problem of low yield and high price; the chemoenzymatic synthesis method of this blood group trisaccharide can simply and directly obtain blood group trisaccharide to obtain the key raw material of carbohydrate-modified compounds, providing new ideas for multi-fields such as glycoprotein / antibody research, conjugated drug development, in vitro diagnosis, and medical device consumables.

[0155] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chemoenzymatic synthesis method of a blood group 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, adding Pd / C, passing hydrogen, stirring sufficiently, filtering to remove Pd / C, concentrating and freeze-drying to obtain solid powdered blood type trisaccharide; Wherein, the substrate is β-benzylgalactose substrate; The structural formula of the substrate is: ； When the glycosylation product is a UDP-Gal solid, the glycosyltransferase is the enzyme GTB; When the glycosylation substance is a UDP-GalNAc solid, the glycosyltransferase is the enzyme BGTA.

2. The chemoenzymatic synthesis method of the blood group trisaccharide according to claim 1, characterized in that, In step S3, when the glycosylated product is a UDP-Gal solid, 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.

3. The chemoenzymatic synthesis method of the blood group trisaccharide according to claim 1, characterized in that, In step S3, when the glycosylated product is UDP-GalNAc solid, 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.

4. The chemoenzymatic synthesis method of the blood group trisaccharide according to claim 1, characterized in that, In step S1, 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.

5. The chemoenzymatic synthesis method of the blood group trisaccharide according to claim 1, wherein 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.

6. The chemoenzymatic synthesis method of the blood group 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.

7. The chemoenzymatic synthesis method of the blood group trisaccharide according to claim 1, wherein Step S5 specifically includes: Step S501: Load the aqueous solution of the second product onto the pretreated C 18 chromatographic column, and elute with an elution solvent to obtain a solution of the second product; Step S502, subjecting the second product solution to rotary evaporation, concentration and freeze-drying to obtain the solid product.

8. The chemoenzymatic synthesis method of the blood group trisaccharide according to claim 7, 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.

9. The chemoenzymatic synthesis method of the blood group 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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