Method for synthesizing blood group antigen NOR1 pentasaccharide by enzyme method

Abstract

The invention belongs to the technical field of carbohydrate synthesis, and relates to a method for synthesizing blood group antigen NOR1 pentasaccharide by an enzyme method. The method comprises the following steps: coupling galactose to lactose or a modifier thereof through an alpha1-4 glucosidic bond by using a one-pot multi-enzyme system to form trisaccharide containing Pk antigen; the preparation method comprises the following steps: coupling trifluoroacetyl galactosamine to trisaccharide containing a Pk antigen through a beta1-4 glucosidic bond to form a tetrasaccharide compound P-TFA; the preparation method comprises the following steps: removing a TFA group on trifluoroacetyl galactosamine under an alkaline condition to form a tetrasaccharide compound P-NH2; the preparation method comprises the following steps: coupling galactose to a tetrasaccharide compound P-NH2 through an alpha1-4 glucosidic bond to form a pentasaccharide compound NOR1-NH2; amino groups on the pentasaccharide compound NOR1-NH2 are changed into acetamido groups, and the pentasaccharide compound NOR1 is formed. The synthesis of the NOR1 pentasaccharide is realized through the interchange of NH2 and NHAc, the steps are simple, the total yield is high, and the NOR1 pentasaccharide can be synthesized on a large scale.

Description

Technical Field

[0001] The invention belongs to the technical field of carbohydrate synthesis and relates to a method for synthesizing blood group antigen NOR1 pentasaccharide by enzymatic method. Background Art

[0002] The blood type antigen NOR1 belongs to the P1PK blood type antigen family. They exist in human red blood cells and other cells in the form of glycolipids, and the sugar chain ends all have Galα1-4 structures. Due to the rarity of the blood type antigen NOR1 in organisms, it is difficult to obtain the blood type antigen by extraction. For chemical synthesis, due to the inherent active polyhydroxy structure of NOR1 itself, cumbersome protection and deprotection steps are required during the synthesis process to ensure regional and stereoselectivity, resulting in many reaction steps and low overall yield. Summary of the invention

[0003] The purpose of the present invention is to provide a method for synthesizing blood group antigen NOR1 pentasaccharide by enzymatic method, which utilizes the substrate specificity principle of glycosyltransferase and realizes the synthesis of blood group antigen NOR1 through the interchange of NH2 and NHAc.

[0004] To achieve the above object, the technical scheme adopted by the present invention is: a method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide, comprising: using a "one-pot multi-enzyme" system to couple galactose to lactose or its modified product via an α1-4 glycosidic bond to form a P-containing k The "one-pot multi-enzyme" system couples trifluoroacetylgalactosamine to the P-containing k The tetrasaccharide compound P-TFA is formed on the trisaccharide of the antigen; the TFA group on trifluoroacetylaminogalactose is removed under alkaline conditions to form a tetrasaccharide compound P-NH2; galactose is coupled to the tetrasaccharide compound P-NH2 via an α1-4 glycosidic bond using a "one-pot multi-enzyme" system to form a pentasaccharide compound NOR1-NH2; the amino group on the pentasaccharide compound NOR1-NH2 is converted into an acetylamino group to form a pentasaccharide compound NOR1; wherein: In the reaction of synthesizing trisaccharides, the "one-pot multi-enzyme" system used includes galactokinase, sugar nucleoside generating enzyme and α1-4 galactosyltransferase; In the reaction of synthesizing tetrasaccharides, the "one-pot multi-enzyme" system used includes N-acetylglucosamine kinase, N-acetylglucosamine-1-P uridyltransferase, and β-1,3-N-acetylgalactosamine transferase; In the reaction of synthesizing pentasaccharides, the "one-pot multi-enzyme" system used includes galactokinase, sugar nucleoside generating enzyme and α1-4 galactosyltransferase.

[0005] Preferably, the synthesis method of the trisaccharide is as follows: lactose or its modified substance is mixed with 1.0-3.0 equivalents of galactose, 1.2-4.0 equivalents of adenine nucleoside triphosphate, 1.2-4.0 equivalents of uridine nucleoside triphosphate, 5-150 mM MgCl2, 100-600 mM Tris-HCl buffer of pH 6-8 to prepare an aqueous solution, the pH of the reaction system is adjusted to between 7.0-8.0, and then galactokinase and UDP-sugar pyrophosphorylase are added, and the reaction is inactivated by boiling after one hour, and α1-4 galactosyltransferase is added, and the reaction time is 2 hours. After the reaction is complete, purification is performed to obtain a product containing P k The reaction route of the trisaccharide compound of the antigen is shown in formula (1): Formula (1).

[0006] Preferably, the synthesis method of tetrasaccharide is: k The trisaccharide of the antigen is mixed with 1.0-3.0 equivalents of trifluoroacetylgalactosamine, 1.2-4.0 equivalents of adenine nucleoside triphosphate, 1.2-4.0 equivalents of uridine nucleoside triphosphate, 5-150 mM MgCl2, and 100-600 mM Tris-HCl buffer at pH 6-8 to prepare an aqueous solution. The pH of the reaction system is adjusted to between 6.0 and 7.0. Then, N-acetylglucosamine kinase and N-acetylglucosamine-1-P uridyltransferase are added. After one hour of reaction, β-1,3-N-acetylgalactosamine transferase is added. The reaction time is 48 hours. After the reaction is complete, the tetrasaccharide compound P-TFA can be obtained by purification. The reaction route is shown in formula (2): Formula (2).

[0007] Preferably, the synthesized tetrasaccharide compound P-TFA aqueous solution is adjusted to pH 12 and reacted overnight to remove the terminal TFA to obtain the tetrasaccharide compound P-NH2 as the acceptor for the next enzyme reaction.

[0008] Preferably, the synthesis method of the pentasaccharide is as follows: P-NH2 is mixed with 1.0-3.0 equivalents of galactose, 1.2-4.0 equivalents of adenine nucleoside triphosphate, 1.2-4.0 equivalents of uridine nucleoside triphosphate, 5-150 mM MgCl2, and 100-600 mM Tris-HCl buffer at pH 6-8 to prepare an aqueous solution, the pH of the reaction system is adjusted to between 7.0 and 8.0, and then galactokinase and UDP-sugar pyrophosphorylase are added, and the mixture is inactivated by boiling after one hour of reaction, and α1-4 galactosyltransferase is added, and the reaction time is 48 hours. After the reaction is complete, the pentasaccharide compound NOR1-NH2 is purified, and the reaction route is shown in Formula (4): Formula (4).

[0009] Preferably, the pentasaccharide compound NOR1-NH2, acetic anhydride and sodium bicarbonate are dissolved in water and stirred for 5 min. After the reaction is complete, purification is performed to obtain the target product NOR1 pentasaccharide compound.

[0010] Preferably, the reaction temperature in the "one pot multi-enzyme" system is 36-38°C and the rotation speed is 120-150 rpm.

[0011] Preferably, the reaction is stopped by boiling the reaction solution in boiling water for 5-15 min to inactivate all proteins, or by adding an equal volume of ice ethanol to the reaction solution and shaking rapidly for 5-15 min.

[0012] Compared with the prior art, the method of the present invention has the following beneficial effects: the present invention adopts a "one-pot multi-enzyme" system to synthesize the blood group antigen NOR1 pentasaccharide, utilizes the substrate specificity principle of glycosyltransferase, and realizes the synthesis of NOR1 antigen through the interchange of NH2 and NHAc. The steps are simple, the overall yield is high, and it can be synthesized in large quantities, reducing the use of chemical reagents, and is environmentally friendly and pollution-free. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : is the H NMR spectrum of the trisaccharide compound synthesized in the embodiment of the present invention; Figure 2 is the C NMR spectrum of the trisaccharide compound synthesized in the embodiment of the present invention; Figure 3 : is the H NMR spectrum of the tetrasaccharide compound P-TFA synthesized in the embodiment of the present invention; Figure 4 : is the NMR spectrum C of the tetrasaccharide compound P-TFA synthesized in the embodiment of the present invention; Figure 5 : is the H NMR spectrum of the tetrasaccharide compound P-NH2 synthesized in the embodiment of the present invention; Figure 6 : is the C NMR spectrum of the tetrasaccharide compound P-NH2 synthesized in the embodiment of the present invention; Figure 7 : is the H NMR spectrum of the pentasaccharide compound NOR1-NH2 synthesized in the embodiment of the present invention; Figure 8 : is the C NMR spectrum of the pentasaccharide compound NOR1-NH2 synthesized in the embodiment of the present invention; Fig. 9 : is the H NMR spectrum of the pentasaccharide compound NOR1 synthesized in the embodiment of the present invention; Fig.10 : This is the C NMR spectrum of the pentasaccharide compound NOR1 synthesized in the examples of the present invention. DETAILED DESCRIPTION

[0014] The present invention is further described below in conjunction with specific embodiments and the accompanying drawings. More details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention is obviously capable of being implemented in a variety of other ways different from the description herein. For those skilled in the art, any substitution, improvement or change made to the embodiments of the present invention is within the protection scope of the present invention, and the protection scope of the present invention should not be limited by the content of this specific embodiment.

[0015] Enzymatic synthesis of NOR1 antigen requires the use of a mutant of human α1-4 galactosyltransferase A4GalT. The inventors of the present invention tried to use the mutant of A4GalT to prepare NOR1 antigen in early experiments, but found that the activity was too weak to achieve large-scale preparation. Subsequently, the substrate specificity principle of glycosyltransferase was used to achieve the synthesis of NOR1 antigen through the exchange of NH2 and NHAc.

[0016] The prokaryotic α1-4 galactosyltransferase LgtC can use oligosaccharides containing Gal at the non-reducing end as receptors to add α1-4Gal to form a Galα1-4Gal structure, but cannot use oligosaccharides with GalNAc at the end as receptors to form NOR1 antigen ( Galα1-4GalNAc βl-4Galαl-4Galβl-4Glc). The present invention is GalNH2 βl-4Galαl-4Galβl-4Glc tetrasaccharide is used as substrate to achieve the synthesis of blood group antigen NOR1 pentasaccharide.

[0017] Example 1 Trisaccharide Compound P k -pro-N3 (Galαl-4Galβl-4GlcβPro-N3), the synthetic route is shown in formula (1): Formula (1).

[0018] The receptor compound Lac-pro-N3 (200 mg), galactose (128 mg), ATP (477 mg), UTP (455 mg), Tris-HCl buffer (100 mM, pH 7.5) and magnesium chloride (50 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, and GalK and AtUSP (1.0-3.0 mg) and NmLgtC (0.5-2.0 mg) were added. After adding double distilled water to a total volume of 50 mL, the reaction system was placed in a shaker and incubated at 37 ° C and 140 r / min for 4 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4:2:1:0.5), it was placed in boiling water and boiled for 10 minutes to terminate the reaction. The reaction solution was then centrifuged at 4 °C and 11000 r / min for 10 min, the supernatant was collected, concentrated by rotary evaporation, and separated by polyacrylamide gel column to obtain a white powder, which was the trisaccharide compound Gb3 (218 mg, 79%). Its H and C NMR spectra were as follows: Figure 1 and Figure 2 As shown, the parameters are as follows: 1 H NMR (600 MHz, D2O) δ 4.88 (d, J = 4.0 Hz, 1H), 4.43 (dd, J = 12.6,7.9 Hz, 2H), 4.31 – 4.25 (m, 1H), 3.99 – 3.90 (m, 4H), 3.89 – 3.80 (m, 2H), 3.76 (ddt, J = 11.3, 8.9, 4.6 Hz, 3H), 3.73 – 3.48 (m, 9H), 3.39 (t, J = 6.7Hz, 2H), 3.26 – 3.20 (m, 1H), 1.84 (p, J = 6.5 Hz, 2H); 13 C NMR (151 MHz, D2O) δ 103.26, 102.08, 100.30, 78.65, 77.35, 75.41,74.80, 74.42, 72.89, 72.15, 70.90, 70.81, 69.12, 68.93, 68.55, 67.35, 60.50,60.36, 60.02, 47.86, 28.22. HRMS (ESI) m / z calculated for C 21 H 37 N3O 16 [M+H]+ 588.2173, found 588.2251.

[0019] Example 2 Synthesis of the tetrasaccharide compound P-TFA-pro-N3, the synthesis route is shown in formula (2): P-TFA (GalNTFAβl-4Galαl-4Galβl-4GlcβPro-N3) Formula (2).

[0020] The receptor compound P k -pro-N3 (100 mg), trifluoroacetylgalactosamine (GalNTFA) (70 mg), ATP (172 mg), UTP (165 mg), Tris-HCl buffer (100 mM, pH 6.5) and magnesium chloride (20 mM (the amount of Tris and MgCl2 is determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, and NahK and AGX1 (1.0-3.0 mg) were added. After one hour of reaction, NmLgtD (0.5-2.0 mg) was added. After adding double distilled water to a total volume of 20 mL, the reaction was placed in a shaker and incubated at 37 °C and 140 r / min for 48 hours. After the reaction was completed by thin layer chromatography (EtOAc:MeOH:H2O:EtOH=4:2:1:0.5), an equal amount of ice ethanol was added and shaken rapidly for 5 min to terminate the reaction. Then, the mixture was incubated at 4 °C and 11000 r / min. The supernatant was collected and concentrated by rotary evaporation, and separated by polyacrylamide gel column to obtain a white compound P-TFA (109 mg, 76%). Its H and C NMR spectra are as follows: Figure 3 and Figure 4 As shown, the parameters are as follows: 1H NMR (600 MHz, D2O) δ 4.90 (d, J = 4.0 Hz, 1H), 4.43 (dd, J = 19.2,7.9 Hz, 2H), 4.36 – 4.29 (m, 1H), 4.22 (dd, J = 3.2, 1.2 Hz, 1H), 4.04 (dd, J= 10.5, 3.2 Hz, 1H), 4.01 – 3.89 (m, 4H), 3.89 – 3.81 (m, 2H), 3.80 – 3.49(m, 13H), 3.39 (t, J = 6.7 Hz, 2H), 3.23 (ddd, J = 16.7, 10.1, 8.2 Hz, 2H),1.85 (td, J = 13.3, 6.7 Hz, 2H); 13 C NMR (151 MHz, D2O) δ 103.33, 102.55, 102.07, 100.49, 79.06,78.86, 77.33, 75.41, 75.04, 74.79, 74.47, 72.91, 72.13, 70.90, HRMS (ESI) m / z calculated for C 29 H 47 F3N4O 21 [M+H] + 845.2684, found845.2760.

[0021] Example 3 Synthesis of the tetrasaccharide compound P-NH2-pro-N3 (GalNH2βl-4Galαl-4Galβl-4GlcβPro-N3), the synthesis route is shown in formula (3): Formula (3).

[0022] Compound P-TFA-pro-N3 (100 mg) was dissolved in a 50 mL centrifuge tube, the pH of the solution was adjusted to 12, and the reaction was carried out at 37 °C overnight to remove TFA to form compound P-NH2. After the reaction was completed by thin layer chromatography (EtOAc:MeOH:H2O:EtOH=4:2:1:0.5), the solution was adjusted to pH 7.5 to start the next step of reaction and partially purify the white compound P-NH2. Its H and C NMR spectra were as follows: Figure 5 and Figure 6 As shown, the parameters are as follows: 1 H NMR (600 MHz, D2O) δ 4.84 (d, J = 4.1 Hz, 2H), 4.43 (ddd, J = 16.6,7.9, 1.2 Hz, 2H), 4.31 (t, J = 6.3 Hz, 1H), 4.20 (d, J = 3.2 Hz, 1H), 4.03 – 3.87 (m, 5H), 3.86 – 3.48 (m, 14H), 3.46 – 3.35 (m, 2H), 3.30 – 3.19 (m, 1H), 1.85 (dq, J = 13.0, 6.1 Hz, 2H). 13 C NMR (151 MHz, D2O) δ 103.20, 102.11,101.25, 100.17, 78.71, 78.43, 77.28, 75.41, 75.35, 74.83, 74.41, 72.94,72.07, 70.88, HRMS (ESI) m / z calculated for C27H48N4O20 [M+H]+749.2861, found 749.2935.

[0023] Example 5 Synthesis of the pentasaccharide compound NOR1-NH2-pro-N3 (Galαl-4GalNH2βl-4Galαl-4Galβl-4GlcβPro-N3), the synthesis route is shown in formula (4): Formula (4).

[0024] The receptor compound NOR1-NH2-pro-N3 (80 mg), galactose (39 mg), ATP (162 mg), UTP (155 mg), Tris-HCl buffer (100 mM, pH 7.5) and magnesium chloride (50 mM) (the amount of Tris and MgCl2 is calculated based on the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, and GalK and AtUSP (1.0-3.0 mg) and NmLgtC (0.5-2.0 mg) were added. Double distilled water was added to a total volume of 15 mL, and the reaction solution was placed in a shaker and incubated at 37 °C and 140 r / min for 48 hours. After the reaction was completed by thin layer chromatography (EtOAc:MeOH:H2O:NH3·H2O=3:2:1:1), it was placed in boiling water and boiled for 10 minutes to terminate the reaction. The mixture was then centrifuged at 4°C and 11000 r / min for 10 min, the supernatant was collected, concentrated by rotary evaporation, and separated by polyacrylamide gel column to obtain a white compound NOR1-NH2 (27 mg, 28%). Its H and C NMR spectra are shown in Figure 7 and Figure 8 As shown, the parameters are as follows: 1 H NMR (600 MHz, D2O) δ 4.95 – 4.88 (m, 3H), 4.43 (dd, J = 19.0, 7.9Hz, 2H), 4.32 (dt, J = 12.5, 6.4 Hz, 2H), 4.19 (d, J = 3.0 Hz, 1H), 1.84 (p, J = 6.5 Hz, 2H); 13C NMR (151 MHz, D2O) δ 103.21, 102.11, 100.42, 100.17, 78.68,78.46, 77.21, 76.70, 75.35, 74.83, 74.41, 72.94, 72.08, 71.10, HRMS (ESI) m / z calculated for C 33 H 58 N4O 25 [M+H] + 911.3390, found911.3465.

[0025] Example 6 Synthesis of the pentasaccharide compound NOR1-pro-N3 (Galαl-4GalNAcβl-4Galαl-4Galβl-4GlcβPro-N3), the synthetic route is shown in formula (5): Formula (5).

[0026] Compound NOR1-NH2 (10 mM), AC2O (15 mM) and NaHCO3 (15 mM) were dissolved in water and stirred overnight. After the reaction was completed, a white compound NOR1 was obtained by thin layer chromatography (EtOAc:MeOH:H2O:NH3·H2O=3:2:1:1). Its H and C NMR spectra were as follows: Fig. 9 and Fig.10 As shown, the parameters are as follows: 1H NMR (600 MHz, D2O) δ 4.92 (d, J = 4.0 Hz, 1H), 4.84 (d, J = 3.7 Hz,2H), 4.43 (ddd, J = 17.1, 7.9, 1.5 Hz, 2H), 4.30 (dt, J = 13.6, 6.4 Hz, 2H),4.17 – 4.13 (m, 1H), 4.01 – 3.92 (m, 5H), 3.91 (td, J = 4.2, 3.6, 1.9 Hz,2H), 3.89 – 3.81 (m, 3H), 3.81 – 3.49 (m, 17H), 3.39 (td, J = 6.7, 1.5 Hz,2H), 3.24 (ddd, J = 9.4, 8.0, 1.6 Hz, 1H), 1.97 (d, J = 1.5 Hz, 3H), 1.84 (p,J = 6.0 Hz, 2H); 13 C NMR (151 MHz, D2O) δ 103.42, 103.28, 102.08, 100.52, 100.38,78.72, 77.15, 77.00, 75.42, 74.94, 74.81, 74.45, 72.92, 72.09, 70.87, 70.83,70.36, 70.30, 69.11, 68.98, 68.95, 68.68, 67.74, 67.36, 60.48, 60.33, 60.31,60.25, 60.04, 52.66, 47.86, 28.22, 22.24. HRMS (ESI) m / z calculated forC 35 H 60 N4O 26 [M+H] + 953.3495, found 953.3570。

Claims

1. A method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide, characterized in that: include: The "one-pot multi-enzyme" system was used to couple galactose to lactose or its modifications via α1-4 glycosidic bonds to form P-containing k The "one-pot multi-enzyme" system couples trifluoroacetylgalactosamine to the P-containing k The tetrasaccharide compound P-TFA is formed on the trisaccharide of the antigen; the TFA group on trifluoroacetylaminogalactose is removed under alkaline conditions to form the tetrasaccharide compound P-NH2; the galactose is coupled to the tetrasaccharide compound P-NH2 via an α1-4 glycosidic bond using a "one-pot multi-enzyme" system to form the pentasaccharide compound NOR1-NH2; the amino group on the pentasaccharide compound NOR1-NH2 is converted into an acetylamino group to form the pentasaccharide compound NOR1; wherein: In the reaction of synthesizing trisaccharides, the "one-pot multi-enzyme" system used includes galactokinase, sugar nucleoside generating enzyme and α1-4 galactosyltransferase; In the reaction of synthesizing tetrasaccharides, the "one-pot multi-enzyme" system used includes N-acetylglucosamine kinase, N-acetylglucosamine-1-P uridyltransferase and β-1,3-N-acetylgalactosamine transferase; In the reaction of synthesizing pentasaccharides, the "one-pot multi-enzyme" system used includes galactokinase, sugar nucleoside generating enzyme and α1-4 galactosyltransferase.

2. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 1, characterized in that: The synthesis method of the trisaccharide is as follows: lactose or its modified substance is mixed with 1.0-3.0 equivalents of galactose, 1.2-4.0 equivalents of adenine nucleoside triphosphate, 1.2-4.0 equivalents of uridine nucleoside triphosphate, 5-150 mM MgCl2, 100-600 mM Tris-HCl buffer of pH 6-8 to prepare an aqueous solution, the pH of the reaction system is adjusted to between 7.0-8.0, and then galactokinase and UDP-sugar pyrophosphorylase are added, and the reaction is inactivated by boiling after one hour, and α1-4 galactosyltransferase is added, and the reaction time is 2 hours. After the reaction is complete, purification is performed to obtain a product containing P k The reaction route of the trisaccharide compound of the antigen is shown in formula (1): Formula (1).

3. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 1, characterized in that: The synthesis method of tetrasaccharide is as follows: k The trisaccharide of the antigen is mixed with 1.0-3.0 equivalents of trifluoroacetylgalactosamine, 1.2-4.0 equivalents of adenine nucleoside triphosphate, 1.2-4.0 equivalents of uridine nucleoside triphosphate, 5-150 mM MgCl2, and 100-600 mM Tris-HCl buffer at pH 6-8 to prepare an aqueous solution. The pH of the reaction system is adjusted to between 6.0 and 7.

0. Then, N-acetylglucosamine kinase and N-acetylglucosamine-1-P uridyltransferase are added. After one hour of reaction, β-1,3-N-acetylgalactosamine transferase is added. The reaction time is 48 hours. After the reaction is complete, the tetrasaccharide compound P-TFA can be obtained by purification. The reaction route is shown in formula (2): Formula (2).

4. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 3, characterized in that: The synthesized tetrasaccharide compound P-TFA aqueous solution was adjusted to pH 12 and reacted overnight to remove terminal TFA to obtain the tetrasaccharide compound P-NH2.

5. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 1, characterized in that: The synthesis method of the pentasaccharide is as follows: P-NH2 is mixed with 1.0-3.0 equivalents of galactose, 1.2-4.0 equivalents of adenine nucleoside triphosphate, 1.2-4.0 equivalents of uridine nucleoside triphosphate, 5-150 mM MgCl2, and 100-600 mM Tris-HCl buffer of pH 6-8 to prepare an aqueous solution, the pH of the reaction system is adjusted to between 7.0-8.0, and then galactokinase and UDP-sugar pyrophosphorylase are added, and the mixture is inactivated by boiling after one hour of reaction, and α1-4 galactosyltransferase is added, and the reaction time is 48 hours. After the reaction is complete, the pentasaccharide compound NOR1-NH2 is purified, and the reaction route is shown in Formula (4): Formula (4).

6. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 5, characterized in that The pentasaccharide compound NOR1-NH2, acetic anhydride and sodium bicarbonate were dissolved in water and stirred for 5 min. After the reaction was complete, purification was performed to obtain the target product NOR1 pentasaccharide compound.

7. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to any one of claims 1 to 6, characterized in that: After the synthesis of trisaccharides, tetrasaccharides and pentasaccharides was completed, thin layer chromatography was used to track the reaction progress. k The developing solvent used for the trisaccharide compound and tetrasaccharide compound P-TFA of the antigen is EA:CH3OH:H2O:HOAc=4:2:1:0.5; the developing solvent used for the tetrasaccharide compound P-NH2, pentasaccharide compounds NOR1-NH2 and NOR1 is EA:CH3OH:H2O:NH3·H20=3:2:1:

1.

8. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to any one of claims 1 to 6, characterized in that: The reaction temperature in the "one-pot multi-enzyme" system is 36-38°C and the rotation speed is 120-150 rpm.

9. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to any one of claims 1, 2, 3 or 5, characterized in that: The reaction can be stopped by boiling the reaction solution in boiling water for 5-15 minutes to inactivate all proteins or by adding an equal volume of ice ethanol to the reaction solution and shaking rapidly for 5-15 minutes.

Images