A method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide

Abstract

The present invention belongs to the technical field of saccharide synthesis, and relates to a method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide. The method includes 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 trisaccharide containing P k antigen; coupling N-acetylgalactosamine to the trisaccharide containing P k antigen via a β1-4 glycosidic bond to form a tetrasaccharide compound P-TFA; removing the TFA group on N-acetylgalactosamine under alkaline conditions to form a tetrasaccharide compound P-NH2; coupling galactose to the tetrasaccharide compound P-NH2 via an α1-4 glycosidic bond to form a pentasaccharide compound NOR1-NH2; converting the amino group on the pentasaccharide compound NOR1-NH2 into an acetamido group to form a pentasaccharide compound NOR1. The present invention realizes the synthesis of NOR1 pentasaccharide through the interchange of NH2 and NHAc, with simple steps, high overall yield, and can be synthesized in large quantities.

Description

Technical Field

[0001] The present invention belongs to the technical field of saccharide synthesis, and relates to a method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide. Background Art

[0002] Blood group antigen NOR1 belongs to the P1PK blood group antigen family. They exist in the form of glycolipids in human red blood cells and other cells, and the sugar chains at the ends all have the Galα1-4 structure. Due to the rarity of blood group antigen NOR1 in organisms, it is difficult to obtain this blood group antigen by extraction. For chemical synthesis, due to the inherently similar multi-hydroxy structure of NOR1 itself, cumbersome protection and deprotection steps are required during the synthesis to ensure regio- and stereo-selectivity, 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 enzymatically synthesizing blood group antigen NOR1 pentasaccharide. By using the substrate specificity principle of glycosyltransferase and through the exchange of NH2 and NHAc, the synthesis of blood group antigen NOR1 is realized.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is: a method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide, including: using a "one-pot multi-enzyme" system to couple galactose to lactose or its modified product through an α1-4 glycosidic bond to form a trisaccharide containing P k antigen; using a "one-pot multi-enzyme" system to couple N-trifluoroacetylgalactosamine to the trisaccharide containing P k antigen through a β1-4 glycosidic bond to form a tetrasaccharide compound P-TFA; removing the TFA group on N-trifluoroacetylgalactosamine under alkaline conditions to form a tetrasaccharide compound P-NH2; using a "one-pot multi-enzyme" system to couple galactose to the tetrasaccharide compound P-NH2 through an α1-4 glycosidic bond to form a pentasaccharide compound NOR1-NH2; changing the amino group on the pentasaccharide compound NOR1-NH2 to an acetylamino group to form a pentasaccharide compound NOR1; wherein:

[0005] In the reaction for synthesizing the trisaccharide, the "one-pot multi-enzyme" system used includes galactokinase, sugar nucleoside generating enzyme, and α1-4 galactosyltransferase;

[0006] In the reaction for synthesizing the tetrasaccharide, the "one-pot multi-enzyme" system used includes N-acetylglucosamine kinase, N-acetylglucosamine-1-P uridyltransferase, and β-1,3-N-acetylgalactosaminyltransferase;

[0007] In the reaction for synthesizing the pentasaccharide, the "one-pot multi-enzyme" system used includes galactokinase, sugar nucleoside generating enzyme, and α1-4 galactosyltransferase.

[0008] Preferably, the method for synthesizing the trisaccharide is as follows: An aqueous solution is prepared by mixing lactose or its modified product with 1.0 - 3.0 equivalents of galactose, 1.2 - 4.0 equivalents of adenosine triphosphate, 1.2 - 4.0 equivalents of uridine triphosphate, 5 - 150 mM of MgCl2, and 100 - 600 mM of Tris-HCl buffer with a pH of 6 - 8. The pH of the reaction system is adjusted to between 7.0 and 8.0, then galactokinase and UDP-sugar pyrophosphorylase are added. After reacting for one hour, the reaction is inactivated by boiling. Then, α1-4 galactosyltransferase is added, and the reaction time is 2 hours. After the reaction is complete, purification can obtain the trisaccharide compound containing P k antigen, and the reaction route is shown in Formula (1):

[0009] Formula (1).

[0010] Preferably, the method for synthesizing the tetrasaccharide is as follows: An aqueous solution is prepared by mixing the trisaccharide containing P k antigen with 1.0 - 3.0 equivalents of N-trifluoroacetylgalactosamine, 1.2 - 4.0 equivalents of adenosine triphosphate, 1.2 - 4.0 equivalents of uridine triphosphate, 5 - 150 mM of MgCl2, and 100 - 600 mM of Tris-HCl buffer with a pH of 6 - 8. 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 reacting for one hour, β-1,3-N-acetylgalactosaminyltransferase is added, and the reaction time is 48 hours. After the reaction is complete, purification can obtain the tetrasaccharide compound P-TFA; the reaction route is shown in Formula (2):

[0011] Formula (2).

[0012] Preferably, the pH of the aqueous solution of the synthesized tetrasaccharide compound P-TFA is adjusted to 12 and reacted overnight to remove the terminal TFA, obtaining the tetrasaccharide compound P-NH2 as the acceptor for the next enzymatic reaction.

[0013] Preferably, the method for synthesizing the pentasaccharide is as follows: An aqueous solution is prepared by mixing P-NH2 with 1.0 - 3.0 equivalents of galactose, 1.2 - 4.0 equivalents of adenosine triphosphate, 1.2 - 4.0 equivalents of uridine triphosphate, 5 - 150 mM of MgCl2, and 100 - 600 mM of Tris-HCl buffer with a pH of 6 - 8. The pH of the reaction system is adjusted to between 7.0 and 8.0, then galactokinase and UDP-sugar pyrophosphorylase are added. After reacting for one hour, the reaction is inactivated by boiling. Then, α1-4 galactosyltransferase is added, and the reaction time is 48 hours. After the reaction is complete, purification can obtain the pentasaccharide compound NOR1-NH2, and the reaction route is shown in Formula (4):

[0014] Formula (4).

[0015] Preferably, dissolve the pentasaccharide compound NOR1-NH2, acetic anhydride and sodium bicarbonate in water and stir for 5 min. After the reaction is complete, purify to obtain the target product, the NOR1 pentasaccharide compound.

[0016] Preferably, in the "one-pot multi-enzyme" system, the reaction temperature is 36 - 38 °C and the rotation speed is 120 - 150 rpm.

[0017] Preferably, the method for stopping the reaction is to boil the reaction solution in boiling water for 5 - 15 min to inactivate all the proteins, or add an equal volume of ice ethanol to the reaction solution and shake rapidly for 5 - 15 min.

[0018] Compared with the prior art, the method of the present invention has the following beneficial effects: The present invention uses a "one-pot multi-enzyme" system to synthesize the blood group antigen NOR1 pentasaccharide. Utilizing the substrate specificity principle of glycosyltransferase, through the exchange of NH2 and NHAc, the synthesis of the NOR1 antigen is achieved. The steps are simple, the overall yield is high, it can be synthesized in large batches, the use of chemical reagents is reduced, and it is environmentally friendly and pollution-free. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 1H NMR spectrum of the trisaccharide compound synthesized in the embodiment of the present invention;

[0020] Figure 2 13C NMR spectrum of the trisaccharide compound synthesized in the embodiment of the present invention;

[0021] Figure 3 1H NMR spectrum of the tetrasaccharide compound P-TFA synthesized in the embodiment of the present invention;

[0022] Figure 4 13C NMR spectrum of the tetrasaccharide compound P-TFA synthesized in the embodiment of the present invention;

[0023] Figure 5 1H NMR spectrum of the tetrasaccharide compound P-NH2 synthesized in the embodiment of the present invention;

[0024] Figure 6 13C NMR spectrum of the tetrasaccharide compound P-NH2 synthesized in the embodiment of the present invention;

[0025] Figure 7 1H NMR spectrum of the pentasaccharide compound NOR1-NH2 synthesized in the embodiment of the present invention;

[0026] Figure 8 13C NMR spectrum of the pentasaccharide compound NOR1-NH2 synthesized in the embodiment of the present invention;

[0027] Figure 91H NMR spectrum of the pentasaccharide compound NOR1 synthesized in the embodiments of the present invention;

[0028] Figure 10 13C NMR spectrum of the pentasaccharide compound NOR1 synthesized in the embodiments of the present invention. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with specific embodiments and drawings. More details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention is clearly capable of being practiced in many other ways different from this description. For those skilled in the art of this technology, any alternative improvements or transformations made to the embodiments of the present invention fall 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.

[0030] For the enzymatic synthesis of the NOR1 antigen, a mutant of human α1-4 galactosyltransferase A4GalT is required. The inventors of the present invention attempted to use the mutant of A4GalT to prepare the NOR1 antigen in previous experiments and found that the activity was too weak to achieve large-scale preparation. Subsequently, based on the substrate specificity principle of glycosyltransferase, the synthesis of the NOR1 antigen was achieved through the exchange of NH2 and NHAc.

[0031] The α1-4 galactosyltransferase LgtC derived from prokaryotes can use an oligosaccharide with Gal at the non-reducing end as a receptor to add α1-4Gal to form the Galα1-4Gal structure, but it cannot use an oligosaccharide with GalNAc at the end as a receptor to form the NOR1 antigen ( Galαl-4GalNAc βl-4Galαl-4Galβl-4Glc). The present invention uses GalNH2 βl-4Galαl-4Galβl-4Glc tetrasaccharide as a substrate to achieve the synthesis of the blood group antigen NOR1 pentasaccharide.

[0032] Example 1 Trisaccharide compound P k -pro-N3 (Galαl-4Galβl-4GlcβPro-N3) was synthesized, and the synthesis route is shown in formula (1):

[0033] Formula (1).

[0034] Dissolve 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 amounts of Tris and MgCl2 are calculated and determined according to the volume of the final reaction solution) in a 50 mL centrifuge tube. Add GalK and AtUSP (1.0 - 3.0 mg) and NmLgtC (0.5 - 2.0 mg). After adding double-distilled water to a total volume of 50 mL, place the reaction system in a shaker and incubate at 37 °C and 140 r / min for 4 hours. After detecting the completion of the reaction by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5), boil it in boiling water for 10 min to terminate the reaction. Then centrifuge the reaction solution at 4 °C and 11,000 r / min for 10 minutes, collect the supernatant, concentrate it by rotary evaporation, separate it through a polyacrylamide gel column, and obtain a white powder, which is the trisaccharide compound Gb3 (218 mg, 79 %). Its 1H NMR and 13C NMR spectra are shown in Figure 1 and Figure 2 respectively, and the parameters are as follows:

[0035] 1 1H 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 = 6.7 Hz, 2H), 3.26 – 3.20 (m, 1H), 1.84 (p, J = 6.5 Hz, 2H);

[0036] 13 13C 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 37N3O 16 [M+H] + 588.2173, found 588.2251。

[0037] Example 2 Synthesis of the tetrasaccharide compound P-TFA-pro-N3. The synthetic route is shown in Formula (2):

[0038] P-TFA (GalNTFAβl-4Galαl-4Galβl-4GlcβPro-N3)

[0039] Formula (2).

[0040] Dissolve the acceptor compound P k -pro-N3 (100 mg), galactosamine trifluoroacetate (GalNTFA) (70 mg), ATP (172 mg), UTP (165 mg), Tris-HCl buffer solution (100 mM, pH 6.5) and magnesium chloride (20 mM (the amounts of Tris and MgCl2 are calculated and determined according to the volume of the final reaction solution)) in a 50 mL centrifuge tube. Add NahK and AGX1 (1.0 - 3.0 mg). After reacting for one hour, add NmLgtD (0.5 - 2.0 mg). After adding double-distilled water to a total volume of 20 mL, place the reaction in a shaker and incubate at 37 °C and 140 r / min for 48 hours. After detecting the completion of the reaction by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5), add an equal volume of ice ethanol and shake rapidly for 5 min to terminate the reaction. Then centrifuge at 4 °C and 11000 r / min for 10 minutes, collect the supernatant, concentrate by rotary evaporation, and separate by a polyacrylamide gel column to obtain a white compound P-TFA (109 mg, 76%). The 1H NMR spectrum and 13C NMR spectrum of it are respectively as Figure 3 and Figure 4 shown, and the parameters are as follows:

[0041] 11H 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);

[0042] 13 13C 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, 70.29, 70.02, 68.89, 67.71, 67.56, 67.36, 61.43, 60.94, 60.37, 60.29, 59.33, 53.40, 47.87, 28.23. HRMS (ESI) m / z calculated for C 29 H 47 F3N4O 21 [M+H] + 845.2684, found 845.2760.

[0043] Example 3 Synthesis of the tetrasaccharide compound P-NH2-pro-N3 (GalNH2β1-4Galα1-4Galβ1-4GlcβPro-N3), and the synthetic route is shown in Formula (3):

[0044] Formula (3).

[0045] Dissolve compound P-TFA-pro-N3 (100 mg) in a 50 mL centrifuge tube, adjust the pH of the solution to 12, and react overnight at 37 °C to remove TFA to form compound P-NH2. After detecting the completion of the reaction by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5), adjust the solution to pH 7.5 to carry out the next reaction and partially purify the white compound P-NH2. The 1H NMR and 13C NMR spectra of it are shown in Figure 5 and Figure 6 respectively, and the parameters are as follows:

[0046] 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, 70.37, 69.67, 68.90, 67.66, 67.55, 67.37, 60.80, 60.36, 60.32,59.99, 53.57, 47.86, 28.22. HRMS (ESI) m / z calculated for C27H48N4O20 [M+H]+749.2861, found 749.2935。

[0047] Example 5 Synthesis of pentasaccharide compound NOR1-NH2-pro-N3 (Galαl-4GalNH2βl-4Galαl-4Galβl-4GlcβPro-N3), and the synthetic route is shown in Formula (4):

[0048] Formula (4).

[0049] Dissolve 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 amounts of Tris and MgCl2 are calculated and determined according to the volume of the final reaction solution) in a 50 mL centrifuge tube. Add GalK and AtUSP (1.0 - 3.0 mg) and NmLgtC (0.5 - 2.0 mg). After adding double-distilled water to a total volume of 15 mL, place the reaction solution in a shaker and incubate at 37 °C and 140 r / min for 48 hours. After detecting the completion of the reaction by thin-layer chromatography (EtOAc:MeOH:H2O:NH3·H2O = 3:2:1:1), boil it in boiling water for 10 min to terminate the reaction. Then centrifuge at 4 °C and 11000 r / min for 10 minutes, collect the supernatant, concentrate it by rotary evaporation, separate it through a polyacrylamide gel column, and obtain the white compound NOR1-NH2 (27 mg, 28%). The 1H NMR and 13C NMR spectra are respectively as follows Figure 7 and Figure 8 shown, and the parameters are as follows:

[0050] 1 H NMR (600 MHz, D2O) δ 4.95 – 4.88 (m, 3H), 4.43 (dd, J = 19.0, 7.9 Hz, 2H), 4.32 (dt, J = 12.5, 6.4 Hz, 2H), 4.19 (d, J = 3.0 Hz, 1H), 4.09 – 4.01 (m, 2H), 4.01 – 3.83 (m, 9H), 3.83 – 3.48 (m, 16H), 3.39 (t, J = 6.7 Hz, 2H), 3.26 (ddd, J = 20.4, 10.2, 8.2 Hz, 2H), 1.84 (p, J = 6.5 Hz, 2H);

[0051] 1313C 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, 70.88, 70.43, 69.10, 68.96, 68.92, 68.54, 67.73, 67.36, 60.61, 60.33, 60.18, 60.00, 47.86, 28.22. HRMS (ESI) m / z calculated for C 33 H 58 N4O 25 [M+H] + 911.3390, found 911.3465。

[0052] Example 6 Synthesis of pentasaccharide compound NOR1-pro-N3 (Galα1-4GalNAcβ1-4Galα1-4Galβ1-4GlcβPro-N3), and the synthetic route is shown in Formula (5):

[0053] Formula (5).

[0054] Dissolve compound NOR1-NH2 (10 mM), AC2O (15 mM) and NaHCO3 (15 mM) in water and stir overnight. After detecting the completion of the reaction by thin-layer chromatography (EtOAc:MeOH:H2O:NH3·H2O = 3:2:1:1), white compound NOR1 is obtained, and its 1H NMR spectrum and 13C NMR spectrum are respectively as Figure 9 and Figure 10 shown, and the parameters are as follows:

[0055] 11H 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);

[0056] 13 13C 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 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: the structural formula of the lactose or its modified product is: 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 tetrasaccharide, the "one-pot multi-enzyme" system used includes N-acetylglucosamine kinase, N-acetylglucosamine-1-P uridine transferase and β-1,3-N-acetylgalactosamine transferase; 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, 5-150mM MgCl2, and 100-600mM Tris-HCl buffer of pH 6-8 to prepare an aqueous solution, and the pH of the reaction system is adjusted to between 6.0 and 7.0, and then N-acetylglucosamine kinase and N-acetylglucosamine-1-P uridyl transferase are added, and β-1,3-N-acetylgalactosamine transferase is added after one hour of reaction, and 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: 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, 5-150mM MgCl2, 100-600mM Tris-HCl buffer of pH6-8 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 performed for one hour before being boiled and inactivated, 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 trisaccharide compound of the antigen, the reaction route is:

3. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 1, 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.

4. 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, 5-150mM MgCl2, and 100-600mM 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 can be obtained by purification, and the reaction route is as follows:

5. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 4, characterized in that The pentasaccharide compound NOR1-NH2, acetic anhydride and sodium bicarbonate are dissolved in water and stirred for 5 minutes. After the reaction is complete, purification is performed to obtain the target product NOR1 pentasaccharide compound.

6. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to any one of claims 1 to 5, 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.

7. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to any one of claims 1 to 5, characterized in that: The reaction temperature in the "one pot multi-enzyme" system is 36-38°C and the rotation speed is 120-150rpm.

8. The method for enzymatically synthesizing blood group antigen NOR1 pentasaccharide according to claim 1, 2 or 4, 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.

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