A method for preparing n-glycoamino acids from n-glycopeptides and n-glycoamino acids

Abstract

The application provides a method for preparing N-glycosyl amino acid from N-glycopeptide and the N-glycosyl amino acid, and belongs to the technical field of protein glycosylation research. In the application, N-glycopeptide is used as raw material, and the N-glycopeptide is subjected to two enzymatic reactions with alkaline protease and carboxypeptidase B in sequence, and the obtained reaction liquid is subjected to filtration and ultrafiltration, and then subjected to anion column purification by using a gradient concentration NaCl solution as an eluent; the sample eluted is subjected to nanofiltration, ultrafiltration, concentration and freeze-drying in sequence, so that the N-glycosyl amino acid is obtained. The purity of the N-glycosyl amino acid prepared by the method reaches more than 96%, and the yield reaches more than 80%. Compared with the prior art, the method has shorter time, lower cost, higher product conversion rate and purity, and is more suitable for industrial large-scale preparation of N-glycosyl amino acid.

Description

Technical Field

[0001] This invention relates to the field of protein glycosylation research technology, specifically to a method for preparing N-glycopeptides into N-glycoamino acids and the N-glycoamino acids obtained by this method. Background Technology

[0002] Glycosylation is a post-translational modification that covalently links sugar chains to specific amino acid sites on a protein. Glycosylation is one of the most common, prevalent, and important post-translational modifications. In the human body, approximately 50%-70% of proteins undergo glycosylation, especially secretory proteins, almost all of which are glycosylated. Based on the different glycopeptide chains, glycosylation can be classified as: (1) N-glycosylation; (2) O-glycosylation; (3) C-glycosylation; (4) Glycosylphosphatidylinositol anchoring (GPI anchoring); (5) O-linked N-glycosylation; and (6) other modification types. N-glycosylation refers to the linkage of sugar chains to the free NH2 of asparagine in proteins. N-glycosylation begins in the endoplasmic reticulum and is completed in the Golgi apparatus. There are over 150 common N-glycoforms in the human body. Usually, the first sugar unit attached is N-acetylglucosamine (GlcNAc), and the attachment site is a specific asparagine. The structural rule of N-glycosylated amino acids is as follows: NXS / T / C (X≠P, that is, X cannot be proline).

[0003] Glycosylation has a profound impact on protein structure and function. For example, it can affect protein folding, stability, distribution, and biological activity. Glycosylation is also a key mechanism for intercellular recognition and signal transduction, participating in the regulation of processes such as immune responses, cell adhesion, and viral infection. Alterations in glycosylation modifications are closely related to many pathological processes, such as the occurrence and metastasis of cancer. Therefore, comprehensive and in-depth research on the types, sites, and content changes of protein glycosylation in cells has significant scientific and clinical translational value.

[0004] The existing technology, "Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder" (DOI: 10.1016 / j.carres.2017.10.001), discloses a method for obtaining N-glycoamino acid A2-Asn with a purity of approximately 90% using sialic acid glycopeptide (SGP) as a raw material and reacting with proteinase E for 5 days. The yield (less than 50%) and purity of N-glycoamino acid A2-Asn prepared by this method are not high enough, and the production cost is relatively high, making it suitable only for laboratory research and not for large-scale industrial production. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for preparing N-glycopeptides from N-glycopeptides. This method uses N-glycopeptides as raw materials and, through a two-step enzymatic reaction, yields N-glycoamino acids with higher purity and yield. This method eliminates the need for highly toxic substances and organic solvents, and the preparation time and cost of N-glycoamino acids are short, making it suitable for large-scale production.

[0006] Specifically, in order to achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing N-glycoamino acids includes the following steps:

[0008] S1. Prepare N-glycopeptide into solution I, add alkaline protease to solution I to carry out the first enzymatic reaction, and obtain reaction intermediate II; boil to terminate the reaction, and obtain reaction solution III;

[0009] S2. Cool the reaction solution III to room temperature, add carboxypeptidase B to the reaction solution III to carry out a second enzymatic reaction, and after the reaction is completed; boil to terminate the reaction, centrifuge to obtain filtrate IV.

[0010] S3. Filter the filtrate IV layer by layer, then perform ultrafiltration, and then purify it using an anion exchange column; use NaCl solution of gradient concentration as the eluent for purification; collect the eluted samples, combine them, and then perform nanofiltration, ultrafiltration, concentration, and lyophilization in sequence to obtain N-glycoamino acids.

[0011] The structural formula of the N-glycopeptide is as follows:

[0012]

[0013] The structural formula of the N-glycoamino acid is as follows:

[0014]

[0015] In a preferred embodiment, the concentration of N-glycopeptide in solution I is 5–30 g / L.

[0016] In a preferred embodiment, the pH of solution I is 7 to 9.

[0017] In a preferred embodiment, solution I contains 5–20 mmol / L of Ca. 2+ .

[0018] In a preferred embodiment, the mass ratio of the alkaline protease to the N-glycopeptide is 1:1 to 1:10.

[0019] In a preferred embodiment, the alkaline protease has an enzyme activity ≥100,000 U / g.

[0020] In a preferred embodiment, the activity of the carboxypeptidase B is >200 U / mg.

[0021] In the preferred embodiment, the temperature of the first enzymatic reaction in step S1 is 20–50°C, and the reaction time is 4–16 h.

[0022] In a preferred embodiment, the mass ratio of carboxypeptidase B to reaction solution III is 1:30-1:5.

[0023] In the preferred embodiment, the temperature of the second enzymatic reaction in step S2 is 20–50°C, and the reaction time is 12–36 h.

[0024] In a preferred embodiment, the boiling time in step S1 is 10 minutes; or / and the boiling time in step S2 is 10 minutes.

[0025] In a preferred embodiment, the concentration of the NaCl solution in step S3 is in the range of 30 mmol / L to 1 mol / L.

[0026] The present invention also provides N-glycoamino acids prepared according to any of the above-described schemes.

[0027] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0028] (1) In this invention, N-glycopeptides are used as raw materials to obtain N-glycoamino acids through two enzymatic reactions. The conversion rate is high, the reaction time is short, and the subsequent purification method is simple, which greatly shortens the process time.

[0029] (2) The enzyme raw materials used in this invention are inexpensive and readily available, which greatly reduces the cost of the preparation process and makes it suitable for preparing N-sugar amino acids on a kilogram-scale basis.

[0030] (3) The preparation method in this invention can obtain N-sugar amino acids with a yield of over 80% and a purity of over 96% through one-step purification, which is more in line with the quality requirements of pharmaceutical raw materials.

[0031] (4) Compared with purification using P2 packed columns or amide columns, the enzyme is removed by membrane separation after the reaction in this invention, which is simple to operate and more efficient; the product is purified by anion exchange column, which is more automated, has a large single processing capacity (P2 packed columns are expensive and have a sample loading capacity of only 200 mg / kg packing material), and has good stability. Attached Figure Description

[0032] Figure 1 The structural formula of N-glycopeptide GP1001, the raw material used in Examples 1 to 3 of this invention;

[0033] Figures 2-18 The structural formulas of the raw materials N-glycopeptides GP1002 to GP1008 used in Examples 4 to 20 of this invention are shown in sequence.

[0034] Figure 19 The structural formulas of the N-glycoamino acids prepared in Examples 1-3 of this invention are shown below.

[0035] Figures 20-36 The structural formulas of the N-glycoamino acids prepared in Examples 4 to 20 of this invention are shown in sequence.

[0036] Figure 37 The general structural formula for N-glycopeptides prepared using the technical solution of this invention is provided.

[0037] Figure 38 To adopt Figure 37 The structural formula of the N-glycopeptide shown is obtained by preparing N-glycoamino acids using the technical solution of this invention;

[0038] Figure 39 The HPLC chromatogram of SGN prepared in Example 3 of the present invention;

[0039] Figure 40 The above is the hydrogen nuclear magnetic resonance (HNMR) spectrum of the SGN prepared in Example 3 of the present invention.

[0040] Figure 41 The mass spectrum (MS) of the SGN prepared in Example 3 of the present invention;

[0041] Figure 42 The HPLC chromatogram of the N-glycoamino acid A2-Asn prepared in Comparative Example 3 is shown.

[0042] In the picture: It represents Neu5Ac (sialic acid); It represents Galactose; This refers to N-acetyl-glucosamine. It refers to Mannose; Fucose represents the sugar; Asn represents asparagine. Detailed Implementation

[0043] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0044] The SGP (sialic acid glycopeptide) used in the following examples was prepared according to the method disclosed in the applicant's earlier invention patent application CN202310655553.X. All other raw materials were common commercially available. The alkaline protease activity only needed to meet the condition of ≥100,000 U / g. The carboxypeptidase B activity only needed to meet the condition of >200 U / mg.

[0045] In the following examples, SGN (full name Sialylglycoasparaginate, structural formula as shown) is... Figure 19 The purity of the amino acid A2-Asn in the sample (shown in the figure) and in Comparative Example 3 was determined by HPLC (High Performance Liquid Chromatography). The detection conditions were as follows:

[0046] Chromatographic column: Waters BEH Amide (250×4.6mm, 5.0μm);

[0047] Mobile phase A: 30 mM ammonium acetate;

[0048] Mobile phase B: Acetonitrile;

[0049] Flow rate: 1 mL / min, column temperature: 70℃;

[0050] Detector: ELSD-G4260B, gas flow rate: 1.6 L / min, drift tube temperature: 40℃;

[0051] Injection volume: 10 μL;

[0052] Maximum pressure limit: 600 bar;

[0053] The gradient elution procedure is shown in Table 1 below:

[0054] Table 1 HPLC gradient elution program

[0055]

[0056]

[0057] In the following examples, N-glycoamino acids were obtained using the freeze-drying procedure in Table 2.

[0058] Table 2 Freeze-drying process

[0059] 01 -40 10 0 02 -25 4 10 03 -20 4 10 04 -15 19 10 05 -10 24 10 06 -5 6 10 07 0 2 10 08 10 1 10 09 20 1 10

[0060] Example 1

[0061] A method for preparing an N-glycoamino acid (GPN-0001) includes the following steps:

[0062] S1, Take SGP(GP-1001, structural formula as follows) Figure 1 The solid powder (as shown) was added to water to prepare solution I with a concentration of 20 g / L, pH 8, and CaCl2 concentration of 15 mM. Alkaline protease (enzyme activity 100,000 U / g) was added to solution I at a mass ratio of alkaline protease to SGP of 1:8. The reaction was carried out at 37°C for 12 h to obtain reaction intermediate II. The reaction was terminated by boiling intermediate II (100°C) for 10 min to obtain reaction solution III.

[0063] S2. Cool reaction solution III to room temperature. Add carboxypeptidase B to reaction solution III at a mass ratio of 1:20. React at 37°C for 36 hours. After the reaction is complete, boil for 10 minutes to terminate the reaction. Centrifuge at 4°C and 8000 rpm for 30 minutes to obtain filtrate IV.

[0064] S3. Filtrate IV was first filtered through a 5 μm pore size membrane, then through a 0.45 μm pore size membrane, followed by ultrafiltration through a 10 nm pore size PES (polyethersulfone) membrane, and then purified by anion exchange column (DEAE Beads 6FF). NaCl was used as the eluent, and impurities and the product were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. The product was eluted in the 50 mM–100 mM gradient. The eluted samples were collected, combined, and then sequentially nanofiltered through a 200 Da nanofiltration membrane, followed by ultrafiltration and desalting through a 1000 Da PES membrane. The solution was concentrated under reduced pressure at 60 °C and lyophilized to obtain N-glycoamino acid GPN-0001 (abbreviated as SGN, structural formula as shown) with a purity of 96%. Figure 19 As shown in the figure, the yield was 81%.

[0065] Example 2

[0066] A method for preparing an N-glycoamino acid (GPN-0001) includes the following steps:

[0067] S1, Take SGP(GP-1001, structural formula as follows) Figure 1 The solid powder (as shown) was added to water to prepare solution I with a concentration of 5 g / L, pH 7, and CaCl2 concentration of 5 mM. Alkaline protease (enzyme activity 100,000 U / g) was added to solution I at a mass ratio of alkaline protease to SGP of 1:10. The reaction was carried out at 37°C for 4 hours to obtain reaction intermediate II. The reaction was terminated by boiling intermediate II (100°C) for 10 minutes to obtain reaction solution III.

[0068] S2. Cool reaction solution III to room temperature. Add carboxypeptidase B to reaction solution III at a mass ratio of 1:10. React at 37°C for 24 hours. After the reaction is complete, boil for 10 minutes to terminate the reaction. Centrifuge at 4°C and 8000 rpm for 30 minutes to obtain filtrate IV.

[0069] S3. Filtrate IV was first filtered through a 5 μm pore size membrane, then through a 0.45 μm pore size membrane, followed by ultrafiltration through a 10 nm pore size PES membrane, and then purified by anion exchange column (DEAE Beads 6FF). NaCl was used as the eluent, and impurities and the product were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. The product was eluted in the 50 mM–100 mM gradient. The eluted samples were collected, combined, and then sequentially nanofiltered through a 200 Da nanofiltration membrane, followed by ultrafiltration and desalting through a 1000 Da PES membrane. The solution was concentrated under reduced pressure at 60 °C and lyophilized to obtain N-glycoamino acid GPN-0001 (abbreviated as SGN, structural formula as shown) with a purity of 98%. Figure 19 (As shown), the yield was 82%.

[0070] Example 3

[0071] A method for preparing an N-glycoamino acid (GPN-0001) includes the following steps:

[0072] S1, Take SGP(GP-1001, structural formula as follows) Figure 1The solid powder (as shown) was added to water to prepare solution I with a concentration of 30 g / L, pH 9, and CaCl2 concentration of 20 mM. Alkaline protease (enzyme activity 100,000 U / g) was added to solution I at a mass ratio of 1:1 to alkaline protease. The reaction was carried out at 37°C for 16 h to obtain reaction intermediate II. The reaction was terminated by boiling intermediate II (100°C) for 10 min to obtain reaction solution III.

[0073] S2. Cool reaction solution III to room temperature. Add carboxypeptidase B to reaction solution III at a mass ratio of 1:5. React at 37°C for 24 hours. After the reaction is complete, boil for 10 minutes to terminate the reaction. Centrifuge at 4°C and 8000 rpm for 30 minutes to obtain filtrate IV (conversion rate of 90% in this step).

[0074] S3. Filtrate IV was first filtered through a 5 μm pore size membrane, then through a 0.45 μm pore size membrane, followed by ultrafiltration through a 10 nm pore size PES membrane, and then purified by anion exchange column (DEAE Beads 6FF). NaCl was used as the eluent, and impurities and the product were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. The product was eluted in the 50 mM–100 mM gradient. The eluted samples were collected, combined, and then sequentially nanofiltered through a 200 Da nanofiltration membrane, followed by ultrafiltration and desalting through a 1000 Da PES membrane. The solution was concentrated under reduced pressure at 60 °C and lyophilized to obtain a purity of 99% (HPLC chromatogram shown). Figure 39 The N-glycoamino acid GPN-0001 (abbreviated as SGN, structural formula as follows) Figure 19 (As shown), the yield was 83%.

[0075] Example 4

[0076] A method for preparing an N-glycoamino acid (GPN-0002) includes the following steps:

[0077] S1. Take N-glycopeptide GP-1002 (structural formula as shown) Figure 2 (As shown) Solid powder was added to water to prepare a solution with a concentration of 10 g / L, pH 8, and Ca... 2+ Solution I was prepared with a concentration of 18 mM. Alkaline protease (enzyme activity 100,000 U / g) was added to solution I at a mass ratio of 1:5 to N-glycopeptide GP-1002. The reaction was carried out at 37°C for 8 hours to obtain reaction intermediate II. The reaction was terminated by boiling intermediate II (100°C) for 10 minutes to obtain reaction solution III.

[0078] S2. Cool reaction solution III to room temperature. Add carboxypeptidase B to reaction solution III at a mass ratio of 1:30. React at 37°C for 36 hours. After the reaction is complete, boil for 10 minutes to terminate the reaction. Centrifuge at 4°C and 8000 rpm for 30 minutes to obtain filtrate IV.

[0079] S3. Filtrate IV was first filtered through a 5 μm pore size membrane, then through a 0.45 μm pore size membrane, followed by ultrafiltration through a 10 nm pore size PES membrane, and then purified by anion exchange column (DEAE Beads 6FF). NaCl was used as the eluent, and impurities and the product were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. The product was eluted in the 50 mM–100 mM gradient. The eluted samples were collected, combined, and then sequentially nanofiltered through a 200 Da nanofiltration membrane, followed by ultrafiltration and desalting through a 1000 Da PES membrane. The solution was concentrated and lyophilized to obtain N-glycoamino acid GPN-0002 (structural formula shown) with a purity of 97%. Figure 20 As shown in the figure, the yield was 81%.

[0080] Example 5

[0081] A method for preparing an N-glycoamino acid (GPN-1003) includes the following steps:

[0082] S1. Take N-glycopeptide GP-1003 (structural formula as shown) Figure 3 The solid powder (as shown) was added to water to prepare solution I with a concentration of 15 g / L, pH 9, and CaCl2 concentration of 10 mM. Alkaline protease (enzyme activity of 100,000 U / g) was added to solution I at a mass ratio of 1:3 to N-glycopeptide GP-1003. The reaction was carried out at 37°C for 10 h to obtain reaction intermediate II. The reaction was terminated by boiling intermediate II (100°C) for 10 min to obtain reaction solution III.

[0083] S2. Cool reaction solution III to room temperature. Add carboxypeptidase B to reaction solution III at a mass ratio of 1:8. React at 37°C for 15 hours. After the reaction is complete, boil for 10 minutes to terminate the reaction. Centrifuge at 4°C and 8000 rpm for 30 minutes to obtain filtrate IV.

[0084] S3. Filtrate IV was first filtered through a 5 μm pore size membrane, then through a 0.45 μm pore size membrane, followed by ultrafiltration through a 10 nm pore size PES membrane, and then purified by anion exchange column (DEAE Beads 6FF). NaCl was used as the eluent, and impurities and the product were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. The product was eluted in the 50 mM–100 mM gradient. The eluted samples were collected, combined, and then sequentially nanofiltered through a 200 Da nanofiltration membrane, followed by ultrafiltration and desalting through a 1000 Da PES membrane. The solution was concentrated under reduced pressure at 60 °C and lyophilized to obtain N-glycoamino acid GPN-0003 (structural formula shown) with a purity of 97%. Figure 21 (As shown), the yield was 82%.

[0085] Examples 6-20

[0086] A method for preparing the corresponding N-glycoamino acids (GPN-0004 to GPN-0018) from N-glycopeptides GP-1004 to GP-1018, respectively, includes the following steps:

[0087] S1. Take N-glycopeptides GP-1004~GP-1018 respectively (structural formulas are as follows) Figures 4-18 The solid powder (as shown) was added to water to prepare solution I with a concentration of 25 g / L, pH 7, and CaCl2 concentration of 12 mM. Alkaline protease (enzyme activity 100,000 U / g) was added to solution I at a mass ratio of alkaline protease to N-glycopeptide of 1:7. The reaction was carried out at 37°C for 14 h to obtain reaction intermediate II. The reaction was terminated by boiling intermediate II (100°C) for 10 min to obtain reaction solution III.

[0088] S2. Cool reaction solution III to room temperature. Add carboxypeptidase B to reaction solution III at a mass ratio of 1:25. React at 37°C for 30 hours. After the reaction is complete, boil for 10 minutes to terminate the reaction. Centrifuge at 4°C and 8000 rpm for 30 minutes to obtain filtrate IV.

[0089] S3. Filtrate IV was first filtered through a 5 μm pore size membrane, then through a 0.45 μm pore size membrane, followed by ultrafiltration through a 10 nm pore size PES membrane, and then purified by anion exchange column (DEAE Beads 6FF). NaCl was used as the eluent, and impurities and products were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. The product was eluted in the 50 mM–100 mM gradient. The eluted samples were collected, combined, and then sequentially nanofiltered through a 200 Da nanofiltration membrane, followed by ultrafiltration and desalting through a 1000 Da PES membrane. The solution was concentrated and lyophilized to obtain N-glycoamino acids (structural formulas as shown below). Figures 22-36 (As shown).

[0090] It should be noted that as long as the following conditions are met... Figure 37 The N-glycopeptides with the structural formulas shown can all be prepared using the methods described in this application to prepare the corresponding N-glycoamino acids (e.g., ...). Figure 38 As shown in the figures, all methods achieved yields of over 80% and purities of over 96%. The above examples only list some methods for preparing N-glycopeptides into N-glycoamino acids; all methods for preparing N-glycoamino acids from N-glycopeptides will not be described in detail here. The yields and purities of Examples 1-20 are shown in Table 3.

[0091] Table 3. Purity and yield of N-glycoamino acids prepared in Examples 1-20

[0092]

[0093]

[0094] Comparative Example 1

[0095] This comparative example provides a method for preparing N-glycoamino acid (GPN-0001), which is basically the same as the method in Example 1, except that the alkaline protease in step S1 is replaced with aminopeptidase (activity 500 U / g). Experimental results showed that an alanine (A) residue remained at the N-terminus of SGP that could not be cleaved.

[0096] Comparative Example 2

[0097] A method for preparing an N-glycoamino acid (GPN-0001) includes the following steps:

[0098] P1. Take SGP(GP-1001, structural formula as follows) Figure 1The solid powder (as shown) was added to water to prepare solution I with a concentration of 30 g / L, pH 9, and CaCl2 concentration of 20 mM. Alkaline protease (100,000 U / g activity) and carboxypeptidase B were added to solution I at a mass ratio of 1:1 (alkaline protease to SGP) and 1:30 (carboxypeptidase B to SGP). The reaction was carried out at 37°C for 72 h, and then stopped by boiling (100°C) for 10 min.

[0099] P2. Centrifuge the reaction solution obtained in step P1 at 4℃ and 8000rpm for 30min to obtain the filtrate. Filter the filtrate first through a 5μm pore size membrane, then through a 0.45μm pore size membrane, then through a 10nm pore size PES membrane for ultrafiltration, and finally through anion exchange column (DEAE Beads 6FF) purification. NaCl was used as the eluent, and impurities and the product were separated using gradient concentrations of 30mM, 50mM, 100mM, and 1M. The product was eluted in the 50mM–100mM gradient. Collect and combine the eluted samples, then sequentially nanofilter using a 200Da nanofiltration membrane, followed by ultrafiltration and desalting using a 1000Da PES membrane. The solution was concentrated and lyophilized to obtain N-glycoamino acid GPN-0001 (abbreviated as SGN, structural formula as shown) with a purity of 96%. Figure 19 (As shown), the yield was 10%.

[0100] Comparative Example 3

[0101] This comparative example uses the method in Section 4.6 of the prior art, "Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder" (DOI: 10.1016 / j.carres.2017.10.001), to prepare N-glycoamino acid A2-Asn (i.e., GPN-1005 in this application) using SGP.

[0102] Product purity testing

[0103] The purity of the N-glycoamino acids prepared in Example 3 and Comparative Example 3 was tested, and the cost, conversion rate, and time of N-glycoamino acid preparation were statistically analyzed. The results are shown in Table 4 below. The purity test results of SGN prepared in Example 3 are shown in Table 4 below. Figure 39 See the nuclear magnetic resonance spectrum. Figure 40 Mass spectrum (see) Figure 41 The purity test results of A2-Asn prepared in Comparative Example 3 are shown in [Figure Number]. Figure 42 .

[0104] Table 4 Comparison of the methods for preparing N-glycoamino acids in this application and existing technologies

[0105]

[0106] from Figure 40 and Figure 41 It can be seen that the preparation method in this application successfully prepared the N-glycoamino acid Sialylglycoasparaginate (structural formula as shown in the image). Figure 19 As shown in Table 4, the method for preparing N-glycoamino acids in this application is lower in cost, shorter in time, and has higher conversion rate and purity than the methods for preparing N-glycoamino acids in the prior art, making it more suitable for large-scale production.

[0107] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of this application and the content of the specification should be included within the scope of protection of this application.

Claims

1. A method for preparing N-glycoamino acids, characterized in that, Includes the following steps: S1. Prepare N-glycopeptide solution I, add alkaline protease to solution I for the first enzymatic reaction to obtain reaction intermediate II; boil to terminate the reaction to obtain reaction solution III; the pH of solution I is 7-9; the temperature of the first enzymatic reaction is 37℃, and the reaction time is 4-16h; the enzyme activity of alkaline protease is ≥100,000 U / g, and the mass ratio of alkaline protease to N-glycopeptide is 1:1 to 1:10; solution I contains 5-20 mmol / L of Ca. 2+ ; S2. Cool the reaction solution III to room temperature, add carboxypeptidase B to the reaction solution III to carry out a second enzymatic reaction, wherein the activity of carboxypeptidase B is >200U / mg, and the mass ratio of carboxypeptidase B to the reaction solution III is 1:5-1:30; the temperature of the second enzymatic reaction is 37℃, and the reaction time is 15~36h; after the reaction is completed, boil to terminate the reaction, centrifuge, and obtain filtrate IV; S3. Filter the filtrate IV layer by layer, then perform ultrafiltration, and then purify it using an anion exchange column; Purification was performed using NaCl solutions of varying concentrations as eluents; The eluted samples were collected, combined, and then subjected to nanofiltration, ultrafiltration, concentration, and lyophilization in sequence to obtain N-sugar amino acids. The structural formula of the N-glycopeptide is shown in any one of formulas GP-1001 to GP-1018: , ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， 。 2. The method for preparing N-glycoamino acids according to claim 1, characterized in that, The concentration of N-glycopeptide in solution I in step S1 is 5~30 g / L.

3. The method for preparing N-glycoamino acids according to claim 1, characterized in that, In step S3, the concentration range of the NaCl solution is 50 mmol / L to 100 mmol / L.

Images