Method for preparing N-sugar amino acid from N-glycopeptide and N-sugar amino acid

Abstract

The invention provides a method for preparing N-sugar amino acid from N-glycopeptide and the N-sugar amino acid, and belongs to the technical field of protein glycosylation research. According to the method, N-glycopeptide is adopted as a raw material, the N-glycopeptide is sequentially subjected to two times of enzymatic reactions with alkaline protease and carboxypeptidase B, an obtained reaction solution is subjected to filtration and ultrafiltration, and then a NaCl solution with gradient concentration is adopted as an eluent for anion column purification; sequentially carrying out nanofiltration, ultrafiltration, concentration and freeze-drying treatment on a sample obtained by elution to obtain the N-sugar amino acid. The purity of the N-sugar amino acid prepared by the method reaches 96% or above, and the yield reaches 80% or above. Compared with the prior art, the method has the advantages of shorter time, lower cost, higher product conversion rate and higher purity, and is more suitable for industrial large-scale preparation of N-sugar amino acid.

Description

Technical Field

[0001] The present invention relates to the technical field of protein glycosylation research, and particularly relates to a method for preparing N-glycoamino acids from N-glycopeptides and the N-glycoamino acids prepared by this method. Background Art

[0002] Glycosylation modification of proteins is a post-translational modification that covalently attaches sugar chains to specific amino acid sites on proteins. Glycosylation modification is one of the most common, prevalent, and important post-translational modifications. In the human body, approximately 50%-70% of proteins can undergo glycosylation modification, especially secreted proteins almost all undergo glycosylation modification. According to the different glycopeptide chains, glycosylation modification can be divided into: (1) N-glycosylation; (2) O-glycosylation; (3) C-glycosylation; (4) glycosylphosphatidylinositol anchor (GPI anchor); (5) O-linked N-glycosylation (O-GlcNAcylation); (6) other modification types. Among them, N-glycosylation (N-Glycans) refers to the sugar chain being linked through the free NH 2 of asparagine in proteins. N-glycosylation begins in the endoplasmic reticulum and is completed in the Golgi apparatus. There are more than 150 common N-glycan types in the human body. Usually, the first sugar unit attached is N-acetylglucosamine (GlcNAc), and the attachment site is specific asparagine. The structural rule of N-glycosylated amino acids is as follows: N-X-S / T / C (X≠P, that is, X cannot be proline).

[0003] Glycosylation has a profound impact on the structure and function of proteins. For example, it can affect protein folding, stability, distribution, and biological activity. Glycosylation is also a key mechanism for cell-cell recognition and signal transduction, and is involved in regulating processes such as immune responses, cell adhesion, and viral infections. Changes in glycosylation modification are closely related to many pathological processes, such as the occurrence and metastasis of cancer. Therefore, conducting a comprehensive and in-depth study on the types, sites, and content changes of protein glycosylation in cells has great scientific research and clinical translation value.

[0004] The prior art "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 about 90% by reacting with protease E for 5 days using sialylglycopeptide (SGP) as a raw material. The yield (less than 50%) and purity of the N-glycoamino acid A2-Asn prepared by this method are not high enough, and the production cost is relatively high, which is only suitable for laboratory research and not suitable for large-scale industrial production. Summary of the Invention

[0005] Based on the above problems, the present invention provides a method for preparing N-glycoamino acids from N-glycopeptides. This method uses N-glycopeptides as raw materials and obtains N-glycoamino acids with higher purity and yield through two-step enzymatic reactions. This method does not require the use of highly toxic substances and organic solvents, has a short preparation time and low cost for N-glycoamino acids, and is suitable for scale-up production.

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

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

[0008] S1. Prepare solution I with N-glycopeptide, 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;

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

[0010] S3. Filter filtrate IV layer by layer, then perform ultrafiltration, and then purify it using an anion column; use a NaCl solution with a gradient concentration as the eluent for purification; collect the eluted samples, and after combining, perform nanofiltration, ultrafiltration, concentration, and freeze-drying treatments 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 7-9.

[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 1:1-1:10.

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

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

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

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

[0023] In a preferred embodiment, in step S2, the temperature of the second enzymatic reaction 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 min; or / and the boiling time in step S2 is 10 min.

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

[0026] The present invention also provides N-glyco amino acids prepared according to the scheme described in any one of the above.

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

[0028] (1) In the present invention, N-glycopeptide is used as the raw material, and N-glyco amino acids are obtained through two-step enzymatic reactions, with high conversion rate, short reaction time, and simple later purification method, which greatly shortens the process time.

[0029] (2) The enzyme raw materials used in the present invention are cheap and easily available. Therefore, the preparation process cost is greatly reduced, and it is suitable for the preparation of N-glyco amino acids on a kilogram scale.

[0030] (3) The preparation method in the present invention can obtain N-glycoamino acid with a yield of more than 80% and a purity of more than 96% through one-step purification, which better meets the quality requirements of drug raw materials.

[0031] (4) Compared with the purification using P2 packing column or amide column, in the present invention, after the reaction, the enzyme is removed by membrane separation method, which has simple operation and higher efficiency; the product is purified by anion column, which has higher automation degree, larger single-treatment volume (the price of P2 packing column is expensive and the sample loading amount is only 200 mg / kg packing), and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the structural formula of the raw material N-glycopeptide GP1001 used in Examples 1-3 of the present invention;

[0033] Figures 2 to 18 They are the structural formulas of the raw material N-glycopeptides GP1002-GP1008 used in Examples 4-20 of the present invention in sequence

[0034] Figure 19 It is the structural formula of the N-glycoamino acid prepared in Examples 1-3 of the present invention;

[0035] Figures 20 to 36 They are the structural formulas of the N-glycoamino acids prepared in Examples 4-20 of the present invention in sequence;

[0036] Figure 37 It is the general structural formula of N-glycopeptide applicable to the preparation of N-glycoamino acid by the technical solution of the present invention;

[0037] Figure 38 It is prepared by using Figure 37 The general structural formula of the N-glycoamino acid prepared by using the N-glycopeptide shown as the raw material by the technical solution of the present invention;

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

[0039] Figure 40 It is the nuclear magnetic resonance hydrogen spectrum (HNMR) diagram of SGN prepared in Example 3 of the present invention;

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

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

[0042] In the figure: It represents Neu5Ac (sialic acid); represents Galactose; represents N-acetyl-glucosamine; represents Mannose; represents Fucose; Asn represents asparagine. Detailed implementation manners

[0043] The following content describes the technical solutions of this application clearly and completely in combination with the embodiments, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are only some preferred embodiments of this application, rather than all embodiments. Any equivalent transformation or substitution made to the following implementation manners by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0044] The SGP (sialoglycopeptide) used in the following examples was prepared according to the method disclosed in the prior invention patent application CN202310655553.X of the applicant of this application, and the remaining raw materials are all common commercially available raw materials. The enzyme activity of alkaline protease only needs to meet the condition of ≥100,000 U / g. The activity of carboxypeptidase B only needs to meet the condition of >200 U / mg.

[0045] In the following examples, the purity of SGN (full name Sialylglycoasparaginate, structural formula as Figure 19 shown) and the purity of N-glycoamino acid A2-Asn in Comparative Example 3 were both detected by HPLC (high performance liquid chromatography), and the detection conditions are as follows:

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

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

[0048] Mobile phase B: acetonitrile;

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

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

[0051] Injection volume: 10 μL;

[0052] Maximum pressure limit: 600 bar;

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

[0054] Table 1 HPLC Gradient Elution Program

[0055]

[0056]

[0057] In the following examples, the freeze-drying procedure in Table 2 was used to obtain N-glycoamino acids.

[0058] Table 2 Freeze-drying Procedure

[0059] Section Temperature / °C Time / h Vacuum degree / Pa 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 preparation method of an N-glycoamino acid (GPN-0001) includes the following steps:

[0062] S1. Take the solid powder of SGP (GP-1001, the structural formula is as Figure 1 shown), add it to water, and prepare a solution I with a concentration of 20 g / L, a pH of 8, and a CaCl 2 concentration of 15 mM. According to the mass ratio of alkaline protease to SGP of 1:8, add alkaline protease (enzyme activity is 100,000 U / g) to solution I. React at 37°C for 12 h to obtain reaction intermediate II. Boil the reaction intermediate II (100°C) for 10 min to terminate the reaction and obtain reaction solution III.

[0063] S2. Cool reaction solution III to room temperature. According to the mass ratio of carboxypeptidase B to reaction solution III of 1:20, add carboxypeptidase B to reaction solution III and react at 37°C for 36 h. After the reaction ends, boil for 10 min to terminate the reaction, centrifuge at 4°C and 8000 rpm for 30 min to obtain filtrate IV.

[0064] S3. First filter filtrate IV with a filter membrane with a pore size of 5 μm, then filter it with a filter membrane with a pore size of 0.45 μm, then perform ultrafiltration with a PES (polyethersulfone) membrane with a pore size of 10 nm, and then perform purification on an anion column (DEAE Beads 6FF). Purification uses NaCl as the eluent, and gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M are used to separate impurities and products. Among them, the product is eluted in the 50 mM - 100 mM gradient. Collect and combine the eluted samples, then perform nanofiltration with a 200 Da nanofiltration membrane in sequence, perform ultrafiltration to remove salt with a 1000 Da PES membrane, concentrate under reduced pressure at 60°C, and freeze-dry to obtain N-glycoamino acid GPN-0001 with a purity of 96% (abbreviated as SGN, the structural formula is as Figure 19 shown), and the yield is 81%.

[0065] Example 2

[0066] A preparation method of N-glyco amino acid (GPN-0001) comprises the following steps:

[0067] S1. Take solid powder of SGP (GP-1001, the structural formula is as Figure 1 shown), add it into water to prepare solution I with a concentration of 5 g / L, a pH of 7, and a CaCl 2 concentration of 5 mM. According to the mass ratio of alkaline protease to SGP of 1:10, add alkaline protease (enzyme activity is 100,000 U / g) to solution I. React at 37°C for 4 h to obtain reaction intermediate II. Boil the reaction intermediate II (100°C) for 10 min to terminate the reaction, and obtain reaction solution III.

[0068] S2. Cool reaction solution III to room temperature. According to the mass ratio of carboxypeptidase B to reaction solution III of 1:10, add carboxypeptidase B to reaction solution III and react at 37°C for 24 h. After the reaction ends, boil for 10 min to terminate the reaction, and centrifuge at 4°C and 8000 rpm for 30 min to obtain filtrate IV.

[0069] S3. Filter filtrate IV first with a filter membrane with a pore size of 5 μm, then with a filter membrane with a pore size of 0.45 μm, then perform ultrafiltration with a PES membrane with a pore size of 10 nm, and then perform purification on an anion column (DEAE Beads6FF). Use NaCl as the eluent for purification, and separate impurities and products using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M respectively. Among them, the product is eluted in the 50 mM - 100 mM gradient. Collect and combine the eluted samples, then perform nanofiltration with a 200 Da nanofiltration membrane, ultrafiltration with a 1000 Da PES membrane to remove salt, concentrate under reduced pressure at 60°C, and freeze-dry to obtain N-glyco amino acid GPN-0001 (abbreviated as SGN, the structural formula is as Figure 19 shown) with a purity of 98% and a yield of 82%.

[0070] Example 3

[0071] A preparation method of N-glyco amino acid (GPN-0001) comprises the following steps:

[0072] S1. Take solid powder of SGP (GP-1001, the structural formula is as Figure 1 shown), add it into water to prepare a solution with a concentration of 30 g / L, a pH of 9, and a CaCl 2Solution I with a concentration of 20 mM. According to the mass ratio of alkaline protease to SGP of 1:1, alkaline protease (enzyme activity of 100,000 U / g) was added to Solution I. React at 37 °C for 16 h to obtain reaction intermediate II. Boil the reaction intermediate II (100 °C) for 10 min to terminate the reaction, and obtain reaction solution III.

[0073] S2. Cool the reaction solution III to room temperature. According to the mass ratio of carboxypeptidase B to reaction solution III of 1:5, add carboxypeptidase B to reaction solution III and react at 37 °C for 24 h. After the reaction, boil for 10 min to terminate the reaction, and centrifuge at 4 °C and 8000 rpm for 30 min to obtain filtrate IV (the conversion rate of this step is 90%).

[0074] S3. First filter the filtrate IV with a filter membrane with a pore size of 5 μm, then filter it with a filter membrane with a pore size of 0.45 μm, then perform ultrafiltration with a PES membrane with a pore size of 10 nm, and then perform purification on an anion column (DEAE Beads6FF). Purification uses NaCl as the eluent, and the impurities and products are separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M respectively. Among them, the product is eluted in the 50 mM - 100 mM gradient. Collect and combine the eluted samples, then perform nanofiltration with a 200 Da nanofiltration membrane in sequence, perform ultrafiltration to remove salt with a 1000 Da PES membrane, concentrate under reduced pressure at 60 °C, and lyophilize to obtain N-glycoamino acid GPN-0001 (abbreviated as SGN, the structural formula is as Figure 39 ) with a purity of 99% (the HPLC chromatogram is shown in Figure 19 ), and the yield is 83%.

[0075] Example 4

[0076] A preparation method of N-glycoamino acid (GPN-002) includes the following steps:

[0077] S1. Take the solid powder of N-glycopeptide GP-1002 (the structural formula is as Figure 2 ), add it to water, and prepare a solution I with a concentration of 10 g / L, a pH of 8, and a Ca 2+ concentration of 18 mM. According to the mass ratio of alkaline protease to N-glycopeptide GP-1002 of 1:5, add alkaline protease (enzyme activity of 100,000 U / g) to solution I. React at 37 °C for 8 h to obtain reaction intermediate II. Boil the reaction intermediate II (100 °C) for 10 min to terminate the reaction, and obtain reaction solution III.

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

[0079] S3. First filter the filtrate IV through a filter membrane with a pore size of 5 μm, then filter it through a filter membrane with a pore size of 0.45 μm, then perform ultrafiltration with a PES membrane with a pore size of 10 nm, and then perform purification on an anion column (DEAE Beads6FF). NaCl is used as the eluent for purification, and the impurities and products are separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M respectively. Among them, the product is eluted in the 50 mM - 100 mM gradient. Collect and combine the eluted samples, then perform nanofiltration with a 200 Da nanofiltration membrane in sequence, perform ultrafiltration and desalting with a 1000 Da PES membrane, concentrate, and lyophilize to obtain N-glycoamino acid GPN-0002 with a purity of 97% (the structural formula is as Figure 20 shown), and the yield is 81%.

[0080] Example 5

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

[0082] S1. Take the solid powder of N-glycopeptide GP-1003 (the structural formula is as Figure 3 shown), add it to water to prepare a solution I with a concentration of 15 g / L, a pH of 9, and a CaCl 2 concentration of 10 mM. Add alkaline protease (enzyme activity: 100,000 U / g) to the solution I at a mass ratio of alkaline protease to N-glycopeptide GP-1003 of 1:3. React at 37 °C for 10 h to obtain reaction intermediate II. Boil the reaction intermediate II at 100 °C for 10 min to terminate the reaction, and obtain reaction solution III.

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

[0084] S3. First, filter the filtrate IV through a filter membrane with a pore size of 5 μm, then through a filter membrane with a pore size of 0.45 μm, and then perform ultrafiltration using a PES membrane with a pore size of 10 nm. Then, perform purification using an anion column (DEAE Beads 6FF). NaCl is used as the eluent for purification, and gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M are used to separate impurities and products. Among them, the product is eluted in the 50 mM - 100 mM gradient. Collect and combine the eluted samples, then perform nanofiltration using a 200 Da nanofiltration membrane, ultrafiltration for desalting using a 1000 Da PES membrane, concentration under reduced pressure at 60 °C, and freeze-dry to obtain N-glycoamino acid GPN-0003 with a purity of 97% (the structural formula is as shown in Figure 21 ), and the yield is 82%.

[0085] Examples 6 - 20

[0086] Methods for preparing corresponding N-glycoamino acids (GPN-0004 - GPN-0018) from N-glycopeptides GP-1004 - GP-1018 respectively include the following steps:

[0087] S1. Respectively take solid powders of N-glycopeptides GP-1004 - GP-1018 (the structural formulas are respectively as shown in Figures 4 to 18 ), add them to water, and prepare solution I with a concentration of 25 g / L, a pH of 7, and a CaCl 2 concentration of 12 mM. According to the mass ratio of alkaline protease to N-glycopeptide of 1:7, add alkaline protease (enzyme activity is 100,000 U / g) to solution I. React at 37 °C for 14 h to obtain reaction intermediate II. Boil the reaction intermediate II at 100 °C for 10 min to terminate the reaction and obtain reaction solution III.

[0088] S2. Cool reaction solution III to room temperature. According to the mass ratio of carboxypeptidase B to reaction solution III of 1:25, add carboxypeptidase B to reaction solution III and react at 37 °C for 30 h. After the reaction ends, boil for 10 min to terminate the reaction, and centrifuge at 4 °C and 8000 rpm for 30 min to obtain filtrate IV.

[0089] S3. First, filter the filtrate IV through a filter membrane with a pore size of 5 μm, then through a filter membrane with a pore size of 0.45 μm, and then perform ultrafiltration using a PES membrane with a pore size of 10 nm. Then, perform purification using an anion column (DEAE Beads6FF). NaCl is used as the eluent for purification, and gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M are used to separate impurities and products. Among them, the product is eluted in the 50 mM - 100 mM gradient. Collect and combine the eluted samples, then perform nanofiltration using a 200 Da nanofiltration membrane, ultrafiltration using a 1000 Da PES membrane to remove salts, concentrate, and lyophilize to obtain N-glycoamino acids (the structural formulas are respectively as Figures 22 to 36 shown).

[0090] It should be noted that as long as the N-glycopeptides satisfying the structural formula as Figure 37 shown can adopt the scheme in this application to prepare the corresponding N-glycoamino acids (as Figure 38 shown), and the yield can reach more than 80% and the purity can reach more than 96%. Only a part of the methods for preparing N-glycoamino acids from N-glycopeptides are listed in the above examples, and all the methods for preparing N-glycoamino acids from N-glycopeptides will not be elaborated one by one 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 an 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 is 500 U / g). The experimental results show that there is still an alanine (A) remaining at the N-terminus of SGP that cannot be removed.

[0096] Comparative Example 2

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

[0098] P1. Take the solid powder of SGP (GP-1001, the structural formula is as Figure 1 shown), add it to water, and prepare a solution with a concentration of 30 g / L, a pH of 9, and CaCl 2Solution I with a concentration of 20 mM. Alkaline protease (enzyme activity: 100,000 U / g) and carboxypeptidase B were added to Solution I at a mass ratio of alkaline protease to SGP of 1:1 and a mass ratio of carboxypeptidase B to SGP of 1:30. The reaction was carried out at 37 °C for 72 h, and then terminated by boiling (100 °C) for 10 min.

[0099] P2. The reaction solution obtained in step P1 was centrifuged at 4 °C and 8000 rpm for 30 min to obtain a filtrate. The filtrate was first filtered through a filter membrane with a pore size of 5 μm, then through a filter membrane with a pore size of 0.45 μm, and then ultrafiltered through a PES membrane with a pore size of 10 nm, and then purified by an anion column (DEAE Beads 6FF). Purification was carried out using NaCl as the eluent, and impurities and products were separated using gradient concentrations of 30 mM, 50 mM, 100 mM, and 1 M. Among them, the product was eluted in the 50 mM - 100 mM gradient. The eluted samples were collected, combined, and then nanofiltrated through a 200 Da nanofiltration membrane, ultrafiltered through a 1000 Da PES membrane to remove salts, concentrated, and freeze-dried to obtain N-glycoamino acid GPN-0001 (abbreviated as SGN, with the structural formula as Figure 19 shown) with a purity of 96% and a yield of 10%.

[0100] Comparative Example 3

[0101] This comparative example adopted 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) from SGP.

[0102] Product purity detection

[0103] The purity of the N-glycoamino acids prepared in Example 3 and Comparative Example 3 was detected, and the cost, conversion rate, and time for preparing the N-glycoamino acids were statistically analyzed. The results are shown in Table 4 below. The purity detection results of SGN prepared in Example 3 are shown in Figure 39 , the nuclear magnetic resonance spectrum is shown in Figure 40 , the mass spectrum is shown in Figure 41 . The purity detection results of A2-Asn prepared in Comparative Example 3 are shown in Figure 42 .

[0104] Table 4 Comparison results of the methods for preparing N-glycoamino acids in this application and the prior art

[0105]

[0106] From Figure 40 and Figure 41 It can be seen that by using the preparation method in the present application, N-glycoamino acid Sialylglycoasparaginate was successfully prepared (the structural formula is as shown in Figure 19 ). It can be seen from Table 4 that the method for preparing N-glycoamino acid in the present application has lower cost, shorter required time, higher conversion rate and purity compared with the method for preparing N-glycoamino acid in the prior art, and is more suitable for large-scale production.

[0107] The above-described embodiments are only preferred embodiments of the present application and are not used to limit the protection scope of the present application. For any person skilled in the art, various changes and modifications can be made to the present application. Any simple equivalent changes and modifications made based on the protection scope of the present application and the content of the specification shall be included in the protection scope of the present application.

Claims

1. A method for preparing N-sugar amino acids, characterized in that: The following steps are involved: S1, preparing the N-glycopeptide into a solution I, adding alkaline protease to the solution I for a first enzymatic reaction to obtain a reaction intermediate II; boiling to terminate the reaction to obtain a reaction solution III; S2, cooling the reaction solution III to room temperature, adding carboxypeptidase B to the reaction solution III for a second enzymatic reaction, and after the reaction is completed; boiling to terminate the reaction, centrifuging, and obtaining a filtrate IV; S3, filtering the filtrate IV layer by layer, then ultrafiltration, and then purifying it using an anion column; The purification used gradient concentration of NaCl solution as eluent; The eluted samples are collected, combined, and sequentially subjected to nanofiltration, ultrafiltration, concentration, and freeze-drying to obtain N-glycoamino acids.

2. The method for preparing N-sugar amino acids according to claim 1, characterized in that: The structural formula of the N-glycopeptide is shown in formula GP-1001 to GP-1018:

3. The method for preparing N-sugar amino acid according to claim 1, characterized in that: The concentration of N-glycopeptide in the solution I is 5 to 30 g / L; or / and the pH of the solution I is 7 to 9; or / and the solution I contains 5 to 20 mmol / L of Ca 2+ .

4. The method for preparing N-sugar amino acids according to claim 1, characterized in that: The mass ratio of the alkaline protease to the N-glycopeptide is 1:1 to 1:

10.

5. The method for preparing N-sugar amino acid according to claim 1, characterized in that: The enzymatic activity of the alkaline protease is ≥ 100,000 U / g; or / and the activity of the carboxypeptidase B is > 200 U / mg.

6. The method for preparing N-sugar amino acids according to claim 1, characterized in that: The temperature of the first enzymatic reaction in step S1 is 20-50° C., and the reaction time is 4-16 hours.

7. The method for preparing N-sugar amino acids according to claim 1, characterized in that: The mass ratio of the carboxypeptidase B to the reaction solution III is 1:30-1:

5.

8. The method for preparing N-sugar amino acids according to claim 1, characterized in that: The temperature of the second enzymatic reaction in step S2 is 20-50° C., and the reaction time is 12-36 hours.

9. The method for preparing N-sugar amino acid according to claim 1, characterized in that: The concentration range of the NaCl solution in step S3 is 30mmol / L to 1mol / L.

10. The N-glycoamino acid prepared according to the preparation method according to any one of claims 1 to 9.

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