Method for chemical enzymatic synthesis of o-glycopeptides and uses thereof

Abstract

This invention discloses a method and application for the chemical enzymatic synthesis of O-glycopeptides, belonging to the field of pharmaceutical technology. The method includes the following steps: assembling a polypeptide intermediate using a solid-phase polypeptide synthesis method, wherein the amino acids at non-glycosylated active sites in the polypeptide intermediate are protected by sterically hindered protecting groups, while the hydroxyl groups on the amino acids at predetermined glycosylation sites are unprotected; glycosylation of the polypeptide intermediate with a glyconucleotide donor in the presence of a glycosyltransferase to obtain a glycopeptide intermediate; and reacting the glycopeptide intermediate with a deprotecting agent to remove the sterically hindered protecting groups, thus obtaining the target glycopeptide. This invention uses amino acids modified with protecting groups as raw materials, selectively protects the active hydroxyl sites in the amino acids, prepares polypeptide intermediates through solid-phase polypeptide synthesis, efficiently and accurately synthesizes the target glycopeptide intermediate with the help of glycosyltransferase synergistic catalysis, and finally obtains the target glycopeptide by removing the protecting groups through a catalyst.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method and application of chemical enzymatic synthesis of O-glycopeptides. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Protein O-glycosylation, as a key post-translational modification, produces O-glycopeptides that play a central role in numerous physiological processes, including cell recognition, immune regulation, and signal transduction. Furthermore, O-glycosylation is closely related to the development and progression of various major diseases, such as tumors, autoimmune diseases, and congenital glycosylation defects. Therefore, obtaining structurally uniform O-glycopeptides with well-defined sites is a crucial prerequisite for elucidating the disease regulatory mechanisms of O-glycosylation and developing glycopeptide vaccines, diagnostic probes, and targeted drugs.

[0004] However, naturally derived O-glycopeptides exhibit inherent microscopic heterogeneity, with the same protein site often occupied by a mixture of various glycoforms, failing to meet the requirements for precise functional research and translational applications. Therefore, artificial synthesis has become the main approach to obtaining homogeneous O-glycopeptides. Currently, the O-glycopeptide synthesis methods developed in this field are mainly divided into three categories: chemical synthesis, enzymatic synthesis, and chemical-enzymatic combined strategies. However, all of them have significant technical bottlenecks, making it difficult to achieve efficient, precise, and large-scale preparation.

[0005] Chemical synthesis methods often involve solid-phase peptide synthesis using pre-prepared glycosylated amino acids. While this ensures a well-defined product structure, the steric hindrance of glycosylated amino acids leads to a sharp decrease in coupling efficiency during peptide chain elongation as the sequence length increases. Furthermore, glycans are prone to side reactions such as hydrolysis and isomerization under strong acid deprotection and cleavage conditions, making them particularly incompatible with the synthesis of complex glycans and long peptide chains, thus limiting their applicability.

[0006] Enzymatic synthesis utilizes the catalytic action of glycosyltransferases to elongate sugar chains, which has the advantages of mild reaction conditions and high regio and stereoselectivity. However, this method has obvious substrate spectrum limitations and it is difficult to introduce non-natural sugar chains. At the same time, the enzyme catalytic efficiency is generally low, and the product yield is usually less than 30%, which is difficult to meet the needs of large-scale preparation.

[0007] The combined chemical-enzyme approach aims to integrate the flexibility of chemical synthesis with the high selectivity of enzymatic methods, and is currently the mainstream direction in O-glycopeptide synthesis research. However, existing chemical-enzyme systems suffer from poor compatibility between chemical synthesis and enzymatic catalysis steps. For example, protecting groups used in chemical synthesis are difficult to remove efficiently without affecting the glycan structure; some strategies require the introduction of specific "tags" or reversible linkers to assist synthesis, and subsequent tag removal steps are cumbersome, inefficient, and prone to generating byproducts. Furthermore, existing systems generally suffer from poor selectivity at glycosylation sites, making it difficult to precisely control the specific sites of glycan linkage, resulting in product heterogeneity and high preparation costs, failing to meet the preclinical research requirements for milligram- to gram-level homogeneous O-glycopeptides. In summary, current technologies cannot efficiently, accurately, and on a large scale synthesize complex O-GalNAc glycopeptides with uniform structure and controllable glycan length and sequence. This problem severely restricts the progress of glycosylation disease biomarker validation, high-specificity antibody screening, glycopeptide vaccine development, and antibody-drug conjugate (ADC) construction.

[0008] In summary, how to overcome the technical bottlenecks of existing methods and develop a universal, efficient, and site-selective O-glycopeptide synthesis strategy to achieve the large-scale preparation of structurally uniform O-glycopeptides has become a key technical problem that urgently needs to be solved in the fields of glycochemistry and glycobiology. Summary of the Invention

[0009] In view of this, the present invention provides a method and application for the chemical enzymatic synthesis of O-glycopeptides.

[0010] In a first aspect, the present invention provides a method for chemically enzymatically synthesizing O-glycopeptides, comprising the following steps: A polypeptide intermediate is assembled using a solid-phase polypeptide synthesis method, wherein the amino acids at non-glycosylated active sites in the polypeptide intermediate are protected by sterically hindered protecting groups, and the hydroxyl groups on the amino acids at preset glycosylation sites are in an unprotected state. In the presence of glycosyltransferase, the polypeptide intermediate is subjected to a glyconucleotide donor for glycosylation to obtain a glycopeptide intermediate. The glycopeptide intermediate is reacted with a deprotecting agent to remove the sterically hindered protecting group, thus obtaining the final product.

[0011] Preferably, the steric hindrance protecting group is selected from one or more of allyl, allyloxycarbonyl, p-allyloxybenzyl, methyl, acetyl, benzoyl, benzyl, and neopentanoyl.

[0012] Preferably, the amino acid is selected from one or more of serine, threonine, hydroxylysine, 4-hydroxyproline, tyrosine, and non-natural amino acids with hydroxyl groups.

[0013] Preferably, the glycosyltransferase is one or more of N-acetylgalactosyltransferase, β1,3-galactosyltransferase, β1,4-galactosyltransferase, core 1 β3-galactosyltransferase 1, α2,3-sialyltransferase, N-acetylgalactosamine α-2,6-sialyltransferase, O-fucosyltransferase, O-mannosyltransferase, and O-glucosyltransferase; The glyconucleotide donor is one or more of UDP-GalNAc, UDP-GlcNAc, UDP-Gal, GDP-Fuc, UDP-Glc, UDP-Xyl, CMP-Neu5Ac, CMP-Neu5Gc, GDP-Man, or their derivatives.

[0014] Preferably, the glycopeptide intermediate undergoes a secondary glycosylation reaction.

[0015] Preferably, the deprotecting agent is selected from one or more of palladium catalysts, basic reagents, Lewis acids, and oxidizing agents.

[0016] The palladium catalyst is selected from at least one of zero-valent palladium complexes, divalent palladium salts, and supported palladium catalysts. The zero-valent palladium complex is Pd(PPh3)4, Pd2(dba)3, or nano-palladium; The divalent palladium salt is Pd(OAc)2, PdCl2, PdI2 or Pd(NO3)2; The supported palladium catalyst is a Pd / C or an ionic liquid-supported palladium catalyst, wherein the ionic liquid-supported palladium catalyst is a Pd / IL / ACC. The alkaline reagent is a sodium methoxide-methanol solution; The Lewis acid is BBr3, AlCl3, or AlBr3; The oxidizing agent is DDQ.

[0017] Preferably, there may be one or more glycosylation sites.

[0018] Preferably, the enzyme-catalyzed reaction conditions are: temperature 4–60℃, pH 4.0–10.0.

[0019] Preferably, the reaction conditions for the removal of protecting groups are: temperature 0-80℃ and pressure 0.1-10 bar.

[0020] Secondly, the present invention provides glycopeptides synthesized by the above-described method.

[0021] Thirdly, the present invention provides the use of the glycopeptides synthesized by the above method in the preparation of medicaments for the prevention or treatment of tumors, autoimmune diseases, infectious diseases, congenital glycosylation defects, IgA nephropathy, inflammatory bowel disease, cystic fibrosis, chronic respiratory diseases or neurodegenerative diseases.

[0022] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention uses amino acids modified with protecting groups as raw materials, selectively orthogonally protects the potential active hydroxyl sites of amino acids, prepares peptide intermediates through solid-phase synthesis of peptides, and then efficiently and accurately synthesizes the target glycopeptide intermediates with the help of glycosyltransferase synergistic catalysis. Finally, the protecting groups are removed by a catalyst to obtain the target glycopeptide, thus realizing the efficient, accurate and large-scale synthesis of complex O-glycopeptides with uniform structure and controllable glycan length and sequence.

[0023] (2) This invention, by leaving the hydroxyl groups at the preset glycosylation sites unprotected during the solid-phase polypeptide synthesis stage and simultaneously protecting the hydroxyl groups at the non-glycosylated active sites with sterically hindered protecting groups, ensures that the subsequent enzymatic glycosylation reaction can be precisely carried out at the preset sites, effectively avoiding the heterogeneity of glycosylation sites and ensuring the site selectivity of the product. Compared with the defects of existing chemical-enzymatic methods, which cannot precisely control the glycan linkage sites and are prone to mixed glycoforms, this invention first protects the sites differently during the solid-phase synthesis stage, combined with the specific catalysis of glycosyltransferases, so that the glycan chains extend only at the preset sites. Verification shows that no isomers are detected in the product, and the structural uniformity can reach more than 95%, which fully meets the stringent requirements for standards in glycobiological functional research and vaccine development.

[0024] (3) The present invention uses a deprotecting agent to remove the protecting group, which is highly compatible with the previous solid-phase synthesis and enzymatic glycosylation steps. On the one hand, the catalytic deprotection reaction conditions are mild, avoiding side reactions such as sugar chain hydrolysis and peptide chain breakage caused by strong acid deprotection in traditional chemical synthesis, and can completely preserve the stereoconfiguration of the sugar chain and the integrity of the peptide chain; on the other hand, there is no need to introduce additional tags or reversible linkers, and the protecting group is removed in one step. Compared with the problems of cumbersome tag removal and easy generation of by-products in existing combined strategies, the process steps are greatly simplified, the synthesis cycle is shortened, and the product conversion rate is improved.

[0025] (4) The operation method of the present invention simplifies the operation steps, has high synthesis efficiency, low cost and is suitable for large-scale production. Attached Figure Description

[0026] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0027] Figure 1 This is a schematic diagram of the chemical enzymatic synthesis method for O-glycopeptides according to the present invention; Figure 2 This is the HPLC spectrum of compound II in Comparative Example 1 of this invention; Figure 3 The HRMS spectrum of compound II in Comparative Example 1 of this invention; Figure 4 This is the HPLC spectrum of compound III in Example 1 of the present invention; Figure 5 The HRMS spectrum of compound III in Example 1 of this invention; Figure 6 This is the HPLC spectrum of compound IV in Comparative Example 1 of this invention; Figure 7 The MALDI spectrum of compound IV in Comparative Example 1 of this invention is shown. Figure 8 This is the HPLC spectrum of compound V in Example 1 of the present invention; Figure 9 The MALDI spectrum of compound V in Example 1 of this invention; Figure 10 This is the HPLC spectrum of compound VI in Example 1 of the present invention; Figure 11 The MALDI spectrum of compound VI in Example 1 of this invention; Figure 12 This is the HPLC spectrum of compound VII in Example 2 of the present invention; Figure 13 The HRMS spectrum of compound VII in Example 2 of this invention; Figure 14 This is the HPLC spectrum of compound VIII in Example 2 of the present invention; Figure 15 This is the MALDI spectrum of compound VIII in Example 2 of the present invention. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] like Figure 1 As shown, the present invention provides a method for chemically enzymatically synthesizing O-glycopeptides, comprising the following steps: A polypeptide intermediate is assembled using a solid-phase polypeptide synthesis method, wherein the amino acids at non-glycosylated active sites in the polypeptide intermediate are protected by sterically hindered protecting groups, and the hydroxyl groups on the amino acids at preset glycosylation sites are in an unprotected state. In the presence of glycosyltransferase, the polypeptide intermediate is subjected to a glyconucleotide donor for glycosylation to obtain a glycopeptide intermediate. The glycopeptide intermediate is reacted with a deprotecting agent to remove the sterically hindered protecting group, thus obtaining the final product.

[0030] In an optional embodiment of the present invention, the solid-phase polypeptide synthesis method includes the following steps: (1) Resin swelling Place Rink Amide MBHA resin into a reaction tube, add DCM / DMF, shake, filter to remove the swollen solution, and wash with DMF; (2) Deprotection Add morpholine / DMF solution, react, filter, wash with DMF to complete the removal of Fmoc from the resin itself; (3) Amino acid coupling Weigh out the first amino acid (Fmoc-AA-OH) and HATU, dissolve them in DMF, add DIPEA, mix well, then add to the resin, shake to react, filter, wash with DMF, and dry. (4) Closed Add a mixed solution of acetic anhydride / pyridine / DMF, seal the mixture, filter, wash with DMF, and dry. (5) Peptide chain elongation The sequence synthesis was completed in cycles (1)–(4), wherein the predetermined non-glycosylated active sites were coupled with amino acids with protecting groups, i.e., Fmoc-Ser(R)-OH / Fmoc-Thr(R)-OH, the remaining glycosylated active sites were coupled with Fmoc-Ser(tBu)-OH / Fmoc-Thr(tBu)-OH, and the inactive sites were coupled with Fmoc-AA-OH; (6) Pyrolysis and purification DMF was replaced with DCM in the resin, TFA / H2O was added, the lysis reaction was carried out, and the lysis buffer was collected. The reaction was repeated once, the lysis buffers were combined and concentrated by nitrogen blowing. Cold diethyl ether was added, centrifuged, and the supernatant was discarded. The ether precipitated, and the precipitate was dried to obtain crude peptide. The peptide was then purified by preparative HPLC and freeze-dried to obtain a polypeptide intermediate.

[0031] In an optional embodiment of the present invention, the polypeptide intermediate has the structural formula shown in Formula I: (I); In this design, R represents a sterically hindered protecting group, and AA represents a natural or non-natural amino acid. The polypeptide intermediate retains free hydroxyl groups only on the amino acid side chains at the pre-defined O-glycosylation site; all other hydroxyl groups are blocked by orthogonal protecting groups of the sterically hindered protecting group to achieve site-specific glycosylation in subsequent applications.

[0032] In an optional embodiment of the present invention, the steric hindrance protecting group is selected from one or more of allyl, allyloxycarbonyl, p-allyloxybenzyl, methyl, acetyl, benzoyl, benzyl, and neopentanoyl.

[0033] In an optional embodiment of the present invention, the amino acid is selected from one or more of serine, threonine, hydroxylysine, 4-hydroxyproline, tyrosine, and non-natural amino acids with hydroxyl groups.

[0034] In an optional embodiment of the present invention, the glycosylation reaction includes the following steps: Add Tris, Tritium X-100, and Mn sequentially to the EP tube. 2+ The polypeptide intermediate and glyconucleotide donor were added, and deionized water was added to make up the volume. Then, glycosyltransferase was added, the reaction was shaken, the reaction was stopped by boiling, centrifugation was performed, and the supernatant was collected to obtain the glycopeptide intermediate.

[0035] In an optional embodiment of the present invention, the removal process of the small steric hindrance protection base includes the following steps: Weigh out the glycopeptide intermediate and N,N-dimethylbarbituric acid, dissolve them in double-distilled water, add DMF solution containing deprotecting agent, and shake the reaction under nitrogen protection. After the reaction is completed, filter the reaction solution through an organic filter membrane, inject it directly into a preparative HPLC for separation and purification, and freeze-dry to obtain a homogeneous O-glycopeptide.

[0036] In an optional embodiment of the present invention, the glycosyltransferase is one or more of N-acetylgalactosyltransferase, β1,3-galactosyltransferase, β1,4-galactosyltransferase, core 1 β3-galactosyltransferase 1, α2,3-sialyltransferase, N-acetylgalactosamine α-2,6-sialyltransferase, O-fucosyltransferase, O-mannosyltransferase, and O-glucosyltransferase.

[0037] The glyconucleotide donor is one or more of UDP-GalNAc, UDP-GlcNAc, UDP-Gal, GDP-Fuc, UDP-Glc, UDP-Xyl, CMP-Neu5Ac, CMP-Neu5Gc, GDP-Man, or their derivatives.

[0038] In an optional embodiment of the present invention, the deprotecting agent is selected from one or more of palladium catalysts, basic reagents, Lewis acids, and oxidizing agents.

[0039] The palladium catalyst is selected from at least one of zero-valent palladium complexes, divalent palladium salts, and supported palladium catalysts; The zero-valent palladium complex is Pd(PPh3)4, Pd2(dba)3, or nano-palladium; wherein, when the zero-valent palladium catalyst is Pd(PPh3)4, a nucleophilic scavenger is optionally added, which is morpholine, formic acid, N,N-dimethylbarbituric acid, or tributyltin hydride. The divalent palladium salt is Pd(OAc)2, PdCl2, PdI2 or Pd(NO3)2; The supported palladium catalyst is a Pd / C or an ionic liquid-supported palladium catalyst, wherein the ionic liquid-supported palladium catalyst is a Pd / IL / ACC. The alkaline reagent is a sodium methoxide-methanol solution; The Lewis acid is BBr3, AlCl3, or AlBr3; The oxidizing agent is DDQ.

[0040] In an optional embodiment of the present invention, there may be one or more glycosylation sites.

[0041] In an optional embodiment of the present invention, the enzyme-catalyzed reaction conditions are: temperature 4-60℃, pH 4.0-10.0.

[0042] In an optional embodiment of the present invention, the reaction conditions for the protecting group removal treatment are: temperature 0-80℃, pressure 0.1-10 bar. When using a palladium catalyst, the reaction must be carried out under an inert gas atmosphere; when using an oxidizing agent, the reaction must be carried out under anhydrous conditions.

[0043] Furthermore, when the sterically hindered protecting group is allyl, allyloxycarbonyl, or benzyl, it is removed using a palladium catalyst under inert gas protection; when the sterically hindered protecting group is p-allyloxybenzyl, it is removed using an oxidizing agent; when the sterically hindered protecting group is methyl, it is removed using a Lewis acid; and when the sterically hindered protecting group is acetyl, benzoyl, or neopentanoyl, it is removed using a basic reagent.

[0044] This invention provides glycopeptides synthesized by the above-described method.

[0045] This invention provides the use of the glycopeptides synthesized by the above method in the preparation of medicaments for the prevention or treatment of tumors, autoimmune diseases, infectious diseases, congenital glycosylation defects, IgA nephropathy, inflammatory bowel disease, cystic fibrosis, chronic respiratory diseases, or neurodegenerative diseases.

[0046] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0047] Example 1 Structure of O-glycopeptide O-glycopeptide comprises a tandem unit peptide having the amino acid sequence given in SEQ ID NO:1 and an O-linked glycan chain; The O-linked glycan is N-acetylgalactosamine (GalNAc) and is linked to either serine or threonine in the tandem unit peptide.

[0048] SEQ ID NO: 1: PVPSTPPTPSPSTPPTPSPS.

[0049] Preparation method of site-specific O-glycopeptides 1. Solid-phase peptide synthesis 1.1 Synthetic routes of polypeptide intermediates

[0050] 1.2 Resin swelling Weigh 100 mg (0.8 mmol / g) of Rink Amide MBHA resin and place it in a 10 mL polypropylene peptide synthesis tube. Add 2 mL of DCM / DMF (volume ratio 1:1) and shake at 25 °C and 110 rpm for 30 min. Filter to remove the swelling solution and wash with DMF.

[0051] 1.3 Deprotection Add 2 mL of 50% (v / v) morpholine / DMF, react at 25℃ and 110 rpm for 20 min, filter, wash with DMF to complete the removal of Fmoc from the resin itself.

[0052] 1.4 Amino acid coupling Weigh 4.0 equivalents of Fmoc-AA-OH and 3.9 equivalents of HATU and dissolve them in 2 mL of DMF. Add 6.0 equivalents of DIPEA and pre-activate at 25°C for 5 min. Add the pre-activated solution to the resin and react at 25°C and 110 rpm for 1 h. Filter, wash with DMF, and dry.

[0053] 1.5 Closed Add 2 mL of 20% acetic anhydride / 20% pyridine / DMF (v / v / v), seal at 25℃ and 110 rpm for 2 h; filter, wash with DMF, and dry.

[0054] 1.6 Peptide chain elongation Sequence synthesis was completed in cycles 1.2–1.4. The predetermined non-glycosylated active sites were coupled with allyl-protected amino acids, i.e., Fmoc-Ser(All)-OH. The polypeptide protecting group only protected positions 4, 18, and 20. The remaining glycosylated active sites were coupled with Fmoc-Ser(tBu)-OH / Fmoc-Thr(tBu)-OH, and the non-active sites were coupled with Fmoc-AA-OH.

[0055] 1.7 Pyrolysis and Purification DMF was replaced with DCM in the resin; 2 mL of 95% TFA / 5% H2O (v / v) was added, and the mixture was lysed at 25 °C and 110 rpm for 1 h, and the lysate was collected; this process was repeated once, the lysates were combined and concentrated by nitrogen blowing; 40 mL of cold diethyl ether was added, and the mixture was centrifuged at 4 °C and 12000 rpm for 30 min, and the supernatant was discarded; the ether precipitate was precipitated three times, and the precipitate was dried to obtain the crude peptide; the peptide was purified by preparative HPLC, and freeze-dried to obtain a polypeptide intermediate with allyl groups protecting the non-reactive site, and the molecular weight was confirmed by HRMS.

[0056] 2. Enzymatic glycosylation 2.1 Synthetic route of glycopeptide intermediates

[0057] The specific steps are as follows: Add the following to a 1.5 mL EP tube sequentially: 500 mM Tris, 2% Trition X-100, and 300 mM Mn. 2+ 2 mM peptide, 13.8 mM UDP-GalNAc, and deionized water were added to make up the volume; ppGalNAc-T2 and ppGalNAc-T4 were added, and the reaction was carried out at 37℃ and 110 rpm for 12 h; the reaction was terminated by boiling at 100℃ for 5 min, centrifuged at 12000 rpm for 5 min, and the supernatant was collected; HPLC detection showed that the substrate / product peak area ratio was ≈1:1, and the conversion rate was ≥95%; the target peak was collected, and the mass change was confirmed by MALDI-TOF to determine the amount of GalNAc added.

[0058] 3. One-time palladium-catalyzed removal of allyl groups Target glycopeptide synthesis route

[0059] The specific steps are as follows: Weigh 2 mg of the above glycopeptide and 1.21 mg of N,N-dimethylbarbituric acid and dissolve them in 10 µL of double-distilled water. Add 30 µL of DMF solution containing 6 mg of Pd(PPh3)4 and react at 40 °C and 220 rpm for 1 h under nitrogen protection. Filter the reaction solution through a 0.22 µm organic filter membrane and directly inject it into a preparative HPLC system for separation and purification. After freeze-drying, a homogeneous O-glycopeptide is obtained. MALDI-TOF shows complete removal of the allyl group, no byproducts, and a purity ≥95%.

[0060] Example 2 The operation steps in this embodiment are basically the same as in embodiment 1, except that the polypeptide protecting group only protects positions 4, 5, 13, 16, 18, and 20.

[0061] Glycopeptide synthesis route diagram:

[0062] Comparative Example 1 Preparation method of non-selective enzymatic O-glycopeptide synthesis 1. Solid-phase peptide synthesis 1.1 Synthetic routes of polypeptide intermediates

[0063] 1.2 Resin swelling Weigh 100 mg (0.8 mmol / g) of Rink Amide MBHA resin and place it in a 10 mL polypropylene peptide synthesis tube. Add 2 mL of DCM / DMF (volume ratio 1:1) and shake at 25 °C and 110 rpm for 30 min. Filter to remove the swelling solution and wash with DMF.

[0064] 1.3 Deprotection Add 2 mL of 50% (v / v) morpholine / DMF, react at 25℃ and 110 rpm for 20 min, filter, wash with DMF to complete the removal of Fmoc from the resin itself.

[0065] 1.4 Amino acid coupling Weigh 4.0 equivalents of Fmoc-AA-OH and 3.9 equivalents of HATU and dissolve them in 2 mL of DMF. Add 6.0 equivalents of DIPEA and pre-activate at 25°C for 5 min. Add the pre-activated solution to the resin and react at 25°C and 110 rpm for 1 h. Filter, wash with DMF, and dry.

[0066] 1.5 Closed Add 2 mL of 20% acetic anhydride / 20% pyridine / DMF (v / v / v), seal at 25℃ and 110 rpm for 2 h; filter, wash with DMF, and dry.

[0067] 1.6 Peptide chain elongation The sequence synthesis was completed in cycles 1.2–1.4, with active sites coupled using Fmoc-Ser(tBu)-OH / Fmoc-Thr(tBu)-OH and inactive sites coupled using Fmoc-AA-OH.

[0068] 1.7 Pyrolysis and Purification DMF was replaced with DCM in the resin; 2 mL of 95% TFA / 5% H2O (v / v) was added, and the mixture was lysed at 25 °C and 110 rpm for 1 h, and the lysate was collected; this process was repeated once, the lysates were combined and concentrated by nitrogen blowing; 40 mL of cold diethyl ether was added, and the mixture was centrifuged at 4 °C and 12000 rpm for 30 min, and the supernatant was discarded; the ether precipitate was precipitated three times, and the precipitate was dried to obtain the crude peptide; the peptide was purified by preparative HPLC, and freeze-dried to obtain a polypeptide intermediate with allyl groups protecting the non-reactive site, and the molecular weight was confirmed by HRMS.

[0069] 2. Enzymatic glycosylation 2.1 Synthetic route of glycopeptide intermediates

[0070] The specific steps are as follows: Add the following to a 1.5 mL EP tube sequentially: 500 mM Tris, 2% Trition X-100, and 300 mM Mn. 2+ 2 mM peptide, 13.8 mM UDP-GalNAc, and deionized water were added to make up the volume; ppGalNAc-T2 and ppGalNAc-T4 were added, and the reaction was carried out at 37℃ and 110 rpm for 12 h; the reaction was terminated by boiling at 100℃ for 5 min, centrifuged at 12000 rpm for 5 min, and the supernatant was collected; the substrate conversion rate was ≥95% by HPLC, but two product peaks were present; the target peak was collected, and the mass change was confirmed by MALDI-TOF to determine the amount of GalNAc added.

[0071] Effect verification The glycopeptides prepared in Examples 1-2 were confirmed by MALDI-TOF and MS / MS to have glycans present only at the preset active sites and have a single glycan form. This product can be directly used for glycan chip spotting, antibody immunogen preparation or ADC linker construction, meeting the requirements of vaccines, diagnostics and targeted delivery systems for structurally uniform glycopeptides.

[0072] The glycopeptide of Comparative Example 1 was confirmed by MALDI-TOF and MS / MS to be a mixed glycopeptide containing 8 and 9 GalNAc modifications, demonstrating the randomness of glycosylation and the heterogeneity of the product without protecting groups, thus highlighting the necessity of the protecting group strategy of the present invention.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing O-glycopeptides using a chemical enzymatic method, characterized in that, Includes the following steps: A polypeptide intermediate is assembled using a solid-phase polypeptide synthesis method, wherein the amino acids at non-glycosylated active sites in the polypeptide intermediate are protected by sterically hindered protecting groups, and the hydroxyl groups on the amino acids at preset glycosylation sites are in an unprotected state. In the presence of glycosyltransferase, the polypeptide intermediate is subjected to a glyconucleotide donor for glycosylation to obtain a glycopeptide intermediate. The glycopeptide intermediate is reacted with a deprotecting agent to remove the sterically hindered protecting group, thus obtaining the final product.

2. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, The small steric hindrance protecting group is selected from one or more of allyl, allyloxycarbonyl, p-allyloxybenzyl, methyl, acetyl, benzoyl, benzyl, and neopentanoyl.

3. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, The amino acid is selected from one or more of serine, threonine, hydroxylysine, 4-hydroxyproline, tyrosine, and non-natural amino acids with hydroxyl groups.

4. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, The glycosyltransferase is one or more of N-acetylgalactosyltransferase, β1,3-galactosyltransferase, β1,4-galactosyltransferase, core 1 β3-galactosyltransferase 1, α2,3-sialyltransferase, N-acetylgalactosamine α-2,6-sialyltransferase, O-fucosyltransferase, O-mannosyltransferase, and O-glucosyltransferase. The glyconucleotide donor is one or more of UDP-GalNAc, UDP-GlcNAc, UDP-Gal, GDP-Fuc, UDP-Glc, UDP-Xyl, CMP-Neu5Ac, CMP-Neu5Gc, GDP-Man, or their derivatives.

5. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, The deprotecting agent is selected from one or more of palladium catalysts, basic reagents, Lewis acids, and oxidizing agents. The palladium catalyst is selected from at least one of zero-valent palladium complexes, divalent palladium salts, and supported palladium catalysts. The zero-valent palladium complex is Pd(PPh3)4, Pd2(dba)3, or nano-palladium; The divalent palladium salt is Pd(OAc)2, PdCl2, PdI2 or Pd(NO3)2; The supported palladium catalyst is a Pd / C or an ionic liquid-supported palladium catalyst, wherein the ionic liquid-supported palladium catalyst is a Pd / IL / ACC. The alkaline reagent is a sodium methoxide-methanol solution; The Lewis acid is BBr3 or AlCl.

3. AlBr3; The oxidizing agent is DDQ.

6. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, There can be one or more glycosylation sites.

7. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, The enzyme-catalyzed reaction conditions are: temperature 4-60℃, pH 4.0-10.

0.

8. The method for synthesizing O-glycopeptides using a chemical enzymatic method as described in claim 1, characterized in that, The reaction conditions for protecting group removal treatment are: temperature 0-80℃, pressure 0.1-10 bar.

9. Claim 1 8. Glycopeptides synthesized by any one of the methods described in the above method.

10. The use of the glycopeptide as described in claim 9 in the preparation of medicaments for the prevention or treatment of tumors, autoimmune diseases, infectious diseases, congenital glycosylation defects, IgA nephropathy, inflammatory bowel disease, cystic fibrosis, chronic respiratory diseases, or neurodegenerative diseases.

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