Uridine diphosphate-n-difluoroacetylglucosamine, preparation method therefor and use thereof

Abstract

A uridine diphosphate-N-difluoroacetylglucosamine, a preparation method therefor and a use thereof. The uridine diphosphate-N-difluoroacetylglucosamine has a similar chemical structure to the natural substrate UDP-GlcNAc, the difference being that the 2-position N-acetyl group thereof is replaced by an N-difluoroacetyl group. The preparation method is as follows: (1) preparing difluoroacetylglucosamine; (2) using difluoroacetylglucosamine as a substrate to prepare uridine diphosphate-N-difluoroacetylglucosamine. A novel, highly active, non-hydrolyzable and artificially synthesizable artificial donor sugar uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) is provided. Compared with the existing donor sugar UDP-GlcNTFA, UDP-GlcNDFA has higher catalytic activity for glycosyltransferases, is stable and not easily hydrolyzed, and can be used as a donor substrate for chemoenzymatic synthesis of heparin oligosaccharide scaffolds, heparin oligosaccharide intermediates and heparin oligosaccharides, as well as hyaluronic acid oligosaccharides, glycoconjugates (glycosylated polypeptides) and polysaccharide antigens.

Description

Uridine diphosphate-N-difluoroacetylglucosamine and its preparation method and application Technical Field

[0001] The present invention relates to uridine diphosphate-N-difluoroacetylglucosamine and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a natural UDP glycosyl donor, has been widely used in various fields, including the synthesis of glycosaminoglycans, glycan antigens, and glycopeptides. Current research on UDP glycosyl donors is not limited to natural UDP-GlcNAc; non-natural GlcNAc analogs have also been extensively studied and applied in practice. For example, in the field of glycosaminoglycans, it has been found that replacing natural GlcNAc with non-natural GlcNAc analogs can produce structurally controllable and uniform target products in the synthesis of polysaccharides and oligosaccharides. Furthermore, non-natural UDP-GlcNAc analogs can serve as effective tools for studying the biosynthesis of glycoconjugates, such as glycosylated peptides. Furthermore, glycan antigens can be labeled with non-natural glycosyl donors, enabling highly selective labeling of cell surface antigens. Therefore, the synthesis and application of novel UDP-glycosyl donors will significantly advance the synthesis of glycoconjugates and the development of new drugs.

[0003] However, it should be noted that although the application results of non-natural UDP sugar donors in various fields have been proven to be reliable and practical, in fact, unlike natural UDP sugar donors, there are inevitably some difficulties in the synthesis process. Among them, the synthesis problem of introducing UDP-GlcNTFA instead of UDP-GlcNAc into the heparin backbone is the most prominent.

[0004] Currently, in the chemoenzymatic synthesis of heparin backbones, GlcA-pNP is used as the starting acceptor substrate, and UDP-GlcNTFA and UDP-GlcA are used as donor substrates to synthesize heparin oligosaccharides. Due to the lack of deacetylating activity of N-deacetyl / N-sulfotransferase (NDST), a non-natural donor sugar of UDP-GlcNTFA is used instead of the natural donor sugar UDP-GlcNAc to introduce the heparin backbone. Subsequently, a weak base is used to remove the trifluoroacetyl group, exposing the amino group before N-sulfation reaction and subsequent modification. However, with the extension of the sugar chain and the increase of reaction time, heparin oligosaccharides containing GlcNTFA are easily hydrolyzed, while oligosaccharides with exposed amino groups at the non-reducing end cannot be further extended by glycosyltransferases, so a more complex purification process is required to prepare and purify oligosaccharides. In addition, the synthesis, use, and storage of the synthetic raw material UDP-GlcNTFA also require special attention. Because the GlcNTFA group is much larger than the naturally occurring GlcNAc, its glycosyltransferase activity is only about 20% of that of GlcNAc. GlcNTFA's low reactivity and easy hydrolysis have led to an urgent need for a new, stable, and reliable group to replace GlcNTFA in the heparin backbone synthesis. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a uridine diphosphate-N-difluoroacetylglucosamine and a preparation method and application thereof.

[0006] Glossary: ​​GlcA-pNP: p-nitrophenyl-β-D-glucuronic acid; UDP-GlcNTFA: uridine diphosphate-N-trifluoroacetylglucosamine; UDP-GlcNDFA: uridine diphosphate-N-difluoroacetylglucosamine; UDP-GlcA: uridine diphosphate-glucuronic acid; PAPS: 3'-phosphoadenosine-5'-phosphosulfate; ES2-VSRA: antiangiogenic peptide; Gal-Glc-pNP: 4-nitrophenyl-β-D-lactoside; PmHS1: heparin backbone synthase 1; PmHS2: heparin backbone synthase 2; NST: N-sulfotransferase; NaKfiA: N-acetylglucosaminyltransferase; PmHAS: hyaluronan synthase; ncOGT: O-GlcNAc glycosyltransferase; NmLgtA: N-acetylglucosaminyltransferase; NahK: N-acetylglucosaminyl 1-kinase; GlmU: UDP-N-acetylglucosamine pyrophosphorylase; PmPPA: inorganic pyrophosphatase.

[0007] The technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides a method for preparing uridine diphosphate-N-difluoroacetylglucosamine, comprising the following steps:

[0009] (1) Dissolve D-glucosamine hydrochloride and anhydrous sodium carbonate in anhydrous methanol, add difluoroethyl acetate, stir and react at 20-30°C for 10-15 hours, evaporate to dryness and purify to obtain difluoroacetylglucosamine (GlcNDFA);

[0010] (2) Disodium adenosine triphosphate, trisodium uridine triphosphate, Mg 2+ and the difluoroacetylglucosamine obtained in step (1) are sequentially added to a Tris-HCl buffer, and the pH is adjusted to 7.0-7.5 to obtain a reaction system; then, N-acetylhexylamine 1-kinase (NahK), UDP-N-acetylglucosamine pyrophosphorylase (GlmU) and inorganic pyrophosphatase (PmPPA) are added to the reaction system, and the reaction is allowed to stand overnight at 35-40° C. for 10-15 hours. After protein removal and purification, uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) is obtained.

[0011] Preferably, according to the present invention, in step (1), the mass ratio of the D-glucosamine hydrochloride to anhydrous sodium carbonate is (1-1.1):1.

[0012] Preferably, according to the present invention, in step (1), the mass volume ratio of anhydrous sodium carbonate to anhydrous methanol is 1:3, unit: g / mL.

[0013] Preferably, according to the present invention, in step (1), the volume ratio of anhydrous methanol to ethyl difluoroacetate is 1:(1-1.2).

[0014] According to the preferred embodiment of the present invention, in step (2), in the reaction system, the final concentration of adenosine triphosphate disodium is 15-25 mM, the final concentration of uridine triphosphate trisodium is 15-25 mM, and the Mg 2+ The final concentration of is 5-15 mM, the final concentration of difluoroacetylglucosamine is 15-25 mM, and the final concentration of Tris-HCl is 45-55 mM.

[0015] According to the present invention, preferably, in step (2), the final concentration of the N-acetylhexamine 1-kinase (NahK) in the reaction system is 0.1 to 0.3 mg / ml; the final concentration of the UDP-N-acetylglucosamine pyrophosphorylase (GlmU) in the reaction system is 0.1 to 0.3 mg / ml; and the final concentration of the inorganic pyrophosphatase (PmPPA) in the reaction system is 0.05 to 0.15 mg / ml.

[0016] According to the preferred embodiment of the present invention, in step (2), the protein removal and purification method is as follows:

[0017] After allowing the reaction to stand overnight, trifluoroacetic acid was added to the reaction system, and the pH was adjusted to 3. Protein was removed by centrifugal filtration. The pH was then adjusted to 6, and the filtrate was concentrated by rotary evaporation and dialyzed for desalination. The dialyzed filtrate was then loaded onto a Q column at a flow rate of 35-45 mL / min and passed through the column in sequence using ultrapure water, 0.1 M NaCl, and 2 M NaCl.

[0018] In a second aspect, the present invention provides uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) prepared by the above method, the structural formula of which is shown below:

[0019] Wherein, R is -CF2H.

[0020] This UDP-GlcNDFA has a chemical structure similar to the natural substrate UDP-GlcNAc, except that the N-acetyl group at position 2 is replaced by an N-difluoroacetyl group. Due to the similarity in their chemical structures, it can be recognized by UDP-GlcNAc glycosyltransferase and can be used as a donor substrate for the chemoenzymatic synthesis of heparin oligosaccharide backbones, heparin oligosaccharide intermediates, and heparin oligosaccharides.

[0021] The third aspect of the present invention provides the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of heparin oligosaccharide backbone.

[0022] The UDP-GlcNDFA can be efficiently transferred to the heparin backbone by glycosyltransferase, thereby synthesizing heparin backbone oligosaccharides having a -GlcNDFA-GlcA- structure. At the same time, since the GlcNDFA structure has very weak hydrolysis activity, a higher yield is achieved in the synthesis process of long oligosaccharides.

[0023] The fourth aspect of the present invention provides the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of heparin oligosaccharide intermediates.

[0024] According to a preferred embodiment of the present invention, the application is: using pNP-GlcA as an acceptor substrate, UDP-GlcA and UDP-GlcNDFA as donor substrates, and using heparin backbone synthase 2 (PmHS2) to repeatedly extend them respectively to synthesize heparin oligosaccharide intermediates of different lengths having a -GlcNDFA-GlcA- structure;

[0025] The structural formula of the heparin oligosaccharide intermediate is shown below:

[0026] Wherein, R1 is -CF2H, n=1-7, and R2 can be a substituted aromatic ring such as p-nitrobenzene, benzene, or substituted phenyl.

[0027] In a fifth aspect, the present invention provides the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of heparin oligosaccharides.

[0028] According to a preferred embodiment of the present invention, the application is: synthesizing a heparin oligosaccharide intermediate according to the above method, removing the difluoroacetyl group from the synthesized heparin oligosaccharide intermediate under the action of lithium hydroxide, thereby generating N-sulfated heparin oligosaccharides under the action of N-sulfotransferase, and then preparing active heparin oligosaccharides after modification.

[0029] In a sixth aspect, the present invention provides the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of hyaluronic acid skeleton.

[0030] The UDP-GlcNDFA can be efficiently transferred to the hyaluronic acid backbone by glycosyltransferase, thereby synthesizing hyaluronic acid backbone oligosaccharides having a -GlcNDFA-GlcA- structure, thereby enriching the diversity of hyaluronic acid oligosaccharides.

[0031] In a seventh aspect, the present invention provides the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of glycosylated polypeptides.

[0032] The UDP-GlcNDFA can be efficiently transferred to polypeptides via glycosyltransferases to synthesize glycosylated polypeptides, while providing an effective tool for the study of the synthesis of sugar complexes.

[0033] In an eighth aspect, the present invention provides the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in antigen synthesis.

[0034] The UDP-GlcNDFA can be efficiently transferred to the glycan antigen via glycosyltransferase, thereby introducing a label and being able to highly selectively label cell surface antigens. Beneficial effects

[0035] 1. The present invention provides a new, highly active, non-hydrolyzable, and artificially synthesizable artificial donor sugar uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA). Compared with the existing donor sugar UDP-GlcNTFA, the UDP-GlcNDFA of the present invention has higher catalytic activity for glycosyltransferases and is stable and non-hydrolyzable. It can be used as a donor substrate for the chemoenzymatic synthesis of heparin oligosaccharide backbones, heparin oligosaccharide intermediates, heparin oligosaccharides, as well as hyaluronic acid oligosaccharides, glycoconjugates (glycosylated polypeptides), glycan antigens, etc.

[0036] 2. The present invention provides a method for preparing uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA). The method has few steps, is simple to operate, is safe, uses conventional raw materials, is green and environmentally friendly, and can be produced on a large scale industrially.

[0037] 3. The heparin oligosaccharide backbone and heparin oligosaccharide intermediate synthesized using uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) of the present invention contain a -GlcNDFA-GlcA- structure, which has extremely high stability in aqueous solution and can effectively avoid the formation of by-products produced by hydrolysis, effectively compensating for the problem of limited initial synthesis efficiency of the heparin backbone.

[0038] 4. The present invention provides a method for synthesizing heparin oligosaccharides using uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA). This method not only enriches the diversity of heparin oligosaccharides but also provides a new approach for heparin backbone synthesis. This method can effectively improve the synthesis efficiency, yield, and output of heparin oligosaccharides. The purity of each heparin oligosaccharide exceeds 95%, and the yield reaches over 85%. This method is expected to promote the development of a process for the rapid, safe, and large-scale preparation of heparin oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG1 is the ESI-MS result of the chemoenzymatic synthesis of UDP-GlcNDFA of the present invention;

[0040] In the figure: the horizontal axis is the mass-to-nuclear ratio m / z, and the vertical axis is the signal intensity.

[0041] FIG2 is a reaction activity determination diagram of UDP-GlcNDFA and UDP-GlcNTFA of the present invention;

[0042] FIG3 is a comparative result of the stability of GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP of the present invention under a reaction environment of pH=7.5.

[0043] FIG4 shows the HPLC chromatographic results of the hydrolysis of GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP under alkaline conditions of the present invention.

[0044] FIG5 is the HPLC chromatographic results of each heparin oligosaccharide synthesized by the present invention;

[0045] In the figure: (1) is heparin disaccharide; (2) is heparin trisaccharide; (3) is heparin tetrasaccharide; (4) is heparin pentasaccharide; (5) is heparin hexasaccharide; (6) is heparin heptasaccharide; (7) is heparin octasaccharide; and (8) is heparin nonasaccharide.

[0046] FIG6 is the HPLC chromatographic results of sulfated modified heparin pentasaccharide, heptaasaccharide, and nonasaccharide synthesized by the present invention;

[0047] In the figure: (1) is sulfated heparin pentasaccharide; (2) is sulfated heparin heptasaccharide; (3) is sulfated heparin nonasaccharide.

[0048] FIG7 is the HPLC chromatography result of the heparin oligosaccharide synthesized by the present invention;

[0049] In the figure: (1) is the catalytic synthesis involving heparin skeleton synthase 1 (PmHS1); (2) is the catalytic synthesis involving heparin skeleton synthase 2 (PmHS2); (3) is the catalytic synthesis involving N-acetylglucosaminyltransferase (NaKfiA);

[0050] FIG8 is the HPLC chromatography results of hyaluronic acid oligosaccharides synthesized in the present invention;

[0051] FIG9 is the HPLC chromatogram results of the glycosylated polypeptide synthesized by the present invention;

[0052] FIG10 is the HPLC chromatography result of the glycan antigen synthesized by the present invention. DETAILED DESCRIPTION

[0053] The technical solutions of the present invention are further described below in conjunction with the embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0054] The GlcA-pNP, UDP-GlcA and UDP-GlcNTFA used in the present invention are available from Sigma.

[0055] The 4-nitrophenyl-β-D-lactoside used in the present invention is available from Macklin.

[0056] The plasmids of the heparin backbone synthase PmHS2 mutants PmHS2(D215N / D217N) and PmHS2(D479N / D481N) and the polypeptide ES2-VSRA used in the examples were commissioned to Nanjing GenScript Biotech Co., Ltd. for artificial synthesis based on the specific sequences.

[0057] Among them, the PmHS2 (D215N / D217N) is a mutation of aspartic acid at positions 215 and 217 in the PmHS2 amino acid sequence (GenBank: AAQ55110.1) to asparagine, that is, the GAT at positions 643 to 645 bp in the PmHS2 nucleotide sequence (GenBank: AY292200.1) is mutated to AAT, and the GAT at positions 649 to 651 bp is mutated to AAT.

[0058] The PmHS2 (D479N / D481N) is a mutation in which the aspartic acid at positions 479 and 481 in the PmHS2 amino acid sequence (GenBank: AAQ55110.1) is mutated to asparagine, that is, the GAT at 1435-1437 bp in the PmHS2 nucleotide sequence (GenBank: AY292200.1) is mutated to AAT, and the GAC at 1441-1443 bp is mutated to AAC.

[0059] That is, PmHS1, PmHS2 and mutants thereof, NST, NaKfiA, PmHAS, ncOGT, NmLgtA, NahK, GlmU and PmPPA used in the present invention are all existing enzymes whose amino acid sequences have been disclosed and can be obtained commercially or artificially synthesized.

[0060] The Escherichia coli BL21 (DE3) competent cells used were purchased from Nanjing Novozymes Biotech Co., Ltd.

[0061] Other raw materials or reagents not described in the present invention can be synthesized according to existing methods or obtained commercially.

[0062] HPLC detection was performed using a YMC amino column, the liquid phase system was produced by Shimadzu Corporation of Japan, and the UV detection system was SPD-20A.

[0063] Example 1: Chemoenzymatic synthesis of the donor substrate uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA)

[0064] 1. A method for preparing uridine diphosphate-N-difluoroacetylglucosamine, comprising the following steps:

[0065] (1) Synthesis of difluoroacetylglucosamine (GlcNDFA):

[0066] 10.2 g of D-glucosamine hydrochloride and 10 g of anhydrous sodium carbonate were dissolved in 30 ml of anhydrous methanol, 35 ml of ethyl difluoroacetate was added, and the mixture was stirred at 25°C for 12 h. The resulting product was then evaporated to dryness to ensure complete anhydrousness during the reaction. The product after rotary evaporation was quickly purified on a silica gel column to obtain the monosaccharide GlcNDFA. The specific reaction formula is as follows:

[0067] (2) Synthesis of uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA):

[0068] According to the system shown in Table 1, adenosine triphosphate disodium (ATP-2Na), uridine triphosphate trisodium (UTP-3Na), Mg 2+and the difluoroacetylglucosamine obtained in step (1) are sequentially added to a Tris-HCl buffer solution, and the pH is adjusted to 7.0-7.5 to obtain a reaction system with a total volume of 1 L (note that stirring is performed while adding, and the pH change is strictly monitored, and the solution cannot be placed in an alkaline condition); then, N-acetylhexylamine 1-kinase (NahK) with a final concentration of 0.2 mg / ml, UDP-N-acetylglucosamine pyrophosphorylase (GlmU) with a final concentration of 0.2 mg / ml, and inorganic pyrophosphatase (PmPPA) with a final concentration of 0.1 mg / ml are added to the reaction system, and the reaction is allowed to stand overnight at 37° C. for 15 h. After protein removal and purification, uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) is obtained. The specific reaction formula is as follows:

[0069] Table 1. Amount of each component in the reaction system

[0070] In step (2), the protein removal and purification methods are as follows:

[0071] After standing overnight to react, trifluoroacetic acid was added to the reaction system at a mass percentage of 0.1%, the pH was adjusted to 3, and the protein was removed by centrifugation; the pH was then adjusted to 6, the filtrate was concentrated by rotary evaporation, and then dialyzed to desalt to a sodium chloride concentration of 0.1 M; then a 2 L column volume Q column was pre-washed with 3 L volume of 0.1 M NaOH, and then rinsed with 4 L ultrapure water (containing 10 mM NaH2PO4) until the pH of the effluent was weakly acidic, and the column was balanced. The dialyzed filtrate was loaded onto the Q column at a flow rate of 40 mL / min, and ultrapure water, 0.1 M NaCl, and 2 M NaCl were used to pass through the column in sequence. The specific purification procedure is shown in Table 2.

[0072] Table 2. Q column purification procedure for UDP-GlcNDFA

[0073] 2. The reaction solution after overnight reaction and before protein removal and purification was subjected to high performance liquid chromatography. The specific liquid chromatography analysis method is shown in Table 3, and the retention time of the main substances is shown in Table 4.

[0074] Liquid phase conditions: A: ultrapure water; B: 1 mol / L KH2PO4; flow rate 0.5 ml / minL; chromatographic column YMC-Pack Polyamine II (250×4.6 mml.D).

[0075] Table 3. Liquid phase detection and analysis methods

[0076] Table 4. Retention time of main substances:

[0077] 3. The UDP-GlcNDFA prepared in this example was subjected to electrospray ionization mass spectrometry analysis. The results are shown in FIG1 .

[0078] As shown in Table 4 and Figure 1, the method provided by the present invention successfully prepared UDP-GlcNDFA.

[0079] Example 2: Feasibility Verification of UDP-GlcNDFA in Heparin Skeleton Synthesis

[0080] 1. Reactivity

[0081] In the case of heparin trisaccharide as acceptor substrate, UDP-GlcNDFA and UDP-GlcNTFA were the Mn-containing 2+ In a Tris-HCl (pH = 7.5) buffer system, PmHS1 and PmHS2 were used to catalyze the reaction respectively. The reaction conditions were as follows: after reacting at 30°C for 1 hour, the reaction was terminated by boiling in a boiling water bath for five minutes. The reaction status was detected by liquid chromatography. Three parallel reactions were set up, and the results are shown in Figure 2.

[0082] As shown in Figure 2, the reaction activity of UDP-GlcNDFA is twice that of UDP-GlcNTFA under the same reaction conditions. This indicates that compared with the existing donor sugar UDP-GlcNTFA, the UDP-GlcNDFA of the present invention has higher catalytic activity for glycosyltransferases and can be used as a donor substrate for the chemoenzymatic synthesis of heparin oligosaccharide backbones, heparin oligosaccharide intermediates, and heparin oligosaccharides.

[0083] 2. Stability

[0084] 0.630 g of p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP) was weighed, and two equivalents of UDP-GlcNDFA and UDP-GlcNTFA were respectively placed in Tris-HCl buffer (50 mmol / L, pH = 7.5, containing 10 mmol / L MnCl2). The volume was diluted to 1 L, and the solution was adjusted to pH 7.0. PmHS2 enzyme mutants (D215N / D217N) were added to a final concentration of 0.1 mg / ml. The reaction was allowed to react at 30°C for 24 hours. The reaction progress was monitored by liquid chromatography and the reaction was terminated when the disaccharide conversion rate exceeded 90%. The resulting reaction solution was adjusted to pH = 3 with trifluoroacetic acid and purified by C18 column chromatography to obtain the heparin disaccharide backbone GlcNDFA-GlcA-pNP / GlcNTFA-GlcA-pNP. Tris-HCl (pH=7.5) was then used to simulate the synthesis reaction environment, and the stability of GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP was monitored in the liquid phase for 15 consecutive days. The results are shown in FIG3 .

[0085] As shown in Figure 3, after 15 days of storage in neutral Tris-HCl (pH = 7.5), GlcNDFA-GlcA-pNP still retained more than 90% of its original substance, while GlcNTFA-GlcA-pNP only retained about 50% of its original substance, with the rest hydrolyzed to GlcNH2-GlcA-pNP. This indicates that GlcNDFA-GlcA-pNP synthesized with UDP-GlcNDFA as a donor substrate is significantly more stable than GlcNTFA-GlcA-pNP synthesized with UDP-GlcNTFA as a donor substrate under near-neutral reaction conditions.

[0086] 3. Hydrolysis

[0087] Lithium hydroxide (final concentration 1 M) was added to the heparin disaccharide backbone obtained in point 2 of this example, the pH was adjusted to 12.0, and the mixture was placed on ice for 30 min. The hydrolysis of DFA / TFA was monitored by liquid phase chromatography, as shown in FIG4 .

[0088] As shown in Figure 4, after incubation on ice for 0.5 h, both GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP were completely hydrolyzed into GlcNH2-GlcA-pNP, which indicates that oligosaccharides with GlcNDFA structure, like oligosaccharides with GlcNTFA structure, can easily expose amino groups to synthesize N-sulfated oligosaccharides.

[0089] Example 3. Chemoenzymatic Synthesis and Purification of Heparin Oligosaccharide Backbone

[0090] 1. Synthesis of heparin oligosaccharide

[0091] a. 0.5 g of the heparin disaccharide backbone obtained in Example 2, 1.5 equivalents of UDP-GlcA, and the PmHS2 enzyme mutant (D479N / D481N) were placed in a Tris-HCl buffer (50 mmol / L, pH=7.5, and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L. The concentration of the PmHS2 enzyme mutant (D479N / D481N) in the reaction system was 0.1 mg / ml. The reaction was then carried out at 30° C. overnight. The reaction progress was monitored by liquid chromatography to determine that the conversion of the trisaccharide was greater than 95%. The reaction was terminated by adjusting the reaction solution to pH=3, and the heparin trisaccharide backbone GlcA-GlcNDFA-GlcA-pNP was purified by C18 column chromatography.

[0092] b. 0.4 g of the heparin trisaccharide backbone obtained in step a, 1.5 equivalents of UDP-GlcNDFA, and a PmHS2 enzyme mutant (D215N / D217N) were placed in a Tris-HCl buffer (50 mmol / L, pH=7.5, and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L. The concentration of the PmHS2 enzyme mutant (D215N / D217N) in the reaction system was 0.1 mg / ml. The reaction was then carried out at 30° C. overnight. The reaction progress was monitored by liquid chromatography to determine that the conversion of the tetrasaccharide was greater than 95%. The reaction solution was adjusted to pH=3 to terminate the reaction, and the mixture was purified by C18 column chromatography to obtain the heparin tetrasaccharide backbone GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0093] c. 0.3 g of the heparin tetrasaccharide backbone obtained in step b, 1.5 equivalents of UDP-GlcA and a PmHS2 enzyme mutant (D479N / D481N) were placed in a Tris-HCl buffer (50 mmol / L, pH=7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L, wherein the concentration of the PmHS2 enzyme mutant (D479N / D481N) in the reaction system was 0.1 mg / ml; the reaction was then carried out at 30° C. overnight, and the reaction progress was monitored by liquid chromatography to determine that the conversion of the pentasaccharide was greater than 95%. The reaction solution was adjusted to pH=3 to terminate the reaction, and the reaction was purified by C18 column chromatography to obtain the heparin pentasaccharide backbone GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP;

[0094] d. Take 0.2 g of the heparin pentasaccharide skeleton obtained in step c, 1.5 equivalents of UDP-GlcNDFA and the PmHS2 enzyme mutant (D215N / D217N), and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L. The concentration of the PmHS2 enzyme mutant (D215N / D217N) in the reaction system is 0.1 mg / ml; then react at 30°C overnight. The reaction progress is monitored by liquid chromatography and the conversion rate of the hexasaccharide is greater than 95%. The reaction solution is adjusted to pH = 3 to terminate the reaction, and the reaction is purified by C18 column chromatography to obtain the heparin hexasaccharide skeleton GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0095] e. Take 0.1 g of the heparin hexasaccharide backbone obtained in step d, 1.5 equivalents of UDP-GlcA and the PmHS2 enzyme mutant (D479N / D481N) and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L, and the concentration of the PmHS2 enzyme mutant (D479N / D481N) in the reaction system is 0.1 mg / ml; then react at 30°C overnight. The reaction progress is monitored by liquid chromatography. The conversion rate of the heptasaccharide is higher than 95%. The reaction solution is adjusted to pH = 3 to terminate the reaction, and the reaction is purified by C18 column chromatography to obtain the heparin heptasaccharide backbone GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDF A-GlcA-pNP.

[0096] f. 0.1 g of the heparin heptasaccharide backbone obtained in step e, 1.5 equivalents of UDP-GlcNDFA, and a PmHS2 enzyme mutant (D215N / D217N) were placed in a Tris-HCl buffer (50 mmol / L, pH = 7.5, and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L. The concentration of the PmHS2 enzyme mutant (D215N / D217N) in the reaction system was 0.1 mg / ml. The reaction was then carried out at 30° C. overnight. The reaction progress was monitored by liquid chromatography, and the conversion of the octasaccharide was greater than 95%. The reaction solution was adjusted to pH = 3 to terminate the reaction, and the mixture was purified by C18 column chromatography to obtain the heparin octasaccharide backbone GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0097] g. Take 0.1 g of the heparin octasaccharide backbone obtained in step f, 1.5 equivalents of UDP-GlcA and the PmHS2 enzyme mutant (D479N / D481N), and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system with a volume of 1 L. The concentration of the PmHS2 enzyme mutant (D479N / D481N) in the reaction system is 0.1 mg / ml; then react at 30°C overnight. The reaction progress is monitored by liquid chromatography. The conversion rate of the nonasaccharide is higher than 95%. The reaction solution is adjusted to pH = 3 to terminate the reaction, and the reaction is purified by C18 column chromatography to obtain the heparin nonasaccharide backbone GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0098] 2. Purification of heparin oligosaccharide

[0099] The above-mentioned heparin oligosaccharide backbones were Purification was performed using a C18-AQ column and AKTA. Prior to purification, the terminated reaction solution was centrifuged at room temperature (8000 rpm, 10 min). The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a C18 column equilibrated with double-distilled water (0.1% TFA). The specific purification procedure is shown in Table 5. The flow rate was 8 ml / min. The results are shown in Figure 5.

[0100] Table 5. Heparin oligosaccharide C18 purification procedure (gradient)

[0101] As shown in Figure 5, the synthesis conversion rate of heparin oligosaccharides was above 95%, the purification yield was above 85%, and the purity was above 95%. This shows that the present invention successfully synthesized heparin oligosaccharide backbone, heparin oligosaccharide intermediates, and heparin oligosaccharides using UDP-GlcNDFA as a substrate through a chemical enzymatic method.

[0102] Example 4: Sulfation modification and purification of heparin oligosaccharide backbone

[0103] 1. Alkaline hydrolysis to remove difluoroacetyl (DFA)

[0104] Lithium hydroxide (final concentration of 1 M) was added to the heparin pentasaccharide skeleton, heparin heptasaccharide skeleton, and heparin nonasaccharide skeleton obtained in Example 3, respectively, and the pH was adjusted to 12.0. The mixture was placed on ice for 30 min, and the hydrolysis of DFA was monitored by liquid phase. After the hydrolysis of DFA was completed, the pH was adjusted to 7.0 using hydrochloric acid.

[0105] 2. Sulfation modification

[0106] 0.1 g of each of the DFA-depleted heparin pentasaccharide backbone, heparin heptaasaccharide backbone, and heparin nonasaccharide backbone, along with 4 equivalents of PAPS and NST, were placed in 50 mM MES buffer to obtain a 200 mL reaction system with a final NST concentration of 0.5 mg / ml. The reaction was then incubated at 37°C overnight. When the reaction progress was monitored by liquid chromatography and the conversion rate exceeded 95%, the reaction solution was adjusted to pH 3 to terminate the reaction, and purification was performed by chromatography on a Q column.

[0107] The specific method for the chromatography purification of the Q column is as follows:

[0108] Prepare solution A: ultrapure water (containing 10 mM NaH2PO4); solution B: 2 M NaCl.

[0109] Before purification, the reaction solution of the terminated reaction was centrifuged at room temperature (8000 rpm, 10 min), and the supernatant was filtered using a 0.22 μm filter membrane and loaded onto a Q column chromatography column that had been equilibrated with ultrapure water (containing 10 mM NaH2PO4) in advance. The specific purification procedure is shown in Table 5, and the flow rate was 10 ml / min.

[0110] Table 6. Q column purification procedure

[0111] The purified sample was rotary evaporated, dialyzed against 200 molecular weight for 2 hours, and concentrated again to a volume of less than 2 ml. The sample was then loaded onto a 2.6 cm x 60 cm P-2 column. Chromatography was performed using 0.1 M ammonium bicarbonate at a flow rate of 0.3 ml / min. Finally, the collected oligosaccharides were analyzed for purity by liquid chromatography, as shown in Figure 6.

[0112] As shown in FIG6 , the conversion rate of the sulfated heparin oligosaccharide synthesis was above 90%, the purification yield was above 85%, and the purity was above 85%.

[0113] Example 5, Synthesis and Application of UDP-GlcNDFA

[0114] 1. Synthesis and application of glycosaminoglycans

[0115] (1) Synthesis of heparin oligosaccharides

[0116] 0.1 g of the heparin trisaccharide backbone in step a of Example 3, 1.5 equivalents of UDP-GlcNDFA and PmHS1 were placed in 200 μL of Tris-HCl buffer (50 mmol / L, pH=7.5 and containing 10 mmol / LMnCl2) to obtain a reaction system in which the concentration of the heparin trisaccharide backbone was 0.2 mM and the concentration of PmHS1 was 0.1 mg / ml. The reaction was then carried out at 30°C for 1 h, and then 0.1% TFA was added to terminate the reaction. The reaction was determined by liquid phase chromatography, and the results are shown in Figure 7 (1).

[0117] 0.1 g of the heparin trisaccharide backbone in step a of Example 3, 1.5 equivalents of UDP-GlcNDFA and the PmHS2 enzyme mutant (D215N / D217N) were placed in 200 μL of Tris-HCl buffer (50 mmol / L, pH=7.5 and containing 10 mmol / LMnCl2) to obtain a reaction system in which the concentration of the heparin trisaccharide backbone was 0.2 mM and the concentration of the PmHS2 enzyme mutant (D215N / D217N) was 0.1 mg / ml; then, after reacting at 30°C for 1 h, 0.1% TFA was added to terminate the reaction, and the reaction was determined by liquid phase analysis. The results are shown in Figure 7 (2).

[0118] 0.1 g of the heparin trisaccharide backbone from step a of Example 3, 1.5 equivalents of UDP-GlcNDFA and NaKfiA were placed in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / LMnCl2) to obtain a reaction system in which the concentration of the heparin trisaccharide backbone was 0.2 mM and the concentration of NaKfiA was 0.1 mg / ml. The reaction was then allowed to proceed at 30°C for 1 h, and then 0.1% TFA was added to terminate the reaction. The reaction was determined by liquid chromatography, and the results are shown in FIG7 (3).

[0119] As shown in Figure 7, UDP-GlcNDFA can be used as a new type of UDP glycosyl donor for the synthesis of heparin oligosaccharides.

[0120] (2) Synthesis of hyaluronic acid oligosaccharides

[0121] 0.1 g of hyaluronic acid trisaccharide backbone, 1.5 equivalents of UDP-GlcNDFA and PmHAS were placed in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system in which the concentration of the hyaluronic acid trisaccharide backbone was 0.2 mM and the concentration of PmHAS was 0.1 mg / ml; then, after reacting at 30°C for 1 hour, 0.1% TFA was added to terminate the reaction, and the reaction was determined by liquid chromatography. The results are shown in Figure 8.

[0122] As shown in Figure 8, UDP-GlcNDFA can be used as a new type of UDP glycosyl donor for the synthesis of hyaluronic acid oligosaccharides.

[0123] 2. Synthesis and application of glycopeptides

[0124] 0.1 g of ES2-VSRA, 4 equivalents of UDP-GlcNDFA and ncOGT were placed in 200 μL of Tris-HCl buffer (25 mmol / L, pH = 7.5 and containing 12.5 mmol / LMgCl2, 0.06 mg / ml BSA, 1 mM DTT) to obtain a reaction system in which the concentration of ES2-VSRA was 0.2 mM and the concentration of ncOGT was 0.2 mg / ml. After reacting at 37°C for 4 hours, 0.1% TFA was added to terminate the reaction. The reaction was determined by liquid chromatography, and the results are shown in Figure 9.

[0125] As shown in Figure 9, UDP-GlcNDFA can be used as a new type of UDP glycosyl donor for the synthesis of glycosylated polypeptides.

[0126] 3. Synthesis and application of antigens

[0127] 0.1 g of 4-nitrophenyl-β-D-lactoside, 1.5 equivalents of UDP-GlcNDFA and NmLgt were placed in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / LMnCl2) to obtain a reaction system, in which the concentration of 4-nitrophenyl-β-D-lactoside in the reaction system was 0.2 mM and the concentration of ncOGT was 0.2 mg / ml; then, the reaction was shaken at 30°C and 200 r / min for 1 hour, and then 0.1% TFA was added to terminate the reaction. The reaction was determined by liquid chromatography, and the results are shown in Figure 10.

[0128] As shown in Figure 10, UDP-GlcNDFA can be used as a new type of UDP glycosyl donor for the synthesis of glycan antigens.

[0129] In summary, the artificial donor sugar uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) provided by the present invention has higher catalytic activity for glycosyltransferases, is stable and not easily hydrolyzed, and can be used as a donor substrate for the chemical enzymatic synthesis of heparin oligosaccharide backbones, heparin oligosaccharide intermediates, heparin oligosaccharides, as well as hyaluronic acid oligosaccharides, glycoconjugates (glycosylated polypeptides), glycan antigens, etc.

Claims

1. A method for preparing uridine diphosphate-N-difluoroacetylglucosamine, characterized in that, It includes the following steps: (1) Dissolve D-glucosamine hydrochloride and anhydrous sodium carbonate in anhydrous methanol, add ethyl difluoroacetate, stir and react at 20 - 30 °C for 10 - 15 h. After evaporation to dryness and purification, obtain glucosamine difluoroacetate (GlcNDFA); (2) Add adenosine disodium triphosphate, uridine triphosphate trisodium, Mg 2+ and the difluoroacetylglucosamine obtained in step (1) to Tris-HCl buffer in sequence, adjust the pH to 7.0 - 7.5 to obtain a reaction system; then add N-acetylhexosamine 1-kinase (NahK), UDP-N-acetylglucosamine pyrophosphorylase (GlmU) and inorganic pyrophosphatase (PmPPA) to the reaction system, and let it stand overnight at 35 - 40 °C for reaction for 10 - 15 h. After protein removal and purification, uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) is obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of the D-glucosamine hydrochloride to the anhydrous sodium carbonate is (1 - 1.1):1; the mass-volume ratio of the anhydrous sodium carbonate to the anhydrous methanol is 1:3, unit: g / mL; The volume ratio of the anhydrous methanol to the ethyl difluoroacetate is 1:(1 - 1.2).

3. The preparation method according to claim 1, characterized in that, In step (2), in the reaction system, the final concentration of disodium adenosine triphosphate is 15 - 25 mM, the final concentration of uridine 5'-triphosphate trisodium salt is 15 - 25 mM, the final concentration of Mg 2+ is 5 - 15 mM, the final concentration of difluoroacetylglucosamine is 15 - 25 mM, and the final concentration of Tris-HCl is 45 - 55 mM; The final concentration of the N-acetylhexosamine 1-kinase (NahK) in the reaction system is 0.1 - 0.3 mg / ml; the final concentration of the UDP-N-acetylglucosamine pyrophosphorylase (GlmU) in the reaction system is 0.1 - 0.3 mg / ml; the final concentration of the inorganic pyrophosphatase (PmPPA) in the reaction system is 0.05 - 0.15 mg / ml; The method for protein removal and purification is as follows: After standing overnight for the reaction, add trifluoroacetic acid to the reaction system, adjust the pH to 3, centrifuge and filter to remove proteins; then adjust the pH to 6, rotary evaporate and concentrate the filtrate, and dialyze to remove salts; then, at a flow rate of 35 - 45 mL / min, load the dialyzed filtrate onto a Q column, and pass through the column successively with ultrapure water, 0.1 M NaCl, and 2 M NaCl.

4. Uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) prepared by the method according to any one of claims 1 to 3, the structural formula of which is shown in the following formula: Among them, R is -CF2H.

5. Use of the uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) according to claim 4 in the synthesis of heparin oligosaccharide backbone.

6. Use of the uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) according to claim 4 in the synthesis of heparin oligosaccharide intermediate; Further preferably, the use is: using pNP-GlcA as the acceptor substrate, UDP-GlcA and UDP-GlcNDFA as the donor substrates, and using heparin backbone synthase 2 (PmHS2) to repeat the extension respectively to synthesize heparin oligosaccharide intermediates with different lengths and having the -GlcNDFA-GlcA- structure; The structural formula of the heparin oligosaccharide intermediate is shown as follows: Among them, R1 is -CF2H, n = 1 - 7, R2 is p-nitrophenyl, phenyl, substituted phenyl.

7. Use of the uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) according to claim 4 in the synthesis of heparin oligosaccharides; Further preferably, the use is: synthesize heparin oligosaccharide intermediates according to the above method, the synthesized heparin oligosaccharide intermediates are de-difluoroacetylated under the action of lithium hydroxide, so as to generate N-sulfated heparin oligosaccharides under the action of N-sulfotransferase, and then after modification, prepare active heparin oligosaccharides.

8. Use of the uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) according to claim 4 in the synthesis of hyaluronic acid backbone.

9. Use of the uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) according to claim 4 in the synthesis of glycosylated polypeptides.

10. Use of uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) as claimed in claim 4 in antigen synthesis.

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