Uridine diphosphate-N-difluoroacetyl glucosamine as well as preparation method and application thereof

Abstract

The invention relates to uridine diphosphate-N-difluoroacetyl glucosamine as well as a preparation method and application thereof. The uridine diphosphate-N-difluoroacetyl glucosamine has a chemical structure similar to that of a natural substrate UDP-GlcNAc, and the difference is that N-acetyl at the site 2 of the uridine diphosphate-N-difluoroacetyl glucosamine is substituted by N-difluoroacetyl. The preparation method comprises the following steps: (1) preparing difluoroacetyl glucosamine; and (2) preparing the uridine diphosphate-N-difluoroacetyl glucosamine by taking the difluoroacetyl glucosamine as a substrate. The invention provides a novel artificial donor glycouridine diphosphate-N-difluoroacetyl glucosamine (UDP-GlcNDFA) which is high in activity, not easy to hydrolyze and capable of being artificially synthesized, and compared with an existing donor sugar UDP-GlcNTFA, the UDP-GlcNDFA has higher catalytic activity on glycosyltransferase, is stable and not easy to hydrolyze, and can be used for preparing glycosyltransferase. The heparin oligosaccharide can be used as a donor substrate for synthesizing a heparin oligosaccharide skeleton, a heparin oligosaccharide intermediate, heparin oligosaccharide, hyaluronic acid oligosaccharide, a sugar complex (glycosylated polypeptide) and a glycan antigen by a chemical enzyme method.

Description

Technical Field

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

[0002] Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) has been widely used in various fields as a natural UDP glycosyl donor, including the synthesis of glycosaminoglycans, the synthesis of glycan antigens, and the synthetic application of glycopeptides, etc. At present, the research progress on UDP glycosyl donors is not limited to natural UDP-GlcNAc, and non-natural GlcNAc analogs have also been better studied and applied in practice. For example, in the direction of glycosaminoglycans, it has been found that in the synthesis of polysaccharides and oligosaccharides, non-natural GlcNAc analogs replacing natural GlcNAc can synthesize target products with controllable structures and good homogeneity. Moreover, non-natural UDP-GlcNAc analogs can also be used as an effective tool to study the biosynthesis of glycoconjugates (such as glycosylated polypeptides), etc. In addition, glycan antigens are labeled by introducing non-natural glycosyl donors, which can highly selectively label cell surface antigens. Therefore, the research on the synthesis and application of novel UDP-glycosyl donors will play an important role in promoting the synthesis of glycoconjugates and the development of new drugs, etc.

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

[0004] Currently, in the process of chemoenzymatic synthesis of heparin backbone, GlcA-pNP is used as the starting acceptor substrate, and UDP-GlcNTFA and UDP-GlcA are used as donor substrates for the synthesis of heparin oligosaccharides. Limited by the lack of deacetylation activity of N-deacetylase / N-sulfotransferase (NDST), the unnatural donor sugar UDP-GlcNTFA is used to replace the natural donor sugar UDP-GlcNAc to introduce the heparin backbone. Subsequently, weak base treatment is used to remove the trifluoroacetyl group, and after the amino group is exposed, N-sulfation reaction and subsequent modifications are carried out. However, with the extension of the sugar chain and the prolongation of the reaction time, the heparin oligosaccharides containing GlcNTFA are easily hydrolyzed, and the oligosaccharides with the amino group exposed at the non-reducing end cannot be extended by glycosyltransferase. Therefore, more complex purification processes are required to prepare and purify the oligosaccharides. In addition, special attention needs to be paid to the synthesis, use and storage of the synthetic raw material UDP-GlcNTFA. Since the size of the GlcNTFA group is much larger than that of the natural GlcNAc, the glycosyltransferase transfer activity of GlcNTFA is only about 20% of that of GlcNAc. The low reactivity and easy hydrolysis of GlcNTFA urgently require a new stable and reliable group to replace GlcNTFA and be applied to the synthesis of heparin backbone in the field of heparin synthesis. Summary of the Invention

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

[0006] Term Explanation:

[0007] GlcA-pNP: p-nitrophenyl-β-D-glucuronide;

[0008] UDP-GlcNTFA: uridine diphosphate-N-trifluoroacetylglucosamine;

[0009] UDP-GlcNDFA: uridine diphosphate-N-difluoroacetylglucosamine;

[0010] UDP-GlcA: uridine diphosphate-glucuronic acid;

[0011] PAPS: 3'-phosphoadenosine-5'-phosphosulfate;

[0012] ES2-VSRA: anti-angiogenic peptide;

[0013] Gal-Glc-pNP: 4-nitrophenyl-β-D-lactoside;

[0014] PmHS1: heparin backbone synthase 1;

[0015] PmHS2: heparin backbone synthase 2;

[0016] NST: N-sulfotransferase;

[0017] NaKfiA: N-acetylglucosaminyltransferase;

[0018] PmHAS: hyaluronan synthase;

[0019] ncOGT: O-GlcNAc transferase;

[0020] NmLgtA: N-acetylglucosamine transferase;

[0021] NahK: N-acetylhexosamine 1-kinase;

[0022] GlmU: UDP-N-acetylglucosamine pyrophosphorylase;

[0023] PmPPA: inorganic pyrophosphatase.

[0024] The technical solution of the present invention is as follows:

[0025] In the first aspect of the present invention, a method for preparing uridine diphosphate-N-difluoroacetylglucosamine is provided, including the following steps:

[0026] (1) Dissolve D-glucosamine hydrochloride and anhydrous sodium carbonate in anhydrous methanol, add ethyl difluoroacetate, and stir and react at 20 - 30 °C for 10 - 15 h. After evaporation to dryness and purification, difluoroacetylglucosamine (GlcNDFA) is obtained;

[0027] (2) Add disodium adenosine triphosphate, uridine 5'-triphosphate trisodium salt, 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 and react overnight at 35 - 40 °C for 10 - 15 h. After protein removal and purification, uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) is obtained.

[0028] Preferably according to the present invention, in step (1), the mass ratio of the D-glucosamine hydrochloride to the anhydrous sodium carbonate is (1 - 1.1):1.

[0029] Preferably according to the present invention, in step (1), the mass-volume ratio of the anhydrous sodium carbonate to the anhydrous methanol is 1:3, unit: g / mL.

[0030] Preferably according to the present invention, in step (1), the volume ratio of the anhydrous methanol to the ethyl difluoroacetate is 1:(1 - 1.2).

[0031] Preferably according to the present invention, 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, Mg 2+ has a final concentration of 5-15 mM, the final concentration of difluoroacetylglucosamine is 15-25 mM, and the final concentration of Tris-HCl is 45-55 mM.

[0032] Preferably according to the present invention, in step (2), the final concentration of N-acetylhexosamine 1-kinase (NahK) in the reaction system is 0.1-0.3 mg / ml; the final concentration of UDP-N-acetylglucosamine pyrophosphorylase (GlmU) in the reaction system is 0.1-0.3 mg / ml; the final concentration of inorganic pyrophosphatase (PmPPA) in the reaction system is 0.05-0.15 mg / ml.

[0033] Preferably according to the present invention, in step (2), the method for removing protein and purification is as follows:

[0034] After standing overnight for reaction, trifluoroacetic acid is added to the reaction system, and then the pH is adjusted to 3, and the protein is removed by centrifugal filtration; then the pH is adjusted to 6, the filtrate is rotary evaporated and concentrated and then dialyzed to remove salts; then, at a flow rate of 35-45 mL / min, the dialyzed filtrate is loaded onto a Q column, and ultrapure water, 0.1 M NaCl and 2 M NaCl are passed through the column in sequence.

[0035] In the second aspect of the present invention, there is provided uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) prepared by the above method, and its structural formula is shown as the following formula:

[0036]

[0037] Among them, R is -CF2H.

[0038] This UDP-GlcNDFA has a chemical structure similar to that of the natural substrate UDP-GlcNAc, except that the N-acetyl group at the 2-position is replaced by an N-difluoroacetyl group. Due to the similarity of its chemical structure, it can be recognized by UDP-GlcNAc glycosyltransferase and can be used as a donor substrate for the chemoenzymatic synthesis of heparin oligosaccharide backbone, heparin oligosaccharide intermediate and heparin oligosaccharide.

[0039] In the third aspect of the present invention, there is provided the application of the above uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of heparin oligosaccharide backbone.

[0040] The UDP-GlcNDFA can be efficiently transferred to the heparin backbone by glycosyltransferase, thereby synthesizing heparin backbone oligosaccharides with a -GlcNDFA-GlcA- structure. At the same time, due to the very weak hydrolytic activity of the GlcNDFA structure, a higher yield is obtained during the synthesis of long oligosaccharides.

[0041] In the fourth aspect of the present invention, there is provided the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of heparin oligosaccharide intermediates.

[0042] Preferably according to the present invention, the use is as follows: 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 a -GlcNDFA-GlcA- structure;

[0043] The structural formula of the heparin oligosaccharide intermediate is shown as follows:

[0044]

[0045] Among them, R1 is -CF2H, n = 1 - 7, and R2 can be a substituted aromatic ring such as p-nitrophenyl, phenyl, substituted phenyl, etc.

[0046] In the fifth aspect of the present invention, there is provided the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of heparin oligosaccharides.

[0047] Preferably according to the present invention, the use is as follows: synthesizing heparin oligosaccharide intermediates according to the above method, and the synthesized heparin oligosaccharide intermediates are deprotected with difluoroacetyl groups under the action of lithium hydroxide, thereby generating N-sulfated heparin oligosaccharides under the action of N-sulfotransferase, and then through modification, active heparin oligosaccharides are prepared.

[0048] In the sixth aspect of the present invention, there is provided the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of hyaluronic acid backbones.

[0049] The UDP-GlcNDFA can be efficiently transferred to the hyaluronic acid backbone by glycosyltransferase, thereby synthesizing hyaluronic acid backbone oligosaccharides with a -GlcNDFA-GlcA- structure, thus enriching the diversity of hyaluronic acid oligosaccharides.

[0050] In the seventh aspect of the present invention, there is provided the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in the synthesis of glycosylated polypeptides.

[0051] This UDP-GlcNDFA can be efficiently transferred to polypeptides by glycosyltransferases to synthesize glycosylated polypeptides, and at the same time provide an effective tool for the study of the synthesis of glycoconjugates.

[0052] In the eighth aspect of the present invention, there is provided the use of the above-mentioned uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) in antigen synthesis.

[0053] This UDP-GlcNDFA can be efficiently transferred to glycan antigens by glycosyltransferases to introduce labels, enabling highly selective labeling of cell surface antigens.

[0054] Beneficial effects

[0055] 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 towards glycosyltransferases and is stable and non-hydrolyzable, and can be used as a donor substrate for the chemoenzymatic synthesis of heparin oligosaccharide skeletons, heparin oligosaccharide intermediates, heparin oligosaccharides, as well as hyaluronic acid oligosaccharides, glycoconjugates (glycosylated polypeptides), glycan antigens, etc.

[0056] 2. The present invention provides a preparation method of uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA). This method has few steps, is simple to operate, safe, uses conventional raw materials, is green and environmentally friendly, and can be mass-produced industrially.

[0057] 3. The heparin oligosaccharide skeletons and heparin oligosaccharide intermediates synthesized using the uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) of the present invention contain the -GlcNDFA-GlcA- structure, which has extremely high stability in aqueous solution, can effectively avoid the generation of by-products caused by hydrolysis, and effectively makes up for the problem of limited initial synthesis efficiency of the heparin skeleton.

[0058] 4. The present invention provides a method for synthesizing heparin oligosaccharides using uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA), which not only enriches the diversity of heparin oligosaccharides, but also provides a new idea for the synthesis of heparin skeletons. Through this method, the synthesis efficiency, yield, and output of heparin oligosaccharides can be effectively improved. The purity of each heparin oligosaccharide reaches over 95%, and the yield reaches over 85%, which is expected to promote the process development of rapid, safe, and large-scale preparation of heparin oligosaccharides. Brief description of the drawings

[0059] Figure 1 ESI-MS result of the chemoenzymatic synthesis of UDP-GlcNDFA of the present invention;

[0060] In the figure: the abscissa is the mass-to-charge ratio m / z, and the ordinate is the signal intensity.

[0061] Figure 2 It is for the determination of the reactivity of UDP-GlcNDFA and UDP-GlcNTFA of the present invention;

[0062] Figure 3 It is the comparison result of the stability of GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP of the present invention in a reaction environment with pH = 7.5.

[0063] Figure 4 It is the HPLC chromatogram result of the hydrolysis of GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP of the present invention under alkaline conditions.

[0064] Figure 5 It is the HPLC chromatogram result of each heparin oligosaccharide synthesized by the present invention;

[0065] 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; (8) is heparin nonasaccharide.

[0066] Figure 6 It is the HPLC chromatogram result of the sulfation modification of heparin pentasaccharide, heptasaccharide and nonasaccharide synthesized by the present invention;

[0067] In the figure: (1) is sulfated heparin pentasaccharide; (2) is sulfated heparin heptasaccharide; (3) is sulfated heparin nonasaccharide.

[0068] Figure 7 It is the HPLC chromatogram result of the heparin oligosaccharide synthesized by the present invention;

[0069] In the figure: (1) is the catalytic synthesis participated by heparin backbone synthase 1 (PmHS1); (2) is the catalytic synthesis participated by heparin backbone synthase 2 (PmHS2); (3) is the catalytic synthesis participated by N-acetylglucosaminyltransferase (NaKfiA);

[0070] Figure 8 It is the HPLC chromatogram result of the hyaluronic acid oligosaccharide synthesized by the present invention;

[0071] Figure 9 It is the HPLC chromatogram result of the glycosylated polypeptide synthesized by the present invention;

[0072] Figure 10 It is the HPLC chromatogram result of the glycan antigen synthesized by the present invention. Detailed implementation manners

[0073] The technical solution of the present invention will be further described below in conjunction with embodiments and the accompanying drawings of the specification, 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 all methods well known to those skilled in the art.

[0074] GlcA-pNP, UDP-GlcA, and UDP-GlcNTFA used in the present invention are available from Sigma.

[0075] 4-Nitrophenyl-β-D-lactoside used in the present invention is available from Macklin.

[0076] 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 all entrusted to Nanjing Genscript Biotech Co., Ltd. for artificial synthesis and production according to the specific sequences.

[0077] Among them, PmHS2(D215N / D217N) is a mutant in which the aspartic acids at positions 215 and 217 in the PmHS2 amino acid sequence (GenBank: AAQ55110.1) are mutated to asparagine, that is, the GAT at positions 643-645 bp and 649-651 bp in the PmHS2 nucleotide sequence (GenBank: AY292200.1) are mutated to AAT.

[0078] PmHS2(D479N / D481N) is a mutant in which the aspartic acids at positions 479 and 481 in the PmHS2 amino acid sequence (GenBank: AAQ55110.1) are mutated to asparagine, that is, the GAT at positions 1435-1437 bp and the GAC at positions 1441-1443 bp in the PmHS2 nucleotide sequence (GenBank: AY292200.1) are mutated to AAT and AAC, respectively.

[0079] That is, PmHS1, PmHS2 and their mutants, NST, NaKfiA, PmHAS, ncOGT, NmLgtA, NahK, GlmU and PmPPA used in the present invention are all existing enzymes with publicly known amino acid sequences, and can be obtained through commercial purchase or artificial synthesis.

[0080] The competent cells of Escherichia coli BL21(DE3) used are available from Nanjing Novoprotein Scientific Inc.

[0081] Other raw materials or reagents not described in the present invention can be synthesized according to existing methods or obtained through commercial purchase.

[0082] HPLC detection was carried out using an amino column of YMC. The liquid phase system was produced by Shimadzu Corporation of Japan, and the ultraviolet detection system was SPD-20A.

[0083] Example 1. Chemical-enzymatic synthesis of donor substrate uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA)

[0084] 1. A preparation method of uridine diphosphate-N-difluoroacetylglucosamine, comprising the following steps:

[0085] (1) Synthesis of difluoroacetylglucosamine (GlcNDFA):

[0086] Dissolve 10.2 g of D-glucosamine hydrochloride and 10 g of anhydrous sodium carbonate in 30 ml of anhydrous methanol, add 35 ml of ethyl difluoroacetate, stir and react at 25 °C for 12 h, then evaporate the obtained product to dryness. Ensure complete anhydrousness during the reaction process. The product after rotary evaporation is quickly purified by a silica gel column to obtain the monosaccharide GlcNDFA. The specific reaction formula is as follows:

[0087]

[0088] (2) Synthesis of uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA):

[0089] According to the system shown in Table 1, add disodium adenosine triphosphate (ATP-2Na), trisodium uridine triphosphate (UTP-3Na), Mg 2+ and the difluoroacetylglucosamine obtained in step (1) to Tris-HCl buffer solution in sequence, adjust the pH to 7.0 - 7.5 to obtain a reaction system with a total volume of 1 L (note to stir while adding, and strictly monitor the change of pH, and do not let the solution be in an alkaline condition); then add N-acetylhexosamine 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 to the reaction system, and let it stand and react overnight at 37 °C for 15 h. After deproteinization and purification, uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) is obtained. The specific reaction formula is as follows:

[0090]

[0091] Table 1. Amounts of each component in the reaction system

[0092]

[0093] In step (2), the methods of deproteinization and purification are specifically as follows:

[0094] After standing overnight for the reaction, trifluoroacetic acid was added to the reaction system at a mass percentage of 0.1% to adjust the pH to 3, and the protein was removed by centrifugation and filtration; then the pH was adjusted to 6, and the filtrate was rotary evaporated and concentrated and then dialyzed to remove salts until the sodium chloride concentration was 0.1 M; then a Q column with a volume of 2 L was first rinsed with 3 L of 0.1 M NaOH, and then rinsed with 4 L of ultrapure water (containing 10 mM NaH2PO4) until the pH of the effluent was weakly acidic, and the column was equilibrated. 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 passed through the column in sequence. The specific purification procedure is shown in Table 2.

[0095] Table 2. Q column purification procedure of UDP-GlcNDFA

[0096]

[0097] 2. Take the reaction solution after standing overnight for the reaction and before protein removal and purification for high-performance liquid chromatography. The specific liquid-phase detection and analysis method is shown in Table 3, and the retention times of the main substances are shown in Table 4.

[0098] Liquid-phase conditions: A: ultrapure water; B: 1 mol / L KH2PO4; flow rate 0.5 ml / min; chromatographic column YMC-Pack Polyamine II (250×4.6 mm i.d.).

[0099] Table 3. Liquid-phase detection and analysis method

[0100]

[0101] Table 4. Retention times of the main substances:

[0102]

[0103]

[0104] 3. The UDP-GlcNDFA prepared in this example was subjected to electrospray ionization mass spectrometry analysis, and the results are as Figure 1 shown.

[0105] As can be seen from Table 4 and Figure 1 it can be known that the method provided by the present invention successfully prepared UDP-GlcNDFA.

[0106] Example 2. Feasibility verification of UDP-GlcNDFA in heparin backbone synthesis

[0107] 1. Reactivity

[0108] In a Tris-HCl (pH = 7.5) buffer system containing Mn, with heparin trisaccharide as the receptor substrate and UDP-GlcNDFA and UDP-GlcNTFA as donor substrates respectively, PmHS1 and PmHS2 were used to catalyze the reaction respectively. The reaction conditions were as follows: after reacting at 30 °C for 1 h, the reaction was terminated by boiling in a boiling water bath for five minutes. The reaction was monitored by liquid phase, and three parallel groups were set up for the reaction. The results are as 2+ shown. Figure 2

[0109] It can be seen from Figure 2 that the reaction activity of UDP-GlcNDFA is 2 times 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 glycosyltransferase and can be used as a donor substrate for the chemoenzymatic synthesis of heparin oligosaccharide skeletons, heparin oligosaccharide intermediates and heparin oligosaccharides.

[0110] 2. Stability

[0111] Weigh 0.630 g of p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP), and respectively measure 2 equivalents of UDP-GlcNDFA and UDP-GlcNTFA and place them in Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2). After diluting the volume to 1 L, adjust the pH of the solution to about 7.0, add the PmHS2 enzyme mutant (D215N / D217N) with a final concentration of 0.1 mg / ml, and let it stand and react at 30 °C for 24 h. The reaction was terminated when the conversion rate of the disaccharide monitored by liquid phase was higher than 90%. The obtained reaction solution was adjusted to pH = 3 with trifluoroacetic acid and purified by C18 column chromatography to obtain the heparin disaccharide skeleton GlcNDFA-GlcA-pNP / GlcNTFA-GlcA-pNP. Then, using Tris-HCl (pH = 7.5) to simulate the synthesis reaction environment, the stability of GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP was continuously monitored by liquid phase for 15 days. The results are as Figure 3 shown.

[0112] It can be seen from Figure 3 ​It can be seen that after being stored for 15 days under the condition of neutral Tris-HCl (pH = 7.5), more than 90% of the original substance of GlcNDFA-GlcA-pNP still remains, while only about 50% of the original substance of GlcNTFA-GlcA-pNP remains, and the rest is hydrolyzed into GlcNH2-GlcA-pNP. This shows that GlcNDFA-GlcA-pNP synthesized with UDP-GlcNDFA as the donor substrate has significantly better stability than GlcNTFA-GlcA-pNP synthesized with UDP-GlcNTFA as the donor substrate under the reaction conditions of near-neutral reaction.

[0113] 3. Hydrolyzability

[0114] Lithium hydroxide (final concentration of 1 M) was added to the heparin disaccharide backbone obtained in the second point of this example, the pH was adjusted to 12.0, and it was placed on ice for 30 min. The hydrolysis of DFA / TFA was monitored by liquid phase, and the hydrolysis situation is as Figure 4 shown.

[0115] It can be Figure 4 seen that after incubating on ice for 0.5 h, both GlcNDFA-GlcA-pNP and GlcNTFA-GlcA-pNP were completely hydrolyzed into GlcNH2-GlcA-pNP, which shows that oligosaccharides with GlcNDFA structure, like oligosaccharides with GlcNTFA structure, can easily expose amino groups to synthesize N-sulfated oligosaccharides.

[0116] Example 3. Chemoenzymatic synthesis and purification of heparin oligosaccharide backbone

[0117] 1. Synthesis of heparin oligosaccharides

[0118] a. Take 0.5 g of the heparin disaccharide backbone obtained in the second point of the example, 1.5-fold equivalent 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 overnight at 30 °C, monitor the reaction progress by liquid phase, the conversion rate of the trisaccharide is higher than 95%, adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin trisaccharide backbone GlcA-GlcNDFA-GlcA-pNP;

[0119] b. Take 0.4 g of the heparin trisaccharide backbone obtained in step a, 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 overnight at 30 °C. Monitor the reaction progress by liquid phase. The conversion rate of the tetrasaccharide is higher than 95%. Adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin tetrasaccharide backbone GlcNDFA-GlcA-GlcNDFA-GlcA-pNP;

[0120] c. Take 0.3 g of the heparin tetrasaccharide backbone obtained in step b, 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 overnight at 30 °C. Monitor the reaction progress by liquid phase. The conversion rate of the pentasaccharide is higher than 95%. Adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin pentasaccharide backbone GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP;

[0121] d. Take 0.2 g of the heparin pentasaccharide backbone 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 overnight at 30 °C. Monitor the reaction progress by liquid phase. The conversion rate of the hexasaccharide is higher than 95%. Adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin hexasaccharide backbone GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0122] 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. The concentration of the PmHS2 enzyme mutant (D479N / D481N) in the reaction system is 0.1 mg / ml;; then react overnight at 30°C. Monitor the reaction progress by liquid phase. The conversion rate of the heptasaccharide is higher than 95%. Adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin heptasaccharide backbone GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0123] f. Take 0.1 g of the heparin heptasaccharide backbone obtained in step e, 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 overnight at 30°C. Monitor the reaction progress by liquid phase. The conversion rate of the octasaccharide is higher than 95%. Adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin octasaccharide backbone GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0124] 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 overnight at 30°C. Monitor the reaction progress by liquid phase. The conversion rate of the nonasaccharide is higher than 95%. Adjust the reaction solution to pH = 3 to terminate the reaction, and purify it by C18 column chromatography to obtain the heparin nonasaccharide backbone GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-GlcNDFA-GlcA-pNP.

[0125] 2. Purification of heparin oligosaccharides

[0126] The above heparin oligosaccharide backbones are used with Purification was performed using a C18-AQ separation column and an AKTA. Before purification, the reaction solution that had terminated the reaction was centrifuged at room temperature (8000 rpm, 10 min). The supernatant was filtered through a 0.22 μm filter membrane and then loaded onto a C18 separation column that had been pre-equilibrated with double-distilled water (0.1% TFA). The specific purification procedure is shown in Table 5, with a flow rate of 8 ml / min. The results are as Figure 5 shown.

[0127] Table 5. C18 purification procedure for heparin oligosaccharides (gradient)

[0128]

[0129] As Figure 5 can be seen, the synthesis conversion rates of heparin oligosaccharides are all above 95%, the purification yields are all above 85%, and the purities are all above 95%. This indicates that the present invention successfully synthesized heparin oligosaccharide skeletons, heparin oligosaccharide intermediates, and heparin oligosaccharides by chemoenzymatic method using UDP-GlcNDFA as a substrate.

[0130] Example 4. Sulfation modification and purification of heparin oligosaccharide skeletons

[0131] 1. Alkaline hydrolysis to remove difluoroacetyl (DFA)

[0132] Lithium hydroxide (final concentration 1 M) was added to the heparin pentasaccharide skeleton, heparin heptasaccharide skeleton, and heparin nonasaccharide skeleton obtained in Example 3 respectively. Then the pH was adjusted to 12.0 and placed on ice for 30 min. 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.

[0133] 2. Sulfation modification

[0134] 0.1 g of the heparin pentasaccharide skeleton, heparin heptasaccharide skeleton, and heparin nonasaccharide skeleton that had been de-DFA in the above step, 4-fold equivalents of PAPS and NST were placed in a 50 mM MES buffer to obtain a reaction system with a volume of 200 mL. The final concentration of NST in the reaction system was 0.5 mg / ml; then it was placed at 37 °C for an overnight reaction. The conversion rate of the reaction progress monitored by liquid phase was higher than 95%. The reaction solution was adjusted to pH = 3 to terminate the reaction and purified by chromatography using a Q column.

[0135] The specific method for chromatography purification using the Q column is as follows:

[0136] Prepare solution A: ultrapure water (containing 10 mM NaH2PO4); solution B: 2 M NaCl.

[0137] Before purification, the reaction solution after terminating the reaction was centrifuged at room temperature (8000 rpm, 10 min). The supernatant was filtered through a 0.22 μm filter membrane and then loaded onto a Q column chromatography column that had been pre-equilibrated with ultrapure water (containing 10 mM NaH2PO4). The specific purification procedure is shown in Table 5, and the flow rate was 10 ml / min.

[0138] Table 6, Q column purification procedure

[0139]

[0140] The purified sample was rotary evaporated, then dialyzed through a 200 molecular weight membrane for 2 h. After being concentrated again to a volume less than 2 ml, it was loaded onto a 2.6 cm * 60 cm P-2 column. Chromatography was carried out at a flow rate of 0.3 ml / min using 0.1 M ammonium bicarbonate. Finally, the collected oligosaccharides were analyzed by liquid chromatography for their purity, and the results are as Figure 6 shown.

[0141] As Figure 6 can be seen, the synthesis conversion rates of heparin oligosaccharides after sulfation modification were all above 90%, the purification yields were all above 85%, and the purities were all above 85%.

[0142] Example 5, Synthesis and Application of UDP-GlcNDFA

[0143] 1. Synthesis and Application of Glycosaminoglycan

[0144] (1) Synthesis of Heparin Oligosaccharides

[0145] Take 0.1 g of the heparin trisaccharide backbone in step a of Example 3, 1.5 equivalents of UDP-GlcNDFA and PmHS1 and place them in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system. The concentration of the heparin trisaccharide backbone in the reaction system was 0.2 mM, and the concentration of PmHS1 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 measured by liquid chromatography. The results are as Figure 7 (1) shown.

[0146] Take 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), and place them in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system. The concentration of the heparin trisaccharide backbone in the reaction system is 0.2 mM, and the concentration of the PmHS2 enzyme mutant (D215N / D217N) is 0.1 mg / ml. Then, after reacting at 30 °C for 1 h, add 0.1% TFA to terminate the reaction, and measure the reaction by liquid phase. The results are as Figure 7 shown in (2).

[0147] Take 0.1 g of the heparin trisaccharide backbone in step a of Example 3, 1.5 equivalents of UDP-GlcNDFA and NaKfiA, and place them in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system. The concentration of the heparin trisaccharide backbone in the reaction system is 0.2 mM, and the concentration of NaKfiA is 0.1 mg / ml. Then, after reacting at 30 °C for 1 h, add 0.1% TFA to terminate the reaction, and measure the reaction by liquid phase. The results are as Figure 7 shown in (3).

[0148] It can be seen from Figure 7 that UDP-GlcNDFA can be used as a novel UDP glycosyl donor for the synthesis of heparin oligosaccharides.

[0149] (2) Synthesis of hyaluronic acid oligosaccharides

[0150] Take 0.1 g of the hyaluronic acid trisaccharide backbone, 1.5 equivalents of UDP-GlcNDFA and PmHAS, and place them in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system. The concentration of the hyaluronic acid trisaccharide backbone in the reaction system is 0.2 mM, and the concentration of PmHAS is 0.1 mg / ml. Then, after reacting at 30 °C for 1 h, add 0.1% TFA to terminate the reaction, and measure the reaction by liquid phase. The results are as Figure 8 shown.

[0151] It can be seen from Figure 8 that UDP-GlcNDFA can be used as a novel UDP glycosyl donor for the synthesis of hyaluronic acid oligosaccharides.

[0152] 2. Synthetic application of glycopeptides

[0153] Take 0.1 g of ES2-VSRA, 4-fold equivalent of UDP-GlcNDFA and ncOGT and place them in 200 μL of Tris-HCl buffer (25 mmol / L, pH = 7.5 and containing 12.5 mmol / L MgCl2, 0.06 mg / ml BSA, 1 mM DTT) to obtain a reaction system. The concentration of ES2-VSRA in the reaction system is 0.2 mM, and the concentration of ncOGT is 0.2 mg / ml. Then, after reacting at 37 °C for 4 h, add 0.1% TFA to terminate the reaction, and measure the reaction by liquid phase. The results are as Figure 9 shown.

[0154] It can be seen from Figure 9 that UDP-GlcNDFA can be used as a novel UDP sugar donor for the synthesis of glycosylated polypeptides.

[0155] 3. Synthetic application of antigen

[0156] Take 0.1 g of 4-nitrophenyl-β-D-lactoside, 1.5-fold equivalent of UDP-GlcNDFA and NmLgt and place them in 200 μL of Tris-HCl buffer (50 mmol / L, pH = 7.5 and containing 10 mmol / L MnCl2) to obtain a reaction system. The concentration of 4-nitrophenyl-β-D-lactoside in the reaction system is 0.2 mM, and the concentration of ncOGT is 0.2 mg / ml. Then, after oscillating and reacting at 30 °C and 200 r / min for 1 h, add 0.1% TFA to terminate the reaction, and measure the reaction by liquid phase. The results are as Figure 10 shown.

[0157] It can be seen from Figure 10 that UDP-GlcNDFA can be used as a novel UDP sugar donor for the synthesis of glycan antigens.

[0158] In summary, the artificial donor 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 chemoenzymatic synthesis of heparin oligosaccharide skeletons, 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, wherein 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 - 3, and its structural formula is as 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 intermediates; Further preferably, the use is: using pNP-GlcA as the acceptor substrate, using 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 as shown in the following formula: 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, and 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 uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) as claimed in claim 4 in the synthesis of hyaluronic acid backbone.

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

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

Images