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

Abstract

The invention relates to uridine diphosphate-N-difluoroacetyl galactosamine as well as a preparation method and application thereof. The chemical structure of the UDP-GalNDFA is similar to that of a natural substrate UDP-GalNAc, and the core difference is that N-acetyl at the site 2 of the UDP-GalNDFA is replaced by N-difluoroacetyl. The preparation method comprises the following steps: (1) preparing difluoroacetyl galactosamine; and (2) preparing the UDP-GalNDFA by taking the difluoroacetyl galactosamine as a substrate. As a novel artificial donor sugar, the UDP-GalNDFA has the characteristics of high activity, difficulty in hydrolysis and capability of being artificially synthesized. Compared with the existing donor sugar UDP-GalNTFA, the glucose UDP-GalNTFA has higher catalytic activity on glycosyl transferase, has stronger stability, is not easy to hydrolyze, and can be used as a donor substrate for synthesizing a chondroitin oligosaccharide skeleton, a chondroitin oligosaccharide intermediate and a chondroitin oligosaccharide derivative by a chemical enzyme method.

Description

Technical Field

[0001] This invention relates to a uridine diphosphate-N-difluoroacetylgalactosamine, its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] Uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc), as a classic natural UDP-glycosyl donor, has demonstrated its application value in various fields such as pharmaceutical research and biosynthesis. Particularly in the field of functional oligosaccharide synthesis, it serves as a core glycosyl donor, providing the foundation for the efficient preparation of various physiologically active sugar molecules. With the continuous advancement of glycobiology research, current exploration of UDP-glycosyl donors has broken through the limitations of natural UDP-GalNAc. A series of non-natural galactose analogs, through structural optimization, introduce special functional groups to endow target sugar products with new physicochemical properties and biological functions. Therefore, the synthesis and application research of novel UDP-glycosyl donors will play a significant role in promoting the synthesis of glycoconjugates and the development of new drugs.

[0003] It is important to note that although non-natural UDP glycosyl donors have proven reliable and practical applications in various fields, unlike natural UDP glycosyl donors, they may be completely unable to enter the active site of enzymes to achieve specific binding, or they may only be partially recognized, resulting in enzymatic catalytic efficiency far lower than that of natural UDP glycosyl donors. Sulfonation at the amino group is a key type of sulfonation, widely found in the important glycosaminoglycan heparin; it is also present in dermatan sulfate, which has a similar association with chondroitin (CS), although dermatan sulfate contains iduronic acid (IdoA) instead of glucuronic acid (GlcA). Given that N-sulfonated chondroitin (CH) polymers possess superior complement-inhibiting activity and other biological activities compared to chondroitin, N-sulfonated chondroitin was further synthesized using a chemical enzymatic method. In this synthesis, the most prominent issue was the substitution of uridine diphosphate-N-trifluoroacetylgalactosamine (UDP-GalNTFA) for uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) in the synthesis of chondroitin oligosaccharide derivatives.

[0004] Currently, in the chemical enzymatic synthesis of chondroitin oligosaccharide derivatives, GlcA-pNP is used as the initiator and acceptor substrate, while UDP-GalNTFA and UDP-GlcA are used as donor substrates to construct the synthetic system for chondroitin oligosaccharide derivatives. However, due to two limitations of N-deacetylation / N-sulfate transferase (NDST): lacking both deacetylation activity and inability to sulfate the N-site of chondroitin oligosaccharides, the non-natural donor sugar UDP-GalNTFA is selected to replace the natural donor sugar UDP-GalNAc and introduced into the chondroitin backbone structure. Subsequently, the trifluoroacetyl group in UDP-GalNTFA needs to be removed by weak base treatment to expose the amino group, followed by chemical sulfation modification of the N-site. It is important to note that the molecular size of the GalNTFA group is much larger than that of the natural GalNAc. This structural difference results in the transfer activity of GalNTFA under the action of chondroitin transferase being only about 30% of that of GalNAc. Furthermore, as the sugar chains extend and the reaction time prolongs, chondroitin oligosaccharides containing GalNTFA are prone to hydrolysis. This necessitates a more complex purification process to achieve efficient preparation and purification of the oligosaccharides. In addition, strict control and extra care must be taken at every stage of the synthesis, use, and storage of the raw material UDP-GalNTFA. In summary, GalNTFA suffers from both low reactivity and easy hydrolysis. These defects make it imperative to develop a novel group that combines stability and reliability to replace GalNTFA in the synthesis of chondroitin oligosaccharide derivatives. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides uridine diphosphate-N-difluoroacetylgalactosamine, its preparation method, and its applications.

[0006] Terminology Explanation: GalNDFA: Difluoroacetylgalactosamine; GlcA- p NP: p-Nitrophenyl-β-D-glucuronic acid; UDP-GalNTFA: Uridine diphosphate-N-trifluoroacetylgalactosamine; UDP-GalNDFA: Uridine diphosphate-N-difluoroacetylgalactosamine; UDP-GlcA: Uridine diphosphate-glucuronic acid; McCS: Chondroitin synthase; NahK: N-acetylhexylamine 1-kinase; GlmU: UDP-N-acetylglucosamine pyrophosphorylase; PmPPA: Inorganic pyrophosphatase; Py·SO3: Sulfur trioxide pyridine.

[0007] The technical solution of this invention is as follows: In a first aspect, this invention provides a method for preparing difluoroacetylgalactosamine, comprising the following steps: D-galactosamine hydrochloride and anhydrous sodium carbonate were dissolved in anhydrous methanol, and ethyl difluoroacetate was added. The mixture was stirred at 20-30°C for 10-15 hours. After evaporation and purification, difluoroacetylgalactosamine (GalNDFA) was obtained.

[0008] According to a preferred embodiment of the present invention, the mass ratio of D-galactosamine hydrochloride to anhydrous sodium carbonate is (1~1.1):1.

[0009] According to a preferred embodiment of the present invention, the mass-to-volume ratio of anhydrous sodium carbonate and anhydrous methanol is 1:3, in g / mL.

[0010] According to a preferred embodiment of the present invention, the volume ratio of anhydrous methanol to ethyl difluoroacetate is 1:(1~1.2).

[0011] In a second aspect, the present invention provides difluoroacetylgalactosamine (GalNDFA) prepared by the above method, the structural formula of which is shown below: .

[0012] This difluoroacetylgalactosamine (GalNDFA) has a similar chemical structure to the existing N-acetylgalactosamine (GalNAc), the main difference being that the N-acetyl group at the second position of GalNAc is replaced by an N-difluoroacetyl group.

[0013] A third aspect of this invention provides a method for preparing uridine diphosphate-N-difluoroacetylgalactosamine, comprising the following steps: Disodium adenosine triphosphate, trisodium uridine triphosphate, and Mg 2+ Urate diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) was added sequentially to Tris-HCl buffer and the pH was adjusted to 7.0-7.5 to obtain the reaction system. Then, N-acetylhexamine 1-kinase (NahK), UDP-N-acetylglucosamine pyrophosphorylase (GlmU) and inorganic pyrophosphorylase (PmPPA) were added to the reaction system and allowed to stand overnight at 35-40°C for 10-15 hours. After protein removal and purification, uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) was obtained.

[0014] According to a preferred embodiment of the present invention, in the reaction system, the final concentration of disodium adenosine triphosphate is 10-20 mM, the final concentration of trisodium uridine triphosphate is 10-20 mM, and the Mg...2+ The final concentration of HCl is 5-15 mM, the final concentration of difluoroacetylgalactosamine is 10-20 mM, and the final concentration of Tris-HCl is 45-55 mM.

[0015] According to a preferred embodiment of the present invention, the final concentration of N-acetylhexylamine 1-kinase (NahK) in the reaction system is 0.8~1 mg / ml; the final concentration of UDP-N-acetylglucosamine pyrophosphorylase (GlmU) in the reaction system is 0.8~1 mg / ml; and the final concentration of inorganic pyrophosphorylase (PmPPA) in the reaction system is 0.05~0.15 mg / ml.

[0016] According to a preferred embodiment of the present invention, the method for protein removal and purification is as follows: After the reaction was allowed to stand overnight, trifluoroacetic acid was added to the reaction system, and the pH was adjusted to 3. The protein was removed by centrifugation and filtration. Then the pH was adjusted to 6, and the filtrate was concentrated by rotary evaporation and dialyzed to remove salt. The dialyzed filtrate was then loaded onto a DEAE column at a flow rate of 10-15 mL / min, and ultrapure water, 0.1 M NaCl, and 2 M NaCl were sequentially passed through the column.

[0017] In a fourth aspect, the present invention provides uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) prepared by the above method, the structural formula of which is shown below: ; Where R is -CF2H.

[0018] This UDP-GalNDFA has a similar chemical structure to the natural substrate UDP-GalNAc, the main difference being that the N-acetyl group at position 2 of UDP-GalNAc is replaced by an N-difluoroacetyl group. Due to the similarity of its chemical structure, it can be recognized by UDP-GalNAc chondroitin synthase and can be used as a donor substrate for the chemoenzymatic synthesis of chondroitin oligosaccharide backbones, chondroitin oligosaccharide intermediates, and chondroitin oligosaccharide derivatives.

[0019] In a fifth aspect, the present invention provides the application of the above-mentioned uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) in the synthesis of chondroitin oligosaccharide backbone.

[0020] This UDP-GalNDFA can be efficiently transferred to the chondroitin backbone via chondroitin synthase, thereby synthesizing a chondroitin oligosaccharide backbone with the -GalNDFA-GlcA- structure. At the same time, due to the very weak hydrolytic activity of the GalNDFA structure, the GalNDFA group is smaller than that of GalNTFA, resulting in a higher yield in the synthesis of long oligosaccharides.

[0021] In a sixth aspect, the present invention provides the application of the above-mentioned uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) in the synthesis of chondroitin oligosaccharide intermediates.

[0022] According to a preferred embodiment of the present invention, the application is: with p Using NP-GlcA as the acceptor substrate and UDP-GlcA and UDP-GalNDFA as donor substrates, chondroitin synthase (McCS) was used to repeatedly extend the enzyme to synthesize chondroitin oligosaccharide intermediates of different lengths with the -GalNDFA-GlcA- structure. The structural formula of the chondroitin oligosaccharide intermediate is shown below:

[0023] Where R1 is -CF2H, n=1~7, and R2 can be a substituted aromatic ring such as p-nitrobenzene, benzene, or substituted phenyl.

[0024] In a seventh aspect, the present invention provides the application of the above-mentioned uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) in the synthesis of chondroitin oligosaccharide derivatives.

[0025] According to a preferred embodiment of the present invention, the application is as follows: the chondroitin oligosaccharide intermediate is de-difluoroacetylated under the action of lithium hydroxide, and then modified by sulfation with Py·SO3 to generate N-sulfated chondroitin oligosaccharide derivative.

[0026] Beneficial effects 1. This invention provides difluoroacetylgalactosamine (GalNDFA) and its preparation method. GalNDFA has a chemical structure similar to existing N-acetylgalactosamine (GalNAc) and can be used as a precursor for the synthesis of uridine diphosphate-N-difluoroacetylgalactose (UDP-GalNDFA). Furthermore, this preparation method has the advantages of mild reaction conditions, fewer side reactions, higher product purity, simpler post-processing, and greater environmental friendliness.

[0027] 2. This invention provides a novel artificial donor, UDP-GalNDFA, which possesses high activity, is not easily hydrolyzed, and can be artificially synthesized. Compared to the existing donor UDP-GalNTFA, UDP-GalNDFA exhibits higher catalytic activity for chondroitin synthase, greater stability, and is less prone to hydrolysis. It can be used as a donor substrate for the chemoenzymatic synthesis of chondroitin oligosaccharide backbones, chondroitin oligosaccharide intermediates, and chondroitin oligosaccharide derivatives.

[0028] 3. This invention provides a method for preparing uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA). This method has the characteristics of fewer steps, simple operation, and high safety. In addition, the raw materials used are conventional and meet the requirements of green environmental protection, which can realize large-scale industrial production.

[0029] 4. The chondroitin oligosaccharide backbone and intermediate synthesized using uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) of this invention both contain the -GalNDFA-GlcA- structure. This structure exhibits extremely high stability in aqueous solution, effectively preventing the formation of hydrolysis byproducts and effectively compensating for the limited initial synthesis efficiency of the chondroitin backbone.

[0030] 5. This invention provides a method for synthesizing chondroitin oligosaccharides using uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA). This method not only effectively improves the synthesis efficiency, yield, and quantity, but also enriches the diversity of chondroitin oligosaccharides. The chondroitin oligosaccharides and derivatives prepared by this method all have a purity of over 90% and a yield exceeding 80%, providing a new technological direction for the rapid, safe, and industrial-scale preparation of chondroitin oligosaccharides and derivatives. Attached Figure Description

[0031] Figure 1 ESI-MS results for the chemical enzymatic synthesis of UDP-GalNDFA; In the figure: the horizontal axis represents the mass-to-nucleus ratio m / z, and the vertical axis represents the signal strength.

[0032] Figure 2 The reactivity of UDP-GalNDFA and UDP-GalNTFA was determined.

[0033] Figure 3 For GalNDFA-GlcA- p NP and GalNTFA-GlcA- p Comparison of NP stability under reaction conditions at pH 7.5.

[0034] Figure 4 For GalNDFA-GlcA- p NP and GalNTFA-GlcA- p HPLC chromatographic results of NP hydrolysis under alkaline conditions.

[0035] Figure 5 The HPLC chromatographic results are shown for the various chondroitin oligosaccharides synthesized in Example 4. In the figure, (1) is chondroitin disaccharide; (2) is chondroitin triaccharide; and (3) is chondroitin tetraaccharide.

[0036] Figure 6The HPLC chromatographic results are for the sulfated modified chondroitin oligosaccharide derivative synthesized in Example 5. Figure 7 The HPLC chromatographic results are for the chondroitin oligosaccharides synthesized in Example 6. In the figure, (1) is the catalytic synthesis involving chondroitin synthase PmCS; (2) is the catalytic synthesis involving chondroitin synthase mutant McCS (D518N / D520N). Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the embodiments and 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 all methods known to those skilled in the art.

[0038] The GlcA used in this invention p NP, UDP-GlcA, and UDP-GalNTFA are available from Sigma.

[0039] The plasmids of the chondroitin synthase mutants McCS (D218N / D220N) and McCS (D518N / D520N) used in the examples were all artificially synthesized by Nanjing Genscript Biotech Co., Ltd. according to the specific sequences.

[0040] The chondroitin synthase mutant McCS (D218N / D220N) is formed by mutating aspartic acid at positions 218 and 220 of the chondroitin synthase McCS amino acid sequence (GenBank: WP_078255998) to asparagine.

[0041] The chondroitin synthase mutant McCS (D518N / D520N) is formed by mutating aspartic acid at positions 518 and 520 of the chondroitin synthase McCS amino acid sequence (GenBank: WP_078255998) to asparagine.

[0042] The chondroitin synthase PmCS, chondroitin synthase McCS and its mutants, NahK, GlmU and PmPPA used in this invention are all existing enzymes whose amino acid sequences have been disclosed and can be obtained through commercial or artificial synthesis.

[0043] The Escherichia coli BL21(DE3) competent cells used are available from Nanjing Novizan Biotechnology Co., Ltd.

[0044] Other raw materials or reagents not described in this invention can be synthesized using existing methods or obtained commercially.

[0045] HPLC detection was performed using an amino column from YMC, with the liquid chromatography system manufactured by Shimadzu Corporation of Japan and the UV detection system being an SPD-20A.

[0046] Example 1: Chemical synthesis of difluoroacetylgalactosamine (GalNDFA) A method for preparing difluoroacetylgalactosamine includes the following steps: 10.2 g of D-galactosamine hydrochloride and 10 g of anhydrous sodium carbonate were dissolved in 30 ml of anhydrous methanol, and 35 ml of difluoroethyl acetate was added. The mixture was stirred at 30 °C for 10 h. The resulting product was then evaporated to dryness, ensuring complete anhydrousity during the reaction. The product after rotary evaporation was rapidly purified by silica gel column chromatography to obtain difluoroacetylgalactosamine (GalNDFA).

[0047] The specific reaction process in this embodiment is as follows; .

[0048] Example 2: Enzymatic synthesis of uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) 1. A method for preparing uridine diphosphate-N-difluoroacetylgalactosamine, comprising the following steps: According to the system shown in Table 1, disodium adenosine triphosphate (ATP-2Na), trisodium uridine triphosphate (UTP-3Na), and Mg... 2+ The difluoroacetylgalactosamine prepared in Example 1 was added sequentially to Tris-HCl buffer, and the pH was adjusted to 7.0-7.5 to obtain a reaction system with a total volume of 0.5 L (note that the mixture was added while stirring, and the pH was strictly monitored to avoid the solution being in alkaline conditions). Then, N-acetylhexamine 1-kinase (NahK), UDP-N-acetylglucosamine pyrophosphorylase (GlmU), and inorganic pyrophosphorylase (PmPPA) with a final concentration of 1 mg / ml were added to the reaction system. The reaction was allowed to proceed overnight at 37°C. After protein removal and purification, uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) was obtained.

[0049] The specific reaction process in this step is as follows; .

[0050] Table 1. Amounts of each component in the reaction system

[0051] The specific methods for protein removal and purification are as follows: After allowing the reaction to stand overnight, trifluoroacetic acid was added to the reaction system at a mass percentage of 0.1% to adjust the pH to 3. The protein was removed by centrifugation and filtration. Then, the pH was adjusted to 6, and the filtrate was concentrated by rotary evaporation and dialyzed to remove salt until the sodium chloride concentration was 0.1M. A 104 mL DEAE column was then pre-washed with 300 mL of 0.1M NaOH, followed by 400 mL of ultrapure water (containing 10 mM NaH2PO4) until the pH of the effluent was weakly acidic. The column was then equilibrated, and the dialyzed filtrate was loaded onto the DEAE column at a flow rate of 10 mL / min. The column was then filtered sequentially with ultrapure water, 0.1M NaCl, and 2M NaCl. The specific purification procedure is shown in Table 2.

[0052] Table 2. DEAE column purification procedure for UDP-GalNDFA

[0053] 2. After the reaction was allowed to stand overnight, the reaction solution before protein purification was subjected to high performance liquid chromatography (HPLC). The specific HPLC detection and analysis methods are shown in Table 3, and the retention times of the main substances are shown in Table 4.

[0054] Liquid chromatography conditions: A: ultrapure water; B: 1 mol / L KH2PO4; flow rate 0.5 ml / min; column YMC-Pack PolyamineII (250 × 4.6 mml. D).

[0055] Table 3. Liquid Chromatography Detection and Analysis Methods

[0056] Table 4. Retention times of major substances

[0057] The UDP-GalNDFA prepared in this embodiment was then analyzed by electrospray ionization mass spectrometry, and the results are as follows: Figure 1 As shown.

[0058] From Table 4 and Figure 1 It is known that the method provided by the present invention successfully prepared uridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA).

[0059] Example 3: Feasibility verification of UDP-GalNDFA in chondroitin scaffold synthesis 1. Reactivity Nitrophenyl-β-D-glucuronic acid (GlcA- p NP) is the acceptor substrate, UDP-GalNDFA is the donor substrate, and a structure containing Mn is constructed. 2+The reaction system was prepared using Tris-HCl (pH=7.5); chondroitin synthase McCs was then added to the reaction system, and the reaction was catalyzed at 30°C for 30 min, followed by boiling in a water bath for 5 min to terminate the reaction. Simultaneously, UDP-GalNTFA was used as the donor substrate, and the catalytic reaction was carried out in the same manner. Three parallel sets were set up for both reaction systems, and liquid chromatography was performed to determine the reaction status. The results are as follows: Figure 2 As shown.

[0060] In the reaction system, the concentration of the acceptor substrate was 0.315 g / L, the donor substrate was 2 equivalents, and Mn 2+ The concentration of the enzyme was 20 mmol / L, and the concentration of Tris-HCl was 50 mM; the final concentration of the chondroitin synthase McCs in the reaction system was 0.2 mg / ml.

[0061] Depend on Figure 2 It is known that UDP-GalNDFA exhibits twice the reactivity of UDP-GalNTFA under the same reaction conditions. This indicates that, compared to the existing donor sugar UDP-GalNTFA, the UDP-GalNDFA of this invention possesses higher catalytic activity towards chondroitin synthase and can be used as a donor substrate for the chemoenzymatic synthesis of chondroitin oligosaccharide backbones, chondroitin oligosaccharide intermediates, and chondroitin oligosaccharide derivatives.

[0062] 2. Stability Weigh 0.315g of p-nitrophenyl-β-D-glucuronic acid (GlcA- p Two equivalents of UDP-GalNDFA and UDP-GalNTFA were separately placed in Tris-HCl buffer (50 mmol / L, pH=7.5, containing 20 mmol / L MnCl2), diluted to 1 L, and the pH of the solution was adjusted to approximately 7.0. Chondroitin synthase mutant McCS (D518N / D520N) was added to a final concentration of 0.2 mg / ml. The reaction was incubated at 30 °C for 12 h. The reaction was terminated when the disaccharide conversion rate exceeded 90% as monitored by liquid chromatography. The resulting reaction solution was adjusted to pH=3 with trifluoroacetic acid and purified by C18 column chromatography to obtain the chondroitin disaccharide backbone GalNDFA-GlcA- p NP / GalNTFA-GlcA- p NP. The reaction environment during the synthesis process was simulated using Tris-HCl (pH=7.5), and GalNDFA-GlcA was continuously monitored by liquid chromatography for 7 days. p NP and GalNTFA-GlcA- p The stability of NP is as follows: Figure 3 As shown.

[0063] As shown in Figure 3, after 7 days of storage in neutral Tris-HCl (pH=7.5), GalNDFA-GlcA-pNP retained over 90% of its original composition, while GalNTFA-GlcA-pNP retained only about 20%, with the remainder hydrolyzed to GalNDFA-GlcA-pNP. This indicates that GalNDFA-GlcA-pNP synthesized using UDP-GalNDFA as the donor substrate exhibits significantly better stability under near-neutral reaction conditions than GalNTFA-GlcA-pNP synthesized using UDP-GalNTFA as the donor substrate.

[0064] 3. Hydrolytic properties Lithium hydroxide (final concentration 1M) was added to the chondroitin bisaccharide backbone obtained in point 2 of this embodiment, 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 chromatography. The hydrolysis status is as follows: Figure 4 As shown.

[0065] Depend on Figure 4 It can be seen that after incubation on ice for 0.5 hours, GalNDFA-GlcA- p NP and GalNTFA-GlcA- p All NPs were hydrolyzed into GlcNH2-GlcA- p The presence of NP indicates that oligosaccharides with the GalNDFA structure, like those with the GalNTFA structure, can easily expose amino groups, providing a basis for subsequent chemical methods to achieve N-sulfation to obtain chondroitin oligosaccharide derivatives.

[0066] Example 4: Chemical enzymatic synthesis and purification of chondroitin oligosaccharide backbone 1. Synthesis of chondroitin oligosaccharides Take 0.25g of the chondroitin disaccharide backbone GalNDFA-GlcA- prepared in point 2 of Example 3. p NP, 1.5 equivalents of UDP-GlcA, and chondroitin synthase mutant McCS (D218N / D220N) were placed in Tris-HCl buffer (50 mmol / L, pH=7.5, containing 20 mmol / L MnCl2) to obtain a 0.5 L reaction system. The concentration of chondroitin synthase mutant McCS (D218N / D220N) in the reaction system was 0.4 mg / mL. The reaction was then carried out overnight at 30 °C. The reaction progress was monitored by liquid chromatography. When the conversion rate of the trisaccharide was higher than 90%, the reaction solution was adjusted to pH=3 to terminate the reaction. The chondroitin trisaccharide skeleton GlcA-GalNDFA-GlcA- was purified by C18 column chromatography. p NP.

[0067] Then, following the same method, using the chondroitin trisaccharide backbone GlcA-GalNDFA-GlcA- p Using NP and UDP-GalNAc as raw materials, and chondroitin synthase mutant McCS (D518N / D520N) at a concentration of 0.2 mg / ml as catalysis, the chondroitin tetrasaccharide backbone GalNDFA-GlcA-GalNDFA-GlcA- was prepared. p NP.

[0068] 2. Purification of chondroitin oligosaccharides The chondroitin disaccharide backbone GalNDFA-GlcA- p NP, chondroitin trisaccharide backbone GlcA-GalNDFA-GlcA- p NP and chondroitin tetrasaccharide skeleton GalNDFA-GlcA-GalNDFA-GlcA- p NP was purified using a SepaFlash® C18-AQ column and AKTA. Before purification, the reaction solution was centrifuged at room temperature (8000 rpm, 10 min), and the supernatant was filtered through a 0.22 μm filter before loading. The C18 column was equilibrated with double-distilled water (containing 0.1% FA) before loading. The specific purification procedure is shown in Table 5, with a flow rate of 8 ml / min. The results are as follows. Figure 5 As shown.

[0069] Table 5. Purification procedure (gradient) for chondroitin oligosaccharides C18

[0070] Depend on Figure 5 It can be seen that the synthesis conversion rate of the chondroitin oligosaccharide backbone is above 90%, the purification yield is above 80%, and the purity is above 90%. This indicates that the present invention successfully synthesized the chondroitin oligosaccharide backbone and chondroitin oligosaccharide intermediates using UDP-GalNDFA as a substrate via a chemical enzymatic method.

[0071] Example 5: Sulfation Modification of Chondroitin Oligosaccharide Backbone Alkaline hydrolysis to dedifluoroacetyl (DFA): Lithium hydroxide (final concentration of 1M) was added to the chondroitin trisaccharide backbone obtained in Example 4, and then 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 chromatography. After the hydrolysis of DFA was completed, the pH was adjusted to 7.0 with hydrochloric acid to obtain the de-DFA chondroitin trisaccharide backbone.

[0072] Sulfation modification: 0.1 g of de-DFA-removed chondroitin trisaccharide backbone was added in two portions (3 equivalents) of Py·SO3 to 50 mM Tris-HCl (pH=9) buffer to obtain a 150 mL reaction system. The system was then incubated overnight at 37 °C. Liquid chromatography monitoring showed a conversion rate exceeding 90%, as shown in the following results. Figure 6 As shown.

[0073] Depend on Figure 6 It can be seen that the conversion rate of the sulfated chondroitin oligosaccharide derivatives is all above 90%.

[0074] Example 6: Synthesis and Application of UDP-GalNDFA 0.1 g of the chondroitin trisaccharide backbone prepared in Example 4, 1.5 equivalents of UDP-GalNDFA, and chondroitin synthase PmCS were placed in 150 mL of Tris-HCl buffer (50 mmol / L, pH=7.5, containing 20 mmol / L MnCl2) to obtain the reaction system. The concentration of the chondroitin trisaccharide backbone in the reaction system was 1 mM, and the concentration of chondroitin synthase PmCS was 0.2 mg / mL. After reacting at 30 °C for 1 h, 0.1% TFA was added to terminate the reaction. The reaction was measured by liquid chromatography, and the results are as follows. Figure 7 As shown in (1).

[0075] 0.1 g of the chondroitin trisaccharide backbone prepared in Example 4, 1.5 equivalents of UDP-GalNDFA, and the chondroitin synthase mutant McCS (D518N / D520N) were placed in 150 mL of Tris-HCl buffer (50 mmol / L, pH=7.5, containing 20 mmol / L MnCl2) to obtain the reaction system. The concentration of the chondroitin trisaccharide backbone in the reaction system was 1 mM, and the concentration of the chondroitin synthase mutant McCS (D518N / D520N) was 0.2 mg / mL. After reacting at 30 °C for 1 h, 0.1% TFA was added to terminate the reaction. The reaction was measured by liquid chromatography, and the results are as follows. Figure 7 As shown in (2).

[0076] Depend on Figure 7 It is known that UDP-GalNDFA can serve as a novel UDP glycosyl donor for the synthesis of chondroitin oligosaccharides.

[0077] In summary, the artificial donor glycosuridine diphosphate-N-difluoroacetylgalactosamine (UDP-GalNDFA) provided by this invention has higher catalytic activity for glycosyltransferases and is stable and not easily hydrolyzed. It can be used as a donor substrate for the chemical enzymatic synthesis of chondroitin oligosaccharide backbone, chondroitin oligosaccharide intermediates, chondroitin oligosaccharide derivatives, etc.

Claims

1. A method for preparing difluoroacetylgalactosamine, characterized by, The method comprises the following steps: D-galactosamine hydrochloride and anhydrous sodium carbonate are dissolved in anhydrous methanol, ethyl difluoroacetate is added, and the reaction is stirred at 20-30 DEG C for 10-15 h, and then the product is obtained after evaporation and purification.

2. The production method according to claim 1, wherein The mass ratio of the D-galactosamine hydrochloride and the anhydrous sodium carbonate is (1-1.1):1; the mass / volume ratio of the anhydrous sodium carbonate and the anhydrous methanol is 1:3, unit: g / mL; and the volume ratio of the anhydrous methanol and the ethyl difluoroacetate is 1:(1-1.2).

3. A difluoroacetylgalactosamine characterized in that, The method is prepared according to any one of claims 1-2, and the structural formula is shown in the following formula: 。 4. A method for the preparation of uridine diphosphate-N- difluoroacetylgalactosamine, characterized in that, The method comprises the following steps: Disodium adenosine triphosphate, trisodium uridine triphosphate, Mg 2+ and the resulting difluoroacetyl galactosamine is sequentially added to Tris-HCl buffer, the pH is adjusted to 7.0~7.5, to obtain a reaction system; then N-acetyl hexamine 1-kinase, UDP-N-acetylglucosamine pyrophosphorylase and inorganic pyrophosphatase are added to the reaction system, and the reaction is carried out at 35~40℃ overnight for 10~15h. After removing protein and purification, uridine diphosphate-N-difluoroacetyl galactosamine is obtained.

5. The production method according to claim 4, wherein The final concentration of adenosine triphosphate disodium in the reaction system is 15-25 mM, the final concentration of uridine triphosphate trisodium is 15-25 mM, the final concentration of Mg 2+ is 5-15 mM, the final concentration of difluoroacetylgalactosamine 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 0.8-1 mg / ml; the final concentration of the UDP-N-acetylglucosamine pyrophosphorylase (GlmU) in the reaction system is 0.8-1 mg / ml; and the final concentration of the inorganic pyrophosphatase (PmPPA) in the reaction system is 0.05-0.15 mg / ml. The method for removing protein and purification is specifically as follows: After the reaction is statically carried out overnight, trifluoroacetic acid is added to the reaction system, the pH is adjusted to 3, and the protein is removed by centrifugal filtration; then the pH is adjusted to 6, the filtrate is concentrated by rotary evaporation, and the salt is removed by dialysis; and then the dialyzed filtrate is loaded onto a DEAE column at a flow rate of 10-15 mL / min, and the column is sequentially eluted with ultrapure water, 0.1M NaCl and 2M NaCl.

6. A uridine diphosphate-N-galactosamine 2-epimer, characterized in that, The method is prepared according to any one of claims 3-4, and the structural formula is shown in the following formula: ； R is -CF2H.

7. The application of the difluoroacetylgalactosamine in the synthesis of uridine diphosphate-N-difluoroacetylgalactosamine.

8. The application of the uridine diphosphate-N-difluoroacetylgalactosamine in the synthesis of a chondroitin oligosaccharide backbone.

9. The application of the uridine diphosphate-N-difluoroacetylgalactosamine in the synthesis of a chondroitin oligosaccharide intermediate. Further preferably, the application is: to synthesize chondroitin oligosaccharide intermediates with different lengths of -GalNDFA-GlcA- structure by using chondroitin synthase repeatedly with NP-GlcA as acceptor substrate and UDP-GlcA and UDP-GalNDFA as donor substrates. p NP-GlcA as acceptor substrate, and UDP-GlcA and UDP-GalNDFA as donor substrates, using chondroitin synthase to extend repeatedly, to synthesize chondroitin oligosaccharide intermediates with different lengths of -GalNDFA-GlcA- structure. The structural formula of the chondroitin oligosaccharide intermediate is shown in the following formula: wherein, R1 is -CF2H, n=1-7, and R2 is p-nitrobenzene, benzene or a substituted benzene.

10. The application of the uridine diphosphate-N-difluoroacetylgalactosamine in the synthesis of a chondroitin oligosaccharide derivative. Further preferably, the application is that the chondroitin oligosaccharide intermediate is subjected to de-difluoroacetylation under the action of lithium hydroxide, and then is subjected to sulfation modification by using Py·SO3 to generate an N-sulfated chondroitin oligosaccharide derivative.

Images