Separation and purification method of uridine-5 '-diphosphate-N-acetyl-glucosamine
Through one-step chromatography purification method, monodisperse microspheres filled with a specific matrix were used to solve the problem of poor purification effect of uridine-5'-diphosphate-N-acetyl-glucosamine in the prior art, and high purity and high yield separation and purification are achieved, which is suitable for large-scale production.
Patent Information
- Application Number
- CN202411839558.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art lacks an effective high-purity and high yield separation and purification process for uridine-5'-diphosphate-N-acetyl-glucosamine, and has a long purification cycle and poor stability.
A one-step chromatographic purification method was used to use a monodispersed microsphere-filled chromatography column with a matrix of polyacrylate or polystyrene-divinylbenzene, and eluted by eluent to achieve high purity and high yield of uridine-5'-diphosphate-N-acetyl-glucosamine separation and purification.
The purity of uridine-5'-diphosphate-N-acetyl-glucosamine and a yield of 65% to 75% were achieved. The purification cycle was short, the stability was high, and the use of organic solvents was not required. It was suitable for large-scale production and reduced production costs.
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Figure CN119661608A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug purification, and in particular to a method for separating and purifying uridine-5'-diphosphate-N-acetyl-glucosamine. Background Art
[0002] Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a sugar nucleotide that is biosynthesized from N-acetylglucosamine-1-phosphate and UTP. It can be used as a donor of N-acetylglucosamine residues for the biosynthesis of peptidoglycan, lipopolysaccharide, polyacetyl glucosamine of fungi, hyaluronic acid, heparin, glycoprotein, and glycolipid of animals.
[0003] The structural formula of uridine-5′-diphospho-N-acetyl-glucosamine is as follows:
[0004]
[0005] The main synthesis process of UDP-GlcNAc is to use microbial cell enzymes. At present, there are few reports on the purification process of uridine-5′-diphospho-N-acetyl-glucosamine in domestic and foreign patents and literature.
[0006] Therefore, in order to obtain high-purity, high-yield uridine-5′-diphosphate-N-acetyl-glucosamine with a short purification cycle and high stability, it is necessary to further study the fine purification process of uridine-5′-diphosphate-N-acetyl-glucosamine. Summary of the invention
[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for separating and purifying uridine-5′-diphosphate-N-acetyl-glucosamine, which only requires one step of chromatography purification to meet the high purity requirement of uridine-5′-diphosphate-N-acetyl-glucosamine, has a high and stable purification yield, a purity of more than 99%, and a yield of 65% to 75%.
[0008] To achieve this object, the present invention adopts the following technical solutions:
[0009] A method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine, comprising the following steps:
[0010] S1. Loading: Filter the crude uridine-5′-diphospho-N-acetyl-glucosamine and load it into a chromatography column, wherein the chromatography column filler is monodisperse microspheres whose matrix is polyacrylate or polystyrene-divinylbenzene;
[0011] S2, elution: eluting the uridine-5′-diphospho-N-acetyl-glucosamine adsorbed in the chromatography column with an eluent;
[0012] S3. Collection and aggregation: The eluted uridine-5′-diphospho-N-acetyl-glucosamine solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.
[0013] The separation and purification method of the present invention only needs one step of chromatography purification to meet the high purity requirement of uridine-5′-diphospho-N-acetyl-glucosamine, and the purification yield is high and stable, with a purity of more than 99%. The purification yield is high and stable. At the same time, the separation method of the present invention is simple and convenient, the purification cycle is short, and no organic solvent is required in the entire purification process, so it can be used for large-scale production, greatly reducing the production cost.
[0014] In step S1, the monodisperse microspheres are microspheres with monodisperse particle sizes and a pore structure.
[0015] Preferably, in step S1, the particle size of the monodisperse microspheres is 20 to 40 μm, for example, the particle size of the monodisperse microspheres is 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm; the pore size is For example, the aperture is
[0016] Preferably, in step S1, the monodisperse microspheres are of model NanoQ-30L, with a particle size of 30 μm and a pore size of
[0017] In the present invention, the chromatographic medium UniQ-30S / NanoQ-30L used in the purification process is a monodisperse microsphere whose matrix bonded with a quaternary amine group is polyacrylate or polystyrene-divinylbenzene. Uridine-5′-diphospho-N-acetyl-glucosamine contains two phosphate groups in its molecular formula. Preferably, under the selected elution conditions, the use of NanoQ-30L for UDP-GlcNAc purification has achieved very good results. At the same time, the skeleton structure of polystyrene-divinylbenzene can tolerate the entire pH range, and the chromatography column can be thoroughly cleaned at a high pH in the later stage, which is convenient for repeated industrial use, has a long filler life, and reduces costs.
[0018] More preferably, in step S1, the particle size of the monodisperse microspheres is 30 μm, and the pore size is
[0019] In step S1, the HPLC purity of the crude uridine-5′-diphospho-N-acetyl-glucosamine is 13.41-20.47%, for example, the HPLC purity of the crude product is 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%.
[0020] In step S1, the column volume of the chromatography column is 4-20 mL, for example, the column volume is 4.15 mL, 4.65 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 15.5 mL, 15.7 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL; the sample loading capacity is 2-5 g / L, for example, the sample loading capacity is 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L.
[0021] In step S2, the eluent contains 0.5 to 2 mol of salt solution, for example, the molar number of the salt solution is 0.5 mol, 0.6 mol, 0.7 mol, 0.8 mol, 0.9 mol, 1 mol, 1.1 mol, 1.2 mol, 1.3 mol, 1.4 mol, 1.5 mol, 1.6 mol, 1.7 mol, 1.8 mol, 1.9 mol, 2 mol, and does not contain an organic solvent, is safe, pollution-free and has very low cost.
[0022] Preferably, the salt solution is one of sodium chloride and ammonium chloride.
[0023] In step S2, the flow rate of the eluent is 120 to 360 cm / h, for example, the flow rate of the eluent is 120 cm / h, 140 cm / h, 160 cm / h, 180 cm / h, 200 cm / h, 220 cm / h, 240 cm / h, 260 cm / h, 280 cm / h, 300 cm / h, 320 cm / h, 340 cm / h, and 360 cm / h.
[0024] In step S2, the elution volume of the eluent is 60 to 80 CV, for example, the elution volume of the eluent is 60CV, 61CV, 62CV, 63CV, 64CV, 65CV, 66CV, 67CV, 68CV, 69CV, 70CV, 71CV, 72CV, 73CV, 74CV, 75CV, 76CV, 77CV, 78CV, 79CV, and 80CV.
[0025] As a preferred embodiment of the present invention, the separation and purification method of uridine-5′-diphospho-N-acetyl-glucosamine comprises the following steps:
[0026] a. Loading: Filter the crude uridine-5′-diphospho-N-acetyl-glucosamine with a purity of 13.41-20.47% and load it into a chromatography column, wherein the column volume of the chromatography column is 4-20 mL, the sample loading capacity is 2-5 g / L, and the chromatography column filler is a monodisperse microsphere NanoQ-30L whose matrix is polystyrene-divinylbenzene;
[0027] b. Elution: eluting the UDP-GlcNAc adsorbed in the chromatography column with an eluent, wherein the eluent contains 0.5 to 2 mol of a salt solution and does not contain an organic solvent, the flow rate of the eluent is 120 to 360 cm / h, and the elution volume of the eluent is 60 to 80 CV;
[0028] c. Collection and aggregation: The eluted uridine-5′-diphospho-N-acetyl-glucosamine solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] 1. The separation and purification method of uridine-5′-diphosphate-N-acetyl-glucosamine of the present invention only requires one step of chromatography purification to meet the high purity requirement of uridine-5′-diphosphate-N-acetyl-glucosamine, and the purification yield is high and stable, the purity is above 99%, and the yield is 64.54-75.24%.
[0031] 2. The separation and purification method of uridine-5′-diphosphate-N-acetyl-glucosamine of the present invention is simple and convenient, with a short purification cycle. In the entire purification process, only a salt solution is used as the mobile phase, and no organic solvent is required. It is safe, pollution-free and has a very low cost. The stationary phase used can be reused and can be used for large-scale production, which greatly reduces environmental pressure and production costs. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 This is a scanning electron microscope image of NanoQ-30L polystyrene-divinylbenzene microspheres used in Example 1 of the method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine of the present invention;
[0033] Figure 2 The chromatographic purification chromatogram of Example 1 of the present invention;
[0034] Figure 3 This is a high performance liquid chromatogram of the crude uridine-5′-diphospho-N-acetyl-glucosamine product before purification in Example 1 of the present invention;
[0035] Figure 4 This is a high performance liquid chromatogram of the purified uridine-5′-diphospho-N-acetyl-glucosamine in Example 1 of the present invention. DETAILED DESCRIPTION
[0036] The following is combined with Figure 1-4 The technical solution of the invention is further described through specific embodiments, but the present invention is not limited to these embodiments.
[0037] The present invention provides a method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine, comprising the following steps:
[0038] S1. Loading: Filter the crude uridine-5′-diphospho-N-acetyl-glucosamine and load it into a chromatography column, wherein the chromatography column filler is a monodisperse microsphere whose matrix is polystyrene-divinylbenzene;
[0039] S2, elution: eluting the UDP-GlcNAc adsorbed in the chromatography column with an eluent;
[0040] S3. Collection and aggregation: The eluted uridine-5′-diphospho-N-acetyl-glucosamine solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.
[0041] Example 1
[0042] Take 45mL of crude uridine-5′-diphospho-N-acetyl-glucosamine (14.28% purity, the sample was diluted 5 times with mobile phase A), filter with a pore size of 0.45μm filter membrane, and collect the filtrate for use. Use a 7.7×100mm pre-packed column and NanoQ-30L strong anion exchange microspheres as the chromatography column filler, with a column volume of 4.65mL and a sample load of 5g / L.
[0043] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.
[0044] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of uridine-5′-diphospho-N-acetyl-glucosamine in the eluate was 99.05% and the yield was 70.20%.
[0045] Figure 1 This is a scanning electron microscope image of NanoQ-30L strong anion exchange resin microspheres. Figure 1 It can be seen that the microspheres exhibit a high degree of particle size uniformity.
[0046] Figure 2 is the HPLC chromatogram of crude uridine-5′-diphospho-N-acetyl-glucosamine before purification. Figure 2 It can be seen that the impurity content in the crude uridine-5′-diphosphate-N-acetyl-glucosamine is high, which does not meet the high purity requirement of uridine-5′-diphosphate-N-acetyl-glucosamine.
[0047] Figure 3 is the HPLC chromatogram of purified uridine-5′-diphospho-N-acetyl-glucosamine, Figure 3 It can be seen that the impurity content in uridine-5′-diphosphate-N-acetyl-glucosamine is reduced after purification, which meets the requirement of high purity of uridine-5′-diphosphate-N-acetyl-glucosamine.
[0048] Example 2
[0049] Take 300mL of crude uridine-5′-diphospho-N-acetyl-glucosamine (15.23% purity, the sample was diluted 5 times with mobile phase A), filter with a pore size of 0.45μm filter membrane, and collect the filtrate for use. Use Tricorn10-200 chromatographic column and NanoQ-30L strong anion exchange microspheres as chromatographic column fillers, with a column volume of 15.7mL and a sample load of 5g / L.
[0050] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.
[0051] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of uridine-5′-diphospho-N-acetyl-glucosamine in the eluate was 99.09% and the yield was 72.24%.
[0052] Example 3
[0053] Take 25mL of crude uridine-5′-diphospho-N-acetyl-glucosamine (13.95% purity, the sample was diluted 5 times with mobile phase A), filter with a pore size of 0.45μm filter membrane, and collect the filtrate for use. Use a 7.7×100mm pre-packed column and NanoQ-30L strong anion exchange microspheres as the chromatography column filler, with a column volume of 4.65mL and a sample load of 2g / L.
[0054] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.
[0055] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of uridine-5′-diphospho-N-acetyl-glucosamine in the eluate was 99.21%, and the yield was 71.57%.
[0056] Example 4
[0057] This example uses another strong anion exchange microsphere UniQ-30S as the chromatography column filler, and other purification conditions remain the same as in Example 1.
[0058] Take 45 mL of crude uridine-5′-diphospho-N-acetyl-glucosamine (14.28% purity, the sample was diluted 5 times with mobile phase A), filter it with a 0.45 μm filter membrane, and collect the filtrate for use. Use a 7.7×100 mm pre-packed column, UniQ-30S strong anion exchange microspheres (polyacrylate matrix, particle size about 36 μm, pore size about ) was used as the chromatography column filler, with a column packing volume of 4.65 mL and a sample loading capacity of 5 g / L.
[0059] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.
[0060] The solution of the target peak was collected in sections, and the component liquids meeting the requirements were collected and analyzed by high performance liquid chromatography. The purity of the purified sample UDPG was 98.9% and the yield was 69.2%.
[0061] In summary, the separation and purification method of uridine-5′-diphosphate-N-acetyl-glucosamine of the present invention only requires one step of chromatography purification to meet the high purity requirement of uridine-5′-diphosphate-N-acetyl-glucosamine, and the purification yield is high and stable, the purity is above 99%, and the yield is 64.54-75.24%.
[0062] In addition, the separation and purification method of uridine-5′-diphosphate-N-acetyl-glucosamine of the present invention is simple and convenient, with a short purification cycle, and only a salt solution is used as a mobile phase during the entire purification process without the need to use any organic solvent. It is safe, pollution-free and has very low cost. The stationary phase used can be reused and can be used for large-scale production, greatly reducing environmental pressure and production costs.
[0063] The present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine, characterized in that , the separation and purification method comprises the following steps: S1. Loading: Filter the crude uridine-5′-diphospho-N-acetyl-glucosamine and load it into a chromatography column, wherein the filler of the chromatography column is monodisperse microspheres whose matrix is polyacrylate or polystyrene-divinylbenzene; S2, elution: eluting the uridine-5′-diphospho-N-acetyl-glucosamine adsorbed in the chromatography column with an eluent; S3. Collection and aggregation: The eluted uridine-5′-diphospho-N-acetyl-glucosamine solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.
2. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 1, characterized in that: In step S1, the monodisperse microspheres are microspheres with monodisperse particle sizes and a pore structure.
3. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 1 or 2, characterized in that: In step S1, the particle size of the monodisperse microspheres is 20 to 40 μm, and the pore size is 4. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 3, characterized in that: In step S1, the model of the monodisperse microspheres is UniQ-30S or NanoQ-30L.
5. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 4, characterized in that: In step S1, the HPLC purity of the crude uridine-5′-diphospho-N-acetyl-glucosamine is 13.41-20.47%.
6. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 5, characterized in that: In step S1, the column volume of the chromatography column is 4-20 mL, and the sample loading capacity is 2-5 g / L.
7. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 6, characterized in that: In step S2, the eluent contains 0.5-2 mol of a salt solution and does not contain an organic solvent.
8. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 7, characterized in that: In step S2, the flow rate of the eluent is 120-360 cm / h.
9. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 8, characterized in that: In step S2, the elution volume of the eluent is 50 to 100 CV.
10. The method for separating and purifying uridine-5′-diphospho-N-acetyl-glucosamine according to claim 9, characterized in that: The separation and purification method comprises the following steps: a. Loading: Filter the crude uridine-5′-diphospho-N-acetyl-glucosamine with a purity of 13.41-20.47% and load it into a chromatography column, wherein the column volume of the chromatography column is 4-20 mL, the sample loading capacity is 2-5 g / L, and the chromatography column filler is a monodisperse microsphere NanoQ-30L whose matrix is polystyrene-divinylbenzene; b. Elution: eluting the uridine-5′-diphospho-N-acetyl-glucosamine adsorbed in the chromatography column with an eluent, wherein the eluent contains 0.5 to 2 mol of a salt solution and does not contain an organic solvent, the flow rate of the eluent is 120 to 360 cm / h, and the elution volume of the eluent is 50 to 100 CV; c. Collection and aggregation: The eluted uridine-5′-diphospho-N-acetyl-glucosamine solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.