Separation and purification method of uridine diphosphate glucose

Abstract

The invention relates to the technical field of medicine purification, and discloses a uridine diphosphate glucose separation and purification method, which comprises the following steps: S1, loading: filtering a uridine diphosphate glucose crude product, loading the filtered uridine diphosphate glucose crude product into a chromatographic column, and taking polystyrene-divinyl benzene monodisperse microspheres as a matrix as a filler of the chromatographic column; s2, elution: eluting the uridine diphosphate glucose adsorbed in the chromatographic column by using an eluent; and S3, collecting and gathering: carrying out segmented collection on the eluted uridine diphosphate glucose solution, and gathering the component liquid meeting the target peak value requirement. The separation and purification method of uridine diphosphate glucose is simple and convenient, the purification period is short, the requirement for high purity of uridine diphosphate glucose can be met only through one-step chromatographic purification, the purity reaches 99% or above, the purification yield is high and stable, and better use prospects are brought.

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 diphosphate glucose. Background Art

[0002] Uridine diphosphate glucose (UDPG or UDP-Glc) is a sugar nucleotide, a compound formed by the dehydration condensation between the hemiacetal hydroxyl group of glucose and the terminal phosphate group of uridine diphosphate. It is synthesized in the body by uridine diphosphate glucose pyrophosphorylase, and the reaction formula is: It exists in plants, animals and microorganisms, and serves as a glucose donor in the synthesis of sucrose, starch, glycogen and other oligosaccharides and polysaccharides. It can also be converted into uridine diphosphate galactose and uridine diphosphate glucuronic acid.

[0003] UDPG has been used in the development of new drugs and new sweeteners. For example, UDPG is used as a glycosyl donor to synthesize the antibiotic BE-7585A through the catalysis of ORF-36-28 enzyme; 14C-UDPG labeled with the hexacarbon as a glycosyl donor is used to synthesize the main glycoside of stevioside through the catalysis of UDP glycosyltransferase. The application of isotope-labeled UDPG has also broadened the research ideas of glycomics. In addition, UDPG is also a precursor for the synthesis of other nucleoside diphosphate monosaccharides, such as uridine diphosphate galactose, uridine diphosphate gluconic acid, and uridine diphosphate xylose.

[0004] The structural formula of UDP-glucose is as follows:

[0005]

[0006] The main synthesis process of UDPG is to use microbial cell enzymes to synthesize it. At present, there are few reports on the purification process of UDP-glucose in domestic and foreign patents and literature.

[0007] Therefore, in order to obtain high-purity, high-yield UDP-glucose with a short purification cycle and high stability, it is necessary to conduct further research on the fine purification process of UDP-glucose. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for separating and purifying UDP-glucose, which only requires one step of chromatography purification to meet the high purity requirements of UDP-glucose, has a high and stable purification yield, a purity of more than 99%, and a yield of 85% to 95%.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for separating and purifying UDP-glucose, the method comprising the following steps:

[0011] S1. Loading: Filter the crude UDP-glucose product and load it into a chromatography column, wherein the chromatography column filler is monodisperse microspheres whose matrix is ​​polyacrylate or polystyrene-divinylbenzene;

[0012] S2, elution: eluting the UDP-glucose adsorbed in the chromatography column with an eluent;

[0013] S3. Collection and aggregation: The eluted UDP-glucose solution is collected in sections, and the component liquids that meet the target peak requirements are aggregated.

[0014] The separation and purification method of the present invention can meet the high purity requirement of UDP-glucose with only one step of chromatography purification, 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 needed in the entire purification process, so it can be used for large-scale production, which greatly reduces the production cost.

[0015] In step S1, the monodisperse microspheres are microspheres with monodisperse particle sizes and a pore structure.

[0016] 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

[0017] Preferably, in step S1, the monodisperse microspheres are of model NanoQ-30L, with a particle size of 30 μm and a pore size of

[0018] 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 a polyacrylate or polystyrene-divinylbenzene. The molecular formula of uridine diphosphate glucose contains two phosphate groups. Preferably, under the selected elution conditions, the use of NanoQ-30L for UDPG 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.

[0019] More preferably, in step S1, the particle size of the monodisperse microspheres is 30 μm, and the pore size is

[0020] In step S1, the HPLC purity of the crude UDP-glucose is 74.28-84.50%, for example, the HPLC purity of the crude product is 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77%, 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%.

[0021] In step S1, the packing volume of the chromatography column is 4-20 mL, for example, the packing 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 5-10 g / L, for example, the sample loading capacity is 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L.

[0022] 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.

[0023] Preferably, the salt solution is one of sodium chloride and ammonium chloride.

[0024] 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.

[0025] 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.

[0026] As a preferred embodiment of the present invention, the method for separating and purifying UDP-glucose comprises the following steps:

[0027] a. Loading: Filter the crude uridine diphosphate glucose with a purity of 74.28-84.5% and load it into a chromatography column, wherein the column volume of the chromatography column is 4-20 mL, the sample loading capacity is 5-10 g / L, and the chromatography column filler is a monodisperse microsphere NanoQ-30L whose matrix is ​​polystyrene-divinylbenzene;

[0028] b. Elution: eluting the UDPG adsorbed in the chromatography column with an eluent, wherein the eluent contains 0.5-2 mol of a salt solution and does not contain an organic solvent, the flow rate of the eluent is 120-360 cm / h, and the elution volume of the eluent is 60-80 CV;

[0029] c. Collection and summary: The eluted UDP-glucose solution is collected in sections, and the component liquids that meet the target peak requirements are summarized.

[0030] Compared with the prior art, the present invention provides a method for separating and purifying UDP-glucose, which has the following beneficial effects:

[0031] 1. The separation and purification method of UDP-glucose of the present invention only needs one step of chromatography purification to meet the high purity requirement of UDP-glucose, and the purification yield is high and stable, the purity is above 99%, and the yield is 89.54-95.24%.

[0032] 2. The separation and purification method of UDP-glucose 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. The method is safe, pollution-free and 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

[0033] 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 UDP-glucose of the present invention;

[0034] Figure 2The chromatographic purification chromatogram of Example 1 of the present invention;

[0035] Figure 3 The HPLC chromatogram of the crude uridine diphosphate glucose product purified from Example 1 of the present invention;

[0036] Figure 4 The figure is a high performance liquid chromatogram of UDP-glucose after purification in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] 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.

[0038] A method for separating and purifying UDP-glucose of the present invention comprises the following steps:

[0039] S1. Loading: Filter the crude UDP-glucose product and load it into a chromatography column, wherein the chromatography column filler is a monodisperse microsphere whose matrix is ​​polystyrene-divinylbenzene;

[0040] S2, elution: eluting the UDPG adsorbed in the chromatography column with an eluent;

[0041] S3. Collection and aggregation: The eluted UDP-glucose solution is collected in sections, and the component liquids that meet the target peak requirements are aggregated.

[0042] Example 1

[0043] Take 45mL of crude UDP-glucose (74.28% purity, the sample was diluted 5 times with mobile phase A), filter with a 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 10g / L.

[0044] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0045] 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 UDP-glucose in the eluate was 99.05% and the yield was 90.20%.

[0046] 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.

[0047] Figure 2is the HPLC chromatogram of crude UDP-glucose before purification. Figure 2 It can be seen that the impurity content in the crude UDP-glucose is high, which does not meet the high purity requirements of UDP-glucose.

[0048] Figure 3 This is the HPLC chromatogram of purified UDP-glucose. Figure 3 It can be seen that the impurity content in UDP-glucose is reduced after purification, which meets the high purity requirement of UDP-glucose.

[0049] Example 2

[0050] Take 300mL of crude UDP-glucose (75.23% purity, the sample was diluted 5 times with mobile phase A), filter with a 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 10g / L.

[0051] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0052] 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 UDP-glucose in the eluate was 99.09% and the yield was 92.24%.

[0053] Example 3

[0054] Take 25mL of crude UDP-glucose (73.95% purity, the sample was diluted 5 times with mobile phase A), filter with a 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.

[0055] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0056] 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 UDP-glucose in the eluate was 99.41%, and the yield was 91.57%.

[0057] Example 4

[0058] 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.

[0059] Take 45 mL of crude UDP-glucose (74.28% 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 later 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 10 g / L.

[0060] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0061] 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 89.2%.

[0062] In summary, the separation and purification method of UDP-glucose of the present invention can meet the high purity requirement of UDP-glucose with only one step of chromatography purification, and the purification yield is high and stable, the purity is above 99%, and the yield is 89.54-95.24%.

[0063] In addition, the separation and purification method of UDP-glucose 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.

[0064] 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 UDP-glucose, characterized in that: The separation and purification method comprises the following steps: S1. Loading: Filter the crude UDP-glucose product and load it into a chromatography column, the filler of which is monodisperse microspheres whose matrix is ​​polyacrylate or polystyrene-divinylbenzene; S2, elution: eluting the UDP-glucose adsorbed in the chromatography column with an eluent; S3. Collection and aggregation: The eluted UDP-glucose solution is collected in sections, and the component liquids that meet the target peak requirements are aggregated.

2. A method for separating and purifying UDP-glucose according to claim 1, characterized in that: In step S1, the monodisperse microspheres are microspheres with monodisperse particle sizes and a pore structure.

3. A method for separating and purifying UDP-glucose according to claim 1 or 2, characterized in that: In step S1, the particle size of the monodisperse microspheres is 20-40 μm, and the pore size is 500- 4. A method for separating and purifying UDP-glucose according to claim 1, characterized in that: In step S1, the model of the monodisperse microspheres is UniQ-30S or NanoQ-30L.

5. A method for separating and purifying UDP-glucose according to claim 4, characterized in that: In step S1, the HPLC purity of the crude UDP-glucose is 74.28-84.5%.

6. A method for separating and purifying UDP-glucose 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 5-10 g / L.

7. A method for separating and purifying UDP-glucose 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. A method for separating and purifying UDP-glucose according to claim 7, characterized in that: In step S2, the flow rate of the eluent is 120-360 cm / h.

9. A method for separating and purifying UDP-glucose 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 UDP-glucose according to claim 9, characterized in that: The separation and purification method comprises the following steps: a. Loading: Filter the crude uridine diphosphate glucose with a purity of 74.28-84.5% and load it into a chromatography column, wherein the column volume of the chromatography column is 4-20 mL, the sample loading capacity is 5-10 g / L, and the chromatography column filler is a monodisperse microsphere NanoQ-30L whose matrix is ​​polystyrene-divinylbenzene; b. Elution: eluting the UDP-glucose 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 summary: The eluted UDP-glucose solution is collected in sections, and the component liquids that meet the target peak requirements are summarized.

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