Crystallization method for separating and purifying deoxyribonucleoside

Abstract

The complex crystallization method is adopted, rapid separation of guanosine impurities can be achieved, the purity of deoxyguanosine reaches 98% or above, and the method is simple, convenient and easy to operate and quite suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification, and particularly relates to a crystallization method for separating and purifying deoxyribonucleosides. Background Art

[0002] As an important raw material and intermediate for synthesizing antiviral and antitumor nucleic acid drugs such as oligodeoxynucleotides, 2'-deoxyguanosine has wide applications in the pharmaceutical industry, and can also be directly used to prepare combined deoxynucleoside drugs or used as a chemical reagent for biochemical research; in addition, it can also be used as an intermediate for synthesizing some antiviral nucleoside drugs and molecular markers.

[0003] At present, the large-scale production of 2'-deoxyguanosine all adopts the method of biocatalysis. Specifically, β-thymidine and disodium guanylate are used as substrates for catalysis to convert into 2'-deoxyguanosine and thymine. In addition to the above products or remaining substrates, a large amount of guanosine impurities will also be produced in this catalytic reaction. Compared with the purification of other nucleosides, it is more difficult to separate and purify 2'-deoxyguanosine by conventional means.

[0004] For guanosine impurities, due to their extremely similar structures to 2'-deoxyguanosine, it is very difficult to use conventional methods for separation and purification, and the separation effect is not ideal. After repeated experiments and condition exploration by the applicant, the method of complexation crystallization is adopted, which can achieve the rapid separation of guanosine impurities, and the purity of deoxyguanosine reaches more than 98%. Moreover, the method itself is simple and easy to operate, and is very suitable for industrial production. Summary of the Invention

[0005] In order to achieve the above object, the present invention provides a crystallization method for separating and purifying deoxyribonucleosides, which is used to separate ribonucleosides in deoxyribonucleosides, and the method at least includes the following steps:

[0006] Step 1, dissolving the raw material of deoxyribonucleoside to be purified into a solution;

[0007] Step 2, adding tetraboric acid or its salt to the solution;

[0008] Step 3, cooling for crystallization.

[0009] Preferably, by mass, the mass fraction of ribonucleoside is 1, and the mass fraction of tetraboric acid or its salt is 2-15, or 3-14, or 4-13, or 4-12, or 4-11, or 4-10, or 5-10, or 6-10, or 7-10, or 7-9.

[0010] Preferably, the tetraborate is sodium tetraborate.

[0011] Preferably, the final temperature of cooling is lower than 20°C, or lower than 10°C, or 0-10°C, or 0-5°C.

[0012] Preferably, the solution concentration in step 1 is: for every 1 ml of solution, 0.01 - 0.1 g, or 0.01 - 0.05 g of deoxyribonucleoside raw material is added.

[0013] Preferably, the deoxyribonucleoside is 2'-deoxyguanosine and the ribonucleoside is guanosine.

[0014] The beneficial effects of the present invention are as follows: by adding tetraborate, it is easier to separate from deoxynucleoside impurities, thereby obtaining a high-purity product. The operation of the present invention is simple and the cost is low, which is very suitable for large-scale production, can greatly improve the impurity removal efficiency, and further improve the production efficiency.

[0015] The present invention is particularly suitable for rapidly separating guanosine from 2'-deoxyguanosine, and the purity and yield after separation are high. Specific Embodiments

[0016] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0017] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all ordinary commercially available products and can be purchased in the market.

[0018] The present invention will be further described below through examples, and these descriptions do not further limit the content of the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention.

[0019] Example 1

[0020] Step 1: Take 0.64 g of 2'-deoxyguanosine raw material (about containing 0.045 g of guanosine impurity), and dissolve it in 40 mL of purified water at 60°C;

[0021] Step 2: Add 0.35 g of sodium tetraborate solid (the mass ratio of sodium tetraborate to guanosine is 7.78:1) to the solution in Step 1, and stir until completely dissolved;

[0022] Step 3: Cool down to 5°C to precipitate solids, filter out the solids, wash them with 5 mL of 90% ethanol, and dry to obtain 0.49 g of solids. The solids are sampled for liquid phase detection.

[0023] The liquid phase detection results of the raw materials and the crystalline products are as follows in the table:

[0024]

[0025] Example 2

[0026] Step 1: Take 4.80 g of 2'-deoxyguanosine raw material (containing approximately 0.061 g of guanosine impurity), and dissolve it in 300 mL of purified water at 60°C;

[0027] Step 2: Add 0.50 g of sodium tetraborate solid to the solution in Step 1 (the mass ratio of sodium tetraborate to guanosine is 8.20:1), and stir until completely dissolved;

[0028] Step 3: Cool down to 5°C to precipitate solids, filter out the solids, wash them with 20 mL of 90% ethanol, and dry to obtain 4.0 g of solids. Take samples of the solids for liquid phase detection.

[0029] The liquid phase detection results of the raw materials and the crystalline products are as follows in the table:

[0030]

[0031] Example 3

[0032] Step 1: Take 93 g of 2'-deoxyguanosine raw material (containing approximately 1.20 g of guanosine impurity), and dissolve it in 2000 mL of purified water at 60°C;

[0033] Step 2: Add 10 g of sodium tetraborate solid to the solution in Step 1 (the mass ratio of sodium tetraborate to guanosine is 8.33:1), and stir until completely dissolved;

[0034] Step 3: Cool down to 5°C to precipitate solids, filter out the solids, wash them with 100 mL of 90% ethanol, and dry to obtain 78 g of solids. Take samples of the solids for liquid phase detection.

[0035] The liquid phase detection results of the raw materials and the crystalline products are as follows in the table:

[0036]

[0037] Example 4

[0038] Step 1: Take 3.02 g of 2'-deoxyguanosine raw material (containing approximately 0.039 g of guanosine impurity), and dissolve it in 100 mL of purified water at 60°C;

[0039] Step 2: Add 0.10 g of sodium tetraborate solid to the solution in Step 1 (the mass ratio of sodium tetraborate to guanosine is 2.56:1), and stir until completely dissolved;

[0040] Step 3: Cool down to 5°C to precipitate solids, filter out the solids, wash them with 10 mL of 90% ethanol, and dry to obtain 2.58 g of solids. Take samples of the solids for liquid phase detection.

[0041] The liquid phase detection results of the raw materials and the crystalline products are as follows in the table:

[0042]

[0043]

[0044] Comparative Example 1

[0045] Step 1: Take 1.28 g of 2'-deoxyguanosine raw material (containing about 0.020 g of guanosine impurities), and dissolve it in 100 mL of purified water at 60 °C;

[0046] Step 2: Cool down to 5 °C to precipitate solids, filter out the solids, wash them with 10 mL of 90% ethanol, and dry to obtain 1.17 g of solids. Take samples of the solids for liquid phase detection.

[0047] The liquid phase detection results of the raw materials and the crystalline products are as follows in the table:

[0048]

[0049] When sodium tetraborate is not added, the content of impurities (GR) increases instead.

[0050] Comparative Example 2

[0051] Step 1: Take 3.05 g of 2'-deoxyguanosine raw material (containing about 0.039 g of guanosine impurities), and dissolve it in 100 mL of purified water at 60 °C;

[0052] Step 2: Add 0.70 g of solid sodium tetraborate (the mass ratio of sodium tetraborate to guanosine is 17.95:1) to the solution in Step 1, and stir until completely dissolved;

[0053] Step 3: Cool down to 5 °C to precipitate solids, filter out the solids, wash them with 10 mL of 90% ethanol, and dry to obtain 1.48 g of solids. Take samples of the solids for liquid phase detection.

[0054] The liquid phase detection results of the raw materials and the crystalline products are as follows in the table:

[0055]

[0056] When the mass ratio of added sodium tetraborate to GR is too high, the crystallization yield is low.

Claims

1. A crystallization method for separating and purifying deoxyribonucleosides, characterized in that: For separating ribonucleosides from deoxyribonucleosides, the method at least includes the following steps: Step 1, dissolving the raw material of deoxyribonucleoside to be purified into a solution; Step 2, adding tetraboric acid or its salt to the solution; Step 3, cooling for crystallization.

2. The crystallization method according to claim 1, characterized in that: Calculated by mass fraction, the mass fraction of ribonucleoside is 1, and the mass fraction of tetraboric acid or its salt is 2 - 15, or 3 - 14, or 4 - 13, or 4 - 12, or 4 - 11, or 4 - 10, or 5 - 10, or 6 - 10, or 7 - 10, or 7 - 9.

3. The crystallization method according to claim 2, characterized in that: The tetraborate is sodium tetraborate.

4. The crystallization method according to claim 1, characterized in that: The final temperature of cooling is lower than 20 °C, or lower than 10 °C, or 0 - 10 °C, or 0 - 5 °C.

5. The crystallization method according to claim 1, characterized in that: The concentration of the solution in Step 1 is: for every 1 ml of solution, 0.01 - 0.1 g, or 0.01 - 0.05 g of the raw material of deoxyribonucleoside is added.

6. The crystallization method according to any one of claims 1 - 5, characterized in that: The deoxyribonucleoside is 2'-deoxyguanosine, and the ribonucleoside is guanosine.

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