Chromatographic method for separating and purifying deoxyribonucleoside

Abstract

The invention provides a chromatographic method for separating and purifying deoxyribonucleoside. The chromatographic method comprises the following steps: 1) preparing a crude product containing deoxyribonucleoside and ribonucleoside into an upper column liquid; (2) enabling the upper column liquid to pass through a chromatographic column; 3) washing the chromatographic column with a tetraboric acid solution; and 4) eluting the chromatographic column with ethanol to obtain an eluent. The impurity removal mode is simple, convenient and easy to operate, low in cost and very suitable for large-scale production, the impurity removal efficiency can be greatly improved, and the production efficiency is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification, and particularly relates to a chromatography 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 a wide range of applications in the pharmaceutical industry. It can also be directly used to prepare combined deoxynucleoside drugs or as a chemical reagent for biochemical research. In addition, it can 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-mentioned products or remaining substrates, a large amount of guanosine impurities will also be generated 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. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a chromatography method for separating and purifying deoxyribonucleosides, including:

[0006] 1), preparing a loading solution by mixing a crude product containing deoxyribonucleosides and ribonucleosides;

[0007] 2), passing the loading solution through a chromatographic column;

[0008] 3), eluting the chromatographic column with a tetraboric acid solution;

[0009] 4), eluting the chromatographic column with ethanol to obtain an eluate.

[0010] Preferably, the chromatographic column is filled with macroporous adsorption resin.

[0011] Preferably, the deoxyribonucleoside is deoxyguanosine, and the ribonucleoside is guanosine.

[0012] Preferably, the tetraboric acid solution is prepared from sodium tetraborate.

[0013] Preferably, the tetraboric acid solution is prepared according to the ratio of adding 0.05 - 0.5 g of sodium tetraborate to 100 ml of solvent.

[0014] Preferably, the tetraboric acid solution is prepared according to the ratio of adding 0.1 - 0.5 g of sodium tetraborate to 100 ml of solvent.

[0015] Preferably, the tetraboric acid solution is prepared by adding 0.1 - 0.4 g of sodium tetraborate to 100 ml of solvent.

[0016] Preferably, the tetraboric acid solution is prepared by adding 0.1 - 0.3 g of sodium tetraborate to 100 ml of solvent.

[0017] Preferably, the tetraboric acid solution is prepared by adding 0.1 - 0.2 g of sodium tetraborate to 100 ml of solvent.

[0018] Preferably, after washing the column with the tetraboric acid solution, the column is washed with water.

[0019] Preferably, after washing the column with water, it is eluted with an ethanol solution.

[0020] Preferably, the resin column is selected from one or a combination of HZ - 816, HZ - 818, LX - T81, LX - 818, LX - 207HF, and H103.

[0021] Preferably, the loading amount on the column is 5 - 25 g / L.

[0022] Preferably, the loading amount on the column is 10 - 20 g / L.

[0023] Preferably, the volume of the tetraboric acid solution for washing the column is 4 - 12 times the volume of the chromatographic column.

[0024] Preferably, the volume concentration of the ethanol solution is 5 - 50%.

[0025] The present invention also provides a chromatographic method for separating and purifying deoxyribonucleosides, comprising:

[0026] 1), preparing a loading solution from a crude product containing deoxyribonucleosides and ribonucleosides;

[0027] 2), mixing the loading solution with a macroporous adsorption resin, and loading it into a chromatographic column after adsorption is completed;

[0028] 3), eluting the chromatographic column with a tetraboric acid solution;

[0029] 4), eluting the chromatographic column with ethanol to obtain an eluate;

[0030] Other chromatographic conditions can be selected and combined according to any of the conditions mentioned above.

[0031] Loading the loading solution directly into the chromatographic column or mixing it with the macroporous adsorption resin for adsorption and then loading it into the chromatographic column can achieve similar effects, and both can adsorb the substances in the loading solution.

[0032] The beneficial effects of the present invention are as follows: By first loading the column with the loading solution and then eluting and removing impurities with a borate solution, the present invention can effectively remove guanosine (GR). However, when the loading solution and the borate solution are first complexed and then chromatographed, guanosine (GR) cannot be effectively removed. In addition, the present invention is suitable for different macroporous resins, and using sodium tetraborate solution to wash impurities can significantly remove guanosine (GR). The impurity removal method of the present invention is simple and easy to operate, with low cost, very suitable for large-scale production, can greatly improve the impurity removal efficiency, and further improve the production efficiency. Detailed implementation manners

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0034] 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.

[0035] The present invention will be further described through examples below, 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.

[0036] Example 1

[0037] Step 1: Take 200 mL of 2'-deoxyguanosine dissolution solution, filter and dilute it to 2000 mL (containing 2.05 g of deoxyguanosine) to prepare a loading solution, and the loading amount on the column is 10.25 g / L;

[0038] Step 2: The loading solution is loaded onto a 200 ml HZ-816 resin column (Flash column) at a flow rate of 10 ml / min;

[0039] Step 3: Wash the column with 1600 mL of 0.1% sodium tetraborate solution;

[0040] Step 4: After washing the column with 600 mL of water, change to 50% ethanol for elution, collect the qualified fractions, and merge them.

[0041] The liquid phase detection results of the loading solution are as follows in the table:

[0042] Serial number Elution time (min) Peak area percentage (%) Substance name 1 3.700 0.25 Guanine (G) 2 3.979 1.17 Guanosine (GR) 3 5.342 97.94 2'-Deoxyguanosine (dG)

[0043] The liquid phase detection results of the elution and merger solution are as follows in the table:

[0044] Serial number Elution time (min) Peak area percentage (%) Substance name 1 / / G 2 3.941 0.02 GR 3 5.282 99.52 dG

[0045] Example 2

[0046] Step 1: Take 500 mL of 2'-deoxyguanosine catalytic solution, filter and dilute it to 3400 mL (containing 3.48 g of deoxyguanosine) to prepare the loading solution, and the loading amount is 17.4 g / L;

[0047] Step 2: Load the loading solution onto a 200 mL LX-T81 resin column (Flash column) at a flow rate of 10 ml / min;

[0048] Step 3: Wash the column with 1800 mL of 0.2% sodium tetraborate solution;

[0049] Step 4: After washing the column with 600 mL of water, change to 10% ethanol for elution, collect the qualified fractions, and combine them.

[0050] The liquid phase detection results of the loading solution are as follows:

[0051] Serial number Elution time (min) Peak area percentage (%) Substance name 1 3.672 1.46 G 2 3.902 5.65 GR 3 5.234 91.66 dG

[0052] The liquid phase detection results of the elution and combined solution are as follows:

[0053] Serial number Elution time (min) Peak area percentage (%) Substance name 1 3.610 0.02 G 2 3.858 0.11 GR 3 5.156 98.62 dG

[0054] Example 3

[0055] Step 1: Take 500 mL of 2'-deoxyguanosine catalytic solution, filter and dilute it to 3400 mL (containing 3.48 g of deoxyguanosine) to prepare the loading solution, and the loading amount is 17.4 g / L;

[0056] Step 2: Load the loading solution onto a 200 mL H-103 resin column (Flash column) at a flow rate of 10 ml / min;

[0057] Step 3: Wash the column with 1000 mL of 0.2% sodium tetraborate solution;

[0058] Step 4: After washing the column with 600 mL of water, change to 10% ethanol for elution, collect the qualified fractions, and combine them.

[0059] The liquid phase detection results of the loading solution are as follows:

[0060] Serial number Elution time (min) Peak area percentage (%) Substance name 1 3.672 1.46 G 2 3.902 5.65 GR 3 5.234 91.66 dG

[0061] The liquid phase detection results of the elution and combined solution are as follows:

[0062] Serial number Elution time (min) Peak area percentage (%) Substance name 1 4.136 0.97 G 2 4.730 1.55 GR 3 5.410 97.34 dG

[0063] Comparative Example 1

[0064] Step 1: Take 800 mL of 2'-deoxyguanosine catalytic solution (containing 1.75 g of deoxyguanosine), add 4.8 g of sodium tetraborate solid, dissolve and complex, filter, and prepare the loading solution. The loading amount on the column is 8.75 g / L;

[0065] Step 2: The loading solution is loaded onto a 200 mL LX-201 resin column at a flow rate of 10 ml / min;

[0066] Step 3: After washing the column with 1600 mL of water, elute with 0.2 M, 0.5 M, and 1 M sodium chloride in three gradients, collect the qualified fractions, and combine them.

[0067] The liquid phase detection results of the loading solution are as follows:

[0068] Serial number Elution time (min) Peak area percentage (%) Substance name 1 3.676 4.33 G 2 3.977 5.24 GR 3 5.321 88.76 dG

[0069] The liquid phase detection results of the elution and combined solution are as follows:

[0070] Serial number Elution time (min) Peak area percentage (%) Substance name 1 3.717 1.06 G 2 4.011 6.71 GR 3 5.360 92.19 dG

[0071] First add sodium tetraborate to the loading solution, and then pass through the ion exchange column. There is no removal effect on the impurity GR.

[0072] Comparative Example 2

[0073] Step 1: Take 500 mL of 2'-deoxyguanosine catalytic solution (containing 1.18 g of deoxyguanosine), add 3.6 g of sodium tetraborate solid, dissolve and complex, filter, and prepare the loading solution. The loading amount on the column is 5.9 g / L;

[0074] Step 2: The loading solution is loaded onto a 200 mL LX-T81 resin column at a flow rate of 10 ml / min;

[0075] Step 3: After washing the column with 1600 mL of water, elute with 10% ethanol, collect the qualified fractions, and combine them.

[0076] The liquid phase detection results of the loading solution are as follows:

[0077] Serial number Elution time (min) Peak area percentage (%) Substance name 1 4.276 1.02 G 2 4.858 1.58 GR 3 5.154 91.48 dG

[0078] The liquid phase detection results of the elution and combined solution are as follows:

[0079] Serial number Elution time (min) Peak area percentage (%) Substance name 1 4.409 0.58 G 2 4.910 1.47 GR 3 5.233 92.42 dG

[0080] First add sodium tetraborate to the loading solution, and then load it onto the macroporous resin column. The removal effect of GR impurities is not obvious.

Claims

1. A chromatographic method for separating and purifying deoxyribonucleosides, characterized in that: It includes: 1), preparing a column loading solution from a crude product containing deoxyribonucleosides and ribonucleosides; 2), passing the column loading solution through a chromatographic column; 3), eluting the chromatographic column with a tetraboric acid solution; 4), eluting the chromatographic column with ethanol to obtain an eluate.

2. The chromatographic method according to claim 1, characterized in that: The chromatographic column is filled with macroporous adsorption resin.

3. The chromatographic method according to claim 2, characterized in that: The deoxyribonucleoside is deoxyguanosine and the ribonucleoside is guanosine.

4. The chromatographic method according to claim 3, characterized in that: The tetraboric acid solution is prepared from sodium tetraborate.

5. The chromatographic method according to claim 3, characterized in that: The tetraboric acid solution is prepared according to the ratio of adding 0.05 - 0.5 g of sodium tetraborate to 100 ml of solvent; Or, the tetraboric acid solution is prepared according to the ratio of adding 0.1 - 0.5 g of sodium tetraborate to 100 ml of solvent; Or, the tetraboric acid solution is prepared according to the ratio of adding 0.1 - 0.4 g of sodium tetraborate to 100 ml of solvent; Or, the tetraboric acid solution is prepared according to the ratio of adding 0.1 - 0.3 g of sodium tetraborate to 100 ml of solvent; Or, the tetraboric acid solution is prepared according to the ratio of adding 0.1 - 0.2 g of sodium tetraborate to 100 ml of solvent.

6. The chromatographic method according to claim 3, characterized in that: The resin column is selected from one or a combination of HZ - 816, HZ - 818, LX - T81, LX - 818, LX - 207HF, H103.

7. The chromatographic method according to claim 3, characterized in that: The column loading amount is 5 - 25 g / L.

8. The chromatographic method according to claim 3, characterized in that: The volume of the tetraboric acid solution for washing the column is 4 - 12 times the volume of the chromatographic column.

9. The chromatographic method according to claim 3, characterized in that: The volume concentration of the ethanol solution is 5 - 50%.

10. A chromatographic method for separating and purifying deoxyribonucleosides, characterized in that: It includes: 1), preparing a column loading solution from a crude product containing deoxyribonucleosides and ribonucleosides; 2), mixing the column loading solution with macroporous adsorption resin, and loading it into a chromatographic column after adsorption; 3), eluting the chromatographic column with a tetraboric acid solution; 4), eluting the chromatographic column with ethanol to obtain an eluate; Other chromatographic conditions are as described in any one of claims 3 - 9.