Method for separating purine and analogues thereof

Abstract

The invention provides a method for separating purine and analogues thereof. The method comprises the following steps: step 1, adding zinc salt into a solution containing purine or / and purine analogues; step 2, adjusting the pH value of the solution to be neutral or above; and step 3, filtering to obtain filtrate. The zinc ion complexing method has the beneficial effects that the zinc ion complexing method is suitable for various catalytic reaction systems, and particularly has a very efficient complexing effect on purine impurities, so that the aim of removing the impurities is fulfilled.

Description

Technical Field

[0001] The present invention relates to the technical field of purification, and in particular to a method for separating purine and its analogs. Background Art

[0002] With the rapid development of small nucleic acid drugs, the demand for raw materials such as nucleosides, such as deoxynucleosides and fluoronucleosides, is increasing. In recent years, with the development of synthetic biology, most nucleosides can be produced by enzyme catalysis. For example, cheap nucleosides can be used to replace the bases on the nucleoside with other bases under the catalysis of enzymes. For example, thymine on β-thymidine is replaced with guanine to form deoxyguanosine, thereby realizing the large-scale production of nucleosides.

[0003] Enzyme-catalyzed production of nucleosides, especially catalytic reactions with purine or its analogs as substrates, will have residues, and the substrate is difficult to separate from the target product in the subsequent purification and separation. The safety and impurity content control of small nucleic acids and mRNA are largely affected by the purity of nucleosides. Since the structure between purines and nucleoside monomers only differs by one furanose (ribose), similar structures often have similar chemical properties. It is difficult to separate purines from purine nucleosides by chromatography or crystallization during separation.

[0004] "Synthesis and characterization of some pyrimidine, purine, amino acid and mixed ligand complexes" discloses that after zinc sulfate is added to adenine, zinc ions can form ligand complexes with adenine. The applicant has found through research that adding zinc ions to the nucleoside reaction solution, and then adjusting the pH and solid-liquid separation can separate purine or purine analogs in a simple and efficient manner to improve the purity of nucleosides. Summary of the invention

[0005] In order to better separate purine or purine analogs, the present invention provides a method for separating purine and its analogs, comprising the following steps:

[0006] Step 1, adding zinc salt to a solution containing purine or / and a purine analog;

[0007] Step 2, adjusting the pH of the solution to neutral or above;

[0008] Step 3, filtering to obtain a filtrate.

[0009] Preferably, the solution in step 1 is obtained by replacing the base in the substrate nucleoside or nucleotide with purine or / and purine analogs under the action of an enzyme, or is obtained by dissolving nucleosides or nucleotides containing purine or / and purine analogs.

[0010] Preferably, the zinc salt is selected from organic zinc salts and / or inorganic zinc salts.

[0011] Preferably, the zinc salt is selected from one or more of zinc sulfate, zinc sulfite, zinc chloride, zinc nitrate, zinc nitrite, zinc acetate, zinc gluconate, and zinc hydroxide.

[0012] Preferably, the zinc salt is selected from zinc sulfate and / or zinc chloride.

[0013] Preferably, in step 2, the pH is adjusted to 7-9, or the pH is adjusted to 7-8.5, or the pH is adjusted to 7-8.4, or the pH is adjusted to 7-8.3, or the pH is adjusted to 7-8.2, or the pH is adjusted to 7-8.1, or the pH is adjusted to 7-8, or the pH is adjusted to 7-7.9, or the pH is adjusted to 7-7.8, or the pH is adjusted to 7-7.7, or the pH is adjusted to 7-7.6, or the pH is adjusted to 7-7.5.

[0014] Preferably, the amount of zinc salt added is such that the molar ratio of zinc ions to purine or / and purine analogs in the solution is 4 to 13:1.

[0015] Preferably, the purine analogue is selected from one or more of adenine, guanine, 2,6-diaminopurine, and 2-amino-6-chloropurine.

[0016] The invention also provides an application of zinc salt in separating purine and its analogs in a solution, wherein the pH of the solution is non-acidic.

[0017] Preferably, the zinc salt is selected from zinc sulfate and / or zinc chloride.

[0018] The zinc salt in the present invention is selected from one or more of the prior art such as zinc sulfate, zinc sulfite, zinc chloride, zinc nitrate, zinc nitrite, zinc acetate, zinc gluconate, zinc hydroxide, etc. In addition, the zinc salt in the present invention also includes the form of hydrates, for example, zinc sulfate refers not only to zinc sulfate itself, but also to the water-containing forms such as zinc sulfate monohydrate and zinc sulfate heptahydrate. The form of water can be bound water or free water.

[0019] The ribose in the nucleoside of the present invention can be ribose or deoxyribose, and the ribose or deoxyribose is obtained by chemical modification, for example, 2' can be fluorine-substituted, methyl-substituted, etc.

[0020] The purine analogs in the present invention refer to purines containing electronegative substituents.

[0021] Purine impurities in the present invention refer to purine and purine analogs.

[0022] In terms of beneficial effects, the zinc ion complexation method is applicable to a variety of catalytic reaction systems, especially for purine impurities, and has a very efficient complexation effect, thereby achieving the purpose of impurity removal.

[0023] The method of the present invention has a stronger impurity removal effect than conventional crystallization or chromatography, and has a high yield, is simpler to operate, and takes a short time, and can be completed within 1-2 hours. Crystallization takes a long time, and chromatography requires packing columns, and the columns need to be treated, and steps such as column washing and elution are required. Relatively speaking, the operation is very cumbersome and takes a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison diagram of the solution containing purine impurities without adjusting the pH (left) and with increasing the pH (right) after adding zinc salt to the solution. DETAILED DESCRIPTION

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0027] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.

[0028] Example 1

[0029] Removal of guanine impurities from deoxyguanosine reaction solution

[0030] Guanine (G) and β-thymidine (dT) are used as substrates, and bases are exchanged under the catalysis of deoxyribosyltransferase to generate thymine (T) and the target product deoxyguanosine (dG). The main impurities in the reaction system are G, T, and dT, and the main purine impurity is unreacted G.

[0031] Take the above catalyst solution, filter and dissolve it to obtain 500 ml of solution (the solution contains about 0.078 g of guanine), add 0.3 g of zinc chloride (the molar ratio of zinc ion to guanine is 4.25:1, the molecular weight of guanine is 151, and the molecular weight of zinc chloride is 136) to the solution, heat to 60°C, adjust the pH to 7.0, stir for 30 minutes and then filter, rinse the filter cake with 20 ml of purified water to obtain a filtrate.

[0032] The solution and the filtrate were detected by HPLC, and the yield of the target product dG was calculated to be 95.58%. The liquid phase detection results of the solution and the filtrate are shown in the following table.

[0033]

[0034] The filtrate from which impurity G is removed is subjected to chromatography, crystallization, etc. to obtain a finished product, which has a heavy metal content including zinc less than 10 ppm after testing.

[0035] Embodiment 2, 3

[0036] Take 1.25g of crude deoxyguanosine containing impurity G, dissolve it in water to 400ml, take two solutions, each containing 200ml (each solution contains 0.026g of guanine), add 0.2g of zinc sulfate heptahydrate (the molar ratio of zinc ion to guanine is 4:1, the molecular weight of guanine is 151, and the molecular weight of zinc sulfate heptahydrate is 287.5) to each, heat to 60℃ to dissolve, adjust pH=7.01 and pH=8.09 respectively, filter, and detect the filtrate by HPLC. The results are shown in the following table.

[0037]

[0038]

[0039] Example 4

[0040] Removal of purine impurities from fluoroguanosine reaction solution

[0041] With 2,6-diaminopurine (2,6-DAP) and 2'-fluoro-2'-deoxyuridine (FDU) as substrates, a deoxyribose transferase and a deaminase undergo a two-step catalytic reaction to generate uracil (U) and a target product 2'-fluoro-2'-deoxyguanosine (FDG). A new impurity guanine (G) is generated in the two-step reaction. The reaction system mainly contains impurities G, 2,6-DAP, U, and FDU, and the main purine impurities are G and 2,6-DAP.

[0042] Take 3500L of the above-mentioned fluoroguanosine catalytic reaction liquid (containing 2.02kg of guanine / purine analogs in total), add 50kg of zinc sulfate heptahydrate (the molar ratio of zinc ion to guanine / purine analogs is 13:1, the molecular weight of guanine is 151, the molecular weight of 2,6-DAP is 150, and the molecular weight of zinc sulfate heptahydrate is 287.5) to the reaction liquid, heat to 60°C, adjust the pH value to 7.5, stir for 30min, and then filter with a plate and frame, rinse the filter cake with 400L of purified water to obtain a filtrate.

[0043] The reaction solution and the filtrate were detected by HPLC, and the yield of the target product FDG was calculated to be 96.32%. The liquid phase detection results of the reaction solution and the filtrate are shown in the following table.

[0044]

[0045] The filtrate from which impurities G and 2,6-DAP are removed is subjected to chromatography, crystallization, etc. to obtain a finished product, which has a heavy metal content including zinc less than 10 ppm according to testing.

[0046] Example 5

[0047] Removal of purine impurities from fluoroadenosine reaction solution

[0048] Adenine (A) and 2'-fluoro-2'-deoxyuridine (FDU) are used as substrates, and uracil (U) and the target product 2'-fluoro-2'-deoxyadenosine (FDA) are generated under the catalysis of deoxyribosyltransferase. The reaction system mainly contains impurities U, A, and FDU, and the main purine impurity is A.

[0049] Take 3000 ml of fluoroadenosine catalytic reaction liquid (containing 1.71 g of adenine), add 40 g of zinc sulfate heptahydrate (the molar ratio of zinc ion to adenine is 11:1, the molecular weight of adenine is 135, and the molecular weight of zinc sulfate heptahydrate is 287.5) to the reaction liquid, heat to 60°C, adjust the pH value to 7.5, stir for 30 minutes and then filter, rinse the filter cake with 300 ml of purified water to obtain a filtrate.

[0050] The reaction solution and the filtrate were detected by HPLC, and the yield of the target product FdA was calculated to be 94.47%.

[0051] The liquid phase detection results of the reaction solution and the filtrate are shown in the following table.

[0052]

[0053] The filtrate from which impurity A is removed is subjected to chromatography, crystallization, etc. to obtain a finished product, which has a heavy metal content including zinc less than 10 ppm after testing.

[0054] Comparative Example 1

[0055] Removal of purine impurities by solution crystallization

[0056] Take 3 g of crude deoxyguanosine containing guanine impurities, dissolve it in 200 ml of water, heat it to 60°C to clarify the solution, cool it to 5°C to crystallize, and filter out the solid.

[0057] The related substances before and after crystallization were detected by HPLC, and the yield of the target product dG was calculated to be 90.58%.

[0058] The liquid phase detection results of crude deoxyguanosine and crystals are shown in the following table.

[0059]

[0060] Comparative Example 2

[0061] Removal of purine impurities by anion resin

[0062] Take 430 ml of the deoxyguanosine reaction solution, filter it, adjust the pH value to 9.4, apply 200 ml of Hz-201 resin (OH type), wash it with water, elute it with sodium chloride, collect the fractions with high main product components, and combine them for treatment.

[0063] The related substances before and after passing through the resin were detected by HPLC, and the yield of the target product dG was calculated to be 91.32%.

[0064] The liquid phase results of the deoxyguanosine reaction solution and the combined solution are shown in the following table.

[0065]

[0066] Purine impurities cannot be removed well by anion resins, and the resin impurity removal method requires a large amount of water washing, and the treatment cycle takes one to several days.

[0067] Comparative Example 3

[0068] Removal of purine impurities by macroporous resin

[0069] Take 1380 ml of the deoxyguanosine reaction solution, filter it, adjust the pH to 8.1, apply 200 ml of LX-818 resin, wash it with water, elute it with 10% ethanol and then with 50% ethanol, collect the fractions with high main product components and combine them.

[0070] The related substances before and after passing through the resin were detected by HPLC, and the yield of the target product dG was calculated to be 93.67%.

[0071] The liquid phase results of the deoxyguanosine reaction solution and the combined solution are shown in the following table.

[0072]

[0073] Purine impurities cannot be removed well by macroporous resins, and the resin impurity removal method requires a large amount of water washing, which requires a treatment cycle ranging from one day to several days.

[0074] Comparative Example 4

[0075] Removal of purine impurities by zinc ions under acidic pH conditions

[0076] Take 1.25g of crude deoxyguanosine containing impurity G, dissolve it in water to 400ml, take two solutions, each containing 200ml (each solution contains 0.026g of guanine), add 0.2g of zinc sulfate heptahydrate (the molar ratio of zinc ion to guanine is 4:1, the molecular weight of guanine is 151, and the molecular weight of zinc sulfate heptahydrate is 287.5), heat to 60℃ to dissolve, do not adjust the pH of the first part (pH=5.04), adjust the pH of the second part to 6.30, filter them separately, and detect the filtrate by HPLC. The results are shown in the following table.

[0077]

[0078]

[0079] The sample without pH adjustment did not produce precipitation, so the impurities in the filtrate did not change.

Claims

1. A method for separating purine and its analogs, characterized in that: The following steps are involved: Step 1, adding zinc salt to a solution containing purine or / and a purine analog; Step 2, adjusting the pH of the solution to neutral or above; Step 3, filtering to obtain a filtrate.

2. The method according to claim 1, characterized in that: The solution in step 1 is obtained by replacing the base in the substrate nucleoside or nucleotide with purine or / and purine analogs under the action of enzymes, or is obtained by dissolving nucleosides or nucleotides containing purine or / and purine analogs.

3. The method according to claim 1, characterized in that: The zinc salt is selected from organic zinc salts and / or inorganic zinc salts.

4. The method according to claim 1, characterized in that: The zinc salt is selected from one or more of zinc sulfate, zinc sulfite, zinc chloride, zinc nitrate, zinc nitrite, zinc acetate, zinc gluconate and zinc hydroxide.

5. The method according to claim 4, characterized in that: The zinc salt is selected from zinc sulfate and / or zinc chloride.

6. The method according to claim 1, characterized in that: In the step 2, the pH is adjusted to 7-9, or the pH is adjusted to 7-8.5, or the pH is adjusted to 7-8.4, or the pH is adjusted to 7-8.3, or the pH is adjusted to 7-8.2, or the pH is adjusted to 7-8.1, or the pH is adjusted to 7-8, or the pH is adjusted to 7-7.9, or the pH is adjusted to 7-7.8, or the pH is adjusted to 7-7.7, or the pH is adjusted to 7-7.6, or the pH is adjusted to 7-7.

5.

7. The method according to claim 1, characterized in that: The amount of zinc salt added is based on the molar ratio of zinc ions to purine or / and purine analogs in the solution = 4 to 13:

1.

8. The method according to claim 1, characterized in that: The purine analogue is selected from one or more of adenine, guanine, 2,6-diaminopurine and 2-amino-6-chloropurine.

9. An application of a zinc salt for separating purine and its analogs in a solution, characterized in that: The pH of the solution is non-acidic.

10. The use according to claim 9, characterized in that: The zinc salt is selected from zinc sulfate and / or zinc chloride.

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