A method for separating purines and analogs thereof

Abstract

The application provides a method for separating purine and analogs thereof, comprising the following steps: step 1, adding a zinc salt into a solution containing purine or / and purine analogs; step 2, adjusting the pH of the solution to be neutral or above; and step 3, filtering to obtain a filtrate. In the beneficial effect aspect, the method of zinc ion complexation is suitable for various catalytic reaction systems, and has very high complexation effect on purine impurities, so that the purpose of impurity removal is achieved.

Description

Technical Field

[0001] This invention relates to the field of purification technology, and more specifically to a method for separating purines and their analogues. Background Technology

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

[0003] Enzymatic production of nucleosides, especially reactions using purines or their analogues as substrates, often leaves residues. These substrates are difficult to separate from the target product during subsequent purification. The safety and impurity control of small nucleic acids and mRNAs are largely influenced by nucleoside purity. Since purines and nucleoside monomers differ in structure by only one furanose (ribose), similar structures often exhibit similar chemical properties. Therefore, it is difficult to separate purines from purine-like nucleosides using methods such as chromatography or crystallization.

[0004] The paper "Synthesis and Characterization of Some Pyrimidine, Purine, Amino Acid, and Mixed Ligand Complexes" discloses that zinc sulfate, when added to adenine, allows zinc ions to form ligand complexes with adenine. The applicant's research has shown that adding zinc ions to the nucleoside reaction solution, followed by pH adjustment and solid-liquid separation, provides a simple and efficient way to separate purines or purine analogues, thereby improving the purity of nucleosides. Summary of the Invention

[0005] To better separate purines or purine analogues, the present invention provides a method for separating purines and their analogues, comprising the following steps:

[0006] Step 1: Add zinc salt to the solution containing purines and / or purine analogues;

[0007] Step 2: Adjust the pH of the solution to neutral or above;

[0008] Step 3: Filter to obtain filtrate.

[0009] Preferably, the solution in step 1 is obtained by replacing the bases in the substrate nucleoside or nucleotide with purines or / and purine analogs under the action of enzymes, or by dissolving nucleosides or nucleotides containing purines 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 7-8.5, or 7-8.4, or 7-8.3, or 7-8.2, or 7-8.1, or 7-8, or 7-7.9, or 7-7.8, or 7-7.7, or 7-7.6, or 7-7.5.

[0014] Preferably, the amount of zinc salt added is based on a molar ratio of zinc ions to purines and / or purine analogs in the solution of 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 present invention also provides an application of zinc salt in the separation of purines and their analogues in a solution with a non-acidic pH.

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

[0018] The zinc salts in this invention are selected from one or more of the existing technologies, such as zinc sulfate, zinc sulfite, zinc chloride, zinc nitrate, zinc nitrite, zinc acetate, zinc gluconate, and zinc hydroxide. Furthermore, the zinc salts in this invention also include hydrated forms; for example, zinc sulfate refers not only to zinc sulfate itself but also to aqueous forms such as zinc sulfate monohydrate and zinc sulfate heptahydrate. The hydrated form can be bound water or free water.

[0019] The ribose in the nucleosides of this invention can be ribose or deoxyribose, which are obtained through chemical modification, such as 2' being fluorine-substituted, methyl-substituted, etc.

[0020] The purine analogues in this invention refer to purines containing electronegative substituents.

[0021] In this invention, purine impurities refer to purines and purine analogues.

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

[0023] The method of this invention has a stronger impurity removal effect and higher yield than conventional crystallization or chromatography. It is also simpler to operate and takes less time, and can be completed within 1-2 hours. Crystallization, on the other hand, requires a longer time, and chromatography requires packing the column with packing material, column treatment, and steps such as washing and elution. Relatively speaking, the operation is very cumbersome and time-consuming. Attached Figure Description

[0024] Figure 1 This is a comparison chart of adding zinc salt to a solution containing purine impurities in this invention, showing the difference between no pH adjustment (left) and pH adjustment (right). Detailed Implementation

[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 commercially available products that can be purchased on the market.

[0027] The present invention will be further described below through embodiments, but these descriptions are not intended to further limit the scope of the invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the present invention still fall within the protection scope of the present invention.

[0028] Example 1

[0029] Removal of guanine impurities from deoxyguanosine reaction solution

[0030] Using guanine (G) and β-thymidine (dT) as substrates, bases are exchanged under the catalysis of deoxyribotransferase to generate thymine (T) and the target product deoxyguanosine (dG). The reaction system mainly contains impurities G, T, and dT, with unreacted G being the main purine impurity.

[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 ions 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℃, adjust the pH to 7.0, stir for 30 min and filter. Rinse the filter cake with 20 ml of purified water to obtain the filtrate.

[0032] The yield of the target product dG was calculated to be 95.58% by HPLC analysis of the solution and filtrate. The HPLC results of the solution and filtrate are shown in the table below.

[0033]

[0034] The filtrate after removing impurity G undergoes chromatography and crystallization to obtain the finished product, which, upon testing, contains less than 10 ppm of heavy metals, including zinc.

[0035] Examples 2 and 3

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

[0037]

[0038]

[0039] Example 4

[0040] Removal of purine impurities from fluoroguanosine reaction solution

[0041] Using 2,6-diaminopurine (2,6-DAP) and 2'-fluoro-2'-deoxyuridine (FDU) as substrates, a two-step catalytic reaction involving a deoxyribotransferase and a deaminase produces uracil (U) and the target product 2'-fluoro-2'-deoxyguanosine (FDG). A new impurity, guanine (G), is generated during the two-step reaction. The reaction system mainly contains impurities G, 2,6-DAP, U, and FDU, with G and 2,6-DAP being the main purine impurities.

[0042] Take 3500L of the above-mentioned fluorinated guanosine catalytic reaction solution (containing 2.02kg of guanine / purine analogue), add 50kg of zinc sulfate heptahydrate (the molar ratio of zinc ions to guanine / purine analogue 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), heat to 60℃, adjust the pH value to 7.5, stir for 30min, filter by plate and frame filter, wash the filter cake with 400L of purified water to obtain the filtrate.

[0043] The yield of the target product FDG was calculated to be 96.32% by HPLC analysis of the reaction solution and filtrate. The HPLC results of the reaction solution and filtrate are shown in the table below.

[0044]

[0045] The filtrate after removing impurities G and 2,6-DAP is subjected to chromatography and crystallization to obtain the finished product, which, after testing, contains less than 10 ppm of heavy metals, including zinc.

[0046] Example 5

[0047] Removal of purine impurities from fluoroadenosine reaction solution

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

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

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

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

[0052]

[0053] The filtrate after removing impurity A undergoes chromatography and crystallization to obtain the finished product, which, upon testing, contains less than 10 ppm of heavy metals, including zinc.

[0054] Comparative Example 1

[0055] Solution crystallization method for removing purine impurities

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

[0057] The yield of the target product dG was calculated to be 90.58% by detecting relevant substances before and after crystallization using HPLC.

[0058] The liquid chromatography results of crude and crystalline deoxyguanosine are shown in the table below.

[0059]

[0060] Comparative Example 2

[0061] Purine impurities are removed using anion exchange resin.

[0062] Take 430 ml of deoxyguanosine reaction solution, filter it, adjust the pH value to 9.4, load it onto 200 ml of Hz-201 resin (OH type), wash with water, elute with sodium chloride, collect the fraction with the highest main product component, and combine them.

[0063] The yield of the target product dG was calculated to be 91.32% by detecting relevant substances before and after resin treatment using HPLC.

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

[0065]

[0066] Anion exchange resins are not effective at removing purine impurities, and resin-based impurity removal requires rinsing with large amounts of water, with treatment cycles ranging from one to several days.

[0067] Comparative Example 3

[0068] Purine impurities removed using macroporous resin

[0069] Take 1380ml of deoxyguanosine reaction solution, filter it, adjust the pH to 8.1, load it onto 200ml of LX-818 resin, wash it with water, elute with 10% ethanol first, then with 50% ethanol, collect the fraction with the higher main product component, and combine them.

[0070] The yield of the target product dG was calculated to be 93.67% by detecting relevant substances before and after resin treatment using HPLC.

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

[0072]

[0073] Macroporous resins cannot effectively remove purine impurities, and resin-based impurity removal requires a large amount of water rinsing, taking anywhere from one to several days for the process.

[0074] Comparative Example 4

[0075] Zinc ions remove purine impurities under acidic pH conditions.

[0076] Take 1.25g of crude deoxyguanosine containing impurity G, dissolve it in water to 400ml, and take two aliquots, each containing 200ml (each aliquot contains 0.026g of guanine). Add 0.2g of zinc sulfate heptahydrate (zinc ion to guanine molar ratio of 4:1, guanine molecular weight 151, zinc sulfate heptahydrate molecular weight 287.5) to each aliquot and heat to 60℃ to dissolve. Do not adjust the pH of the first aliquot (pH=5.04), and adjust the pH of the second aliquot to 6.30. Filter both aliquots and analyze the filtrates by HPLC. The results are shown in the table below.

[0077]

[0078]

[0079] No precipitate was formed in the unadjusted pH sample, therefore the impurities in the filtrate remained unchanged.

Claims

1. A method for separating nucleosides from impurities, characterized in that: Includes the following steps: Step 1: Add zinc salt to the solution containing nucleosides and impurities; Step 2: Adjust the pH of the solution to 7-8.1; Step 3: Filter to obtain filtrate, in which the impurities are separated in solid form; The impurity is a purine analogue; The bases of the nucleoside are derived from purine analogs. The zinc salt is selected from zinc sulfate and / or zinc chloride. The amount of zinc salt added should be based on a molar ratio of zinc ions to purine analogs in the solution of 4~13:

1. The purine analogue is selected from one or more of adenine, guanine, 2,6-diaminopurine, and 2-amino-6-chloropurine.

2. An application of a zinc salt in separating nucleosides from impurities in solution, characterized in that: The solution contains nucleosides and impurities, and the pH of the solution is adjusted to 7-8.1 after the addition of zinc salt; The impurity is a purine analogue; The bases of the nucleoside are derived from purine analogs; The impurities are separated in solid form. The zinc salt is selected from zinc sulfate and / or zinc chloride. The amount of zinc salt added should be based on a molar ratio of zinc ions to purine analogs in the solution of 4~13:

1. The purine analogue is selected from one or more of adenine, guanine, 2,6-diaminopurine, and 2-amino-6-chloropurine.

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