Pharmaceutical Nucleic Acid Synthesis Process

Abstract

The present invention discloses a pharmaceutical nucleic acid synthesis process. In the synthesis process, the present invention temporarily protects unnecessary groups as much as possible. After a round of coupling reaction, the protecting groups on the previous round of groups are selectively removed to form a specific phosphodiester bond, thereby reducing side reactions as much as possible, improving the yield of the target product, and reducing the difficulty of purification. The present invention can achieve no transfer of the synthesis column during the aminolysis process by simply transforming the synthesis column, online aminolysis, elution of the oligonucleic acid product loaded on the surface of the solid phase support in the synthesis column, collection of the eluate containing the oligonucleic acid product and subsequent purification process. Since the synthesis column does not need to be offline during the whole process, the difficulty and complexity of the work are greatly reduced on the one hand, and the exhaust gas leakage during the disassembly and installation process is significantly reduced on the other hand.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical nucleic acid synthesis process. Background Art

[0002] Medicinal nucleic acids are widely used in the development of small nucleic acid drugs and new vaccine adjuvants. In recent years, the third generation of drugs based on nucleic acid therapy has developed rapidly. The characteristics of nucleic acid drugs are that they can directly bind to pathogenic genes and prevent their translation, thereby preventing the production of pathogenic proteins. Compared with small molecule drugs and protein antibody drugs, nucleic acid drugs can be precisely guided to reach the target directly and are easy to scale up industrial production.

[0003] In the production process of pharmaceutical nucleic acids, the backbone is a 3'→5' phosphodiester bond between adjacent nucleotides. Since nucleotides are molecules with multiple functional groups and the artificial chemical synthesis of nucleic acid molecules is a multi-step continuous reaction, there are many side reactions during the reaction process, which will significantly reduce the yield of the target product, increase the difficulty of purification, and reduce production efficiency and product quality. Therefore, in the actual production process, it is necessary to inhibit the occurrence of side reactions to achieve the effect of increasing the yield of the target product, reducing the difficulty of purification, and improving production efficiency. In order to solve the above problems, the present invention provides the following technical solutions. Summary of the invention

[0004] The purpose of the present invention is to provide a pharmaceutical nucleic acid synthesis process to solve the problem that many by-products in the pharmaceutical nucleic acid preparation process in the prior art affect the purification results and purification efficiency.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A process for synthesizing pharmaceutical nucleic acid, comprising the following steps:

[0007] The first step is to add the prepared raw materials into a fully automatic synthesizer and perform the synthesis operation under normal temperature and fully closed conditions. The entire synthesis cycle is 24 hours;

[0008] The chain extension reaction process extends from 3'-5' direction, including four steps of deprotection, activation coupling, sulfurization / oxidation, and capping. After the four steps are completed, the next round of chain extension reaction is carried out;

[0009] The deprotection reaction comprises: removing the protecting group DMT on the nucleotide with dichloroacetic acid to obtain the 5'-hydroxyl end, i.e., the nucleotide monomer, for the next coupling step;

[0010] The activated coupling reaction comprises: before coupling, the 5'-hydroxyl end protected by phosphoramidite is mixed with an activating agent ethylthiotetrazolyl to obtain a nucleoside phosphorous acid activated intermediate, whose 3' end is activated, the 5'-hydroxyl is protected by DMT, and a condensation reaction occurs with a free 5'-hydroxyl in the solution;

[0011] The sulfurization / oxidation reaction comprises: under the action of a sulfurizing agent, phenylacetyl disulfide, or an oxidizing agent, iodine solution, the phosphorous acid form is converted into a phosphate triester;

[0012] The capping reaction comprises: terminating the subsequent reaction of the 5'-hydroxyl group with acetic anhydride;

[0013] The second step is amine washing;

[0014] The third step is aminolysis;

[0015] Step 4: purification;

[0016] Step 5: Ultrafiltration;

[0017] Step 6: freeze-drying to obtain the finished product.

[0018] As a further embodiment of the present invention, the vulcanization reaction and the oxidation reaction are not carried out simultaneously in a single chain extension reaction process, and the reaction times are the same in a single batch production process.

[0019] As a further embodiment of the present invention, in the activated coupling reaction, ethylthiotetrazolyl is used in excess.

[0020] As a further solution of the present invention, the product is washed with diethylamine to remove the protective group cyanoethyl. The washing time of the diethylamine and acetonitrile mixed solution in the amine washing process is about 10 minutes.

[0021] As a further solution of the present invention, after the amine washing is completed, acetonitrile is used to clean the synthesizer and the synthesis column in the synthesizer.

[0022] As a further scheme of the present invention, the aminolysis is to transfer the reaction product in the synthesis column together with the solid phase carrier into an aminolysis tank, use nitrogen to pressurize 25% ammonia water to the aminolysis tank, elute the oligonucleic acid synthesized on the solid phase support, and then press the ammonia water containing the oligonucleic acid product into a mobile tank through a nitrogen closed pipeline.

[0023] As a further solution of the present invention, a water inlet and a water outlet are respectively installed at both ends of the synthesis column, and a valve is respectively set at both ends of the synthesis column. Valves are also set on the water inlet and the water outlet. When aminolysis is carried out, the valves at both ends of the synthesis column are closed, and 25% ammonia water is pressed from the storage barrel to the synthesis column by nitrogen to elute the oligonucleic acid synthesized on the solid support. Then, the ammonia water containing the oligonucleic acid product is discharged through the water outlet of the synthesis column and pressed into the mobile tank.

[0024] As a further embodiment of the present invention, the purification method includes ion exchange method and reverse phase chromatography method.

[0025] As a further solution of the present invention, a 16% by mass NaOH solution is used to rinse the ultrafiltration membrane before ultrafiltration, and after rinsing, injection water is used for displacement desalination.

[0026] Beneficial effects of the present invention:

[0027] (1) The basic skeleton of nucleic acid molecules is the 3'→5' phosphodiester bond between adjacent nucleotides. Since nucleotides are molecules with multiple functional groups and the artificial chemical synthesis of nucleic acid molecules is a multi-step continuous reaction, the present invention temporarily protects unnecessary groups as much as possible during the synthesis process. After a round of coupling reaction, the protecting groups on the groups in the previous round are selectively removed to form a specific phosphodiester bond, thereby minimizing side reactions, improving the yield of the target product, and reducing the difficulty of purification.

[0028] (2) The present invention can achieve online aminolysis by simply modifying the synthesis column, thereby achieving no need to transfer the synthesis column during the aminolysis process, eluting the oligonucleic acid product loaded on the surface of the solid phase carrier in the synthesis column, collecting the eluate containing the oligonucleic acid product and performing subsequent purification steps. Since the synthesis column does not need to be taken offline during the entire process, on the one hand, the difficulty and complexity of the work are greatly reduced, and on the other hand, the leakage of waste gas during the disassembly and installation process is significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings.

[0030] Figure 1 It is a schematic diagram of the structure of the synthetic board of the present invention;

[0031] Figure 2 It is a schematic diagram of the structure of the synthetic board of the present invention;

[0032] Figure 3 It is a structural schematic diagram of the regulating connecting pipe of the present invention;

[0033] In the figure: 1. upper sealing plate; 2. lower sealing plate; 3. air pressure regulating area; 4. synthesis column structure; 5. regulating connecting pipe; 11. feeding hole; 51. ventilation pipe; 52. pipe body; 53. sealing retaining ring; 54. threaded area. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] A process for synthesizing pharmaceutical nucleic acid, comprising the following steps:

[0036] In the first step, the prepared raw materials are added into a fully automatic synthesizer and the synthesis operation is performed under normal temperature and fully closed conditions. The entire synthesis cycle is 24 hours.

[0037] The synthesizer is equipped with synthesis columns of 350mm, 600mm and 800mm;

[0038] The chain extension reaction process is to extend from 3' to 5', which mainly includes four steps of reaction: deprotection, activation coupling, sulfurization / oxidation, and capping. After the four steps are completed, the next round of chain extension reaction is carried out, and the number of chain extension reactions is set according to demand.

[0039] Wherein the vulcanization reaction and the oxidation reaction are not carried out simultaneously in a single chain extension reaction process, and the number of reactions in a single batch production process is the same;

[0040] The deprotection reaction comprises: removing the protecting group DMT (dimethoxytrityl) on the nucleotide with dichloroacetic acid to obtain the 5'-hydroxyl end (nucleotide monomer) for the next coupling step;

[0041] The equation is as follows: nucleoside monomer + dichloroacetic acid → 5'-hydroxyl end + dimethoxytrityl + dichloroacetate;

[0042]

[0043] The activation coupling reaction comprises: before coupling, nucleotide monomers are mixed with tetrazole and enter into a synthesis column, at which time tetrazole provides a proton to the N atom of the diisopropylamine group on the phosphate, and the protonated diisopropylamine is a good free group, which forms an active intermediate of phosphoramidite tetrazole with tetrazole, and the excess of tetrazole ensures sufficient activation.

[0044] The phosphoramidite-protected 5'-hydroxyl end is mixed with the activator ethylthiotetrazolyl to obtain a nucleoside phosphorous acid activated intermediate, whose 3' end is activated and the 5'-hydroxyl is still protected by DMT, and condenses with the free 5'-hydroxyl in the solution;

[0045] The equation is as follows: activator 5'-hydroxyl end + phosphoramidite → nucleoside phosphorous acid activated intermediate + NR2 (alkylamine);

[0046]

[0047] The sulfurization / oxidation reaction comprises: under the action of a sulfurizing agent phenylacetyl disulfide (PADS) or an oxidizing agent iodine solution, the phosphorous acid form is converted into a more stable phosphotriester (nucleic acid intermediate), that is, trivalent phosphorus is sulfurized into pentavalent phosphorus, and the byproduct is benzoic acid;

[0048] The equation is as follows: Nucleoside phosphorous acid activated intermediate + sulfurizing agent (PADS) → phosphotriester + benzoic acid;

[0049]

[0050] The capping reaction includes: a very small number of 5'-hydroxyl groups may not participate in the reaction (less than 2%) in the condensation reaction, and the reaction continues after being terminated by acetic anhydride and N-methylimidazole (as a solvent, not participating in the reaction), and such short fragments can be separated during purification;

[0051] The equation is as follows: 5'-hydroxyl end + acetic anhydride → nucleic acid intermediate + acetic acid;

[0052]

[0053] In one embodiment of the present invention, when a synthesis reaction is carried out by a fully automatic synthesizer, the synthesis column structure 4 is supported by a synthesis plate;

[0054] like Figures 1 to 3 As shown, the synthetic plate includes an upper sealing plate 1 and a lower sealing plate 2, a certain distance is maintained between the upper sealing plate 1 and the lower sealing plate 2, an air pressure regulating area 3 is formed between the upper sealing plate 1 and the lower sealing plate 2, and a plurality of regulating connecting pipes 5 are arranged between the upper sealing plate 1 and the lower sealing plate 2 corresponding to the synthetic column structure 4;

[0055] The regulating connecting pipe 5 comprises a pipe body 52, which is a hollow tubular structure with two ends opened. The pipe walls at both ends of the pipe body 52 are seamlessly and hermetically connected to the upper sealing plate 1 and the lower sealing plate 2 respectively.

[0056] A feeding circular hole 11 is provided on the upper sealing plate 1 at one end corresponding to the tube body 52, and materials can be fed into the synthesis column structure 4 through the feeding circular hole 11;

[0057] A vent pipe 51 is provided on the side wall of the tube body 52, and a flow regulating valve is provided on the vent pipe 51, and the opening and closing state of the flow regulating valve can be remotely adjusted during operation;

[0058] A sealing ring 53 is provided on the inner wall of the tube body 52. ​​The inner diameter of the sealing ring 53 matches the inner diameter of the feed port of the synthesis column structure 4. The sealing ring 53 is a structure in which a layer of corrosion-resistant rubber is coated on the surface of a stainless steel liner.

[0059] When the synthetic column structure 4 is assembled, the top of the synthetic column structure 4 is in close contact with the surface of the sealing retainer ring 53, thus achieving a sealing effect;

[0060] In one embodiment of the present invention, the inner wall of the tube body 52 is provided with a threaded area 54 matching the synthetic column structure 4 below the sealing ring 53;

[0061] When the present invention is working, all the feeding holes 11 are sealed and blocked, and the air pressure regulating area 3 is pressurized or evacuated to form a local high-pressure area or negative-pressure area, and then the flow regulating valve on the vent pipe 51 is adjusted to control the opening and closing state and the opening and closing degree of the controller, so as to achieve the expected effect;

[0062] For example, when it is necessary to empty the reagents of each synthetic column structure 4, all the feed holes 11 are sealed and blocked, and the air pressure regulating area 3 is pressurized to form a local high-pressure area, and then the flow regulating valve on the regulating connecting pipe 5 that is not connected to the synthetic column structure 4 is closed to start the emptying process. When the emptying progress of each synthetic column structure 4 is different, the flow regulating valve corresponding to the synthetic column structure 4 that has been emptied can be closed first, or the opening of the flow regulating valve can be adjusted when the emptying is not completed.

[0063] Step 2: Amine Washing

[0064] The product and the solid phase carrier in the synthesizer are subjected to amine washing. The product is washed with diethylamine, so that the diethylamine reacts with the protective group cyanoethyl to achieve the purpose of removing the protective group cyanoethyl. The washing time of the diethylamine and acetonitrile mixed solution in the amine washing process is about 10 minutes, and the waste liquid generated by the washing is connected to the waste liquid storage tank through a pipeline;

[0065] After the amine wash is completed, the synthesizer is rinsed with acetonitrile to remove impurities in the synthesizer, and the synthesis column in the synthesizer is cleaned to remove residues in the synthesis column. The organic waste liquid generated during the cleaning process is transported to the waste liquid storage tank through a closed pipeline for temporary storage;

[0066] The third step is aminolysis

[0067] After the synthesis is completed, the aminolysis process is carried out;

[0068] In one embodiment of the present invention, the synthesis column that has completed amine washing in the second step is transferred to the ammonolysis workshop, the reaction product in the synthesis column and the solid phase carrier are transferred to the ammonolysis tank together, and 25% ammonia water is pressed from the storage barrel to the ammonolysis tank using nitrogen, and the operating temperature is 65°C (ethylene glycol is used as a cold and hot medium, ethylene glycol vapor is used for indirect heating, and chilled water is used for cooling), the oligonucleic acid synthesized on the solid phase support is eluted, and then the ammonia water containing the oligonucleic acid product is pressed into a mobile tank through a nitrogen sealed pipeline, and a filter is installed in the pipeline to filter out the solid phase carrier. In this process, a small amount of waste gas is generated during the feeding of ammonia water and the opening of the ammonolysis kettle, and a small amount of organic waste gas is volatilized during the opening of the synthesis column, and waste solid phase carriers and waste filters are generated;

[0069] In another embodiment of the present invention, a water inlet and a water outlet are respectively installed at both ends of the synthesis column, and a valve is respectively set at both ends of the synthesis column. The water inlet and the water outlet are also provided with valves. In the process of amine washing and the like, the valves on the water inlet and the water outlet are in a normally closed state. When performing the aminolysis operation, the valves at both ends of the synthesis column are closed, and 25% ammonia water is pressed from a storage barrel to the synthesis column by nitrogen, and the operating temperature is 65° C. (ethylene glycol is used as a cold and hot medium, ethylene glycol vapor is used for indirect heating, and chilled water is used for cooling and cooling), the oligonucleic acid synthesized on the solid support is eluted, and then the ammonia water containing the oligonucleic acid product is discharged through the water outlet of the synthesis column and pressed into a mobile tank. A filter is set at the water outlet of the synthesis column to prevent the solid phase carrier from being separated from the synthesis column. Ammonia and a small amount of organic waste gas will be generated during the whole process;

[0070] Step 4: Purification

[0071] Purification methods include ion exchange and reverse phase chromatography;

[0072] By removing by-products and impurities in the product through purification, the purity of the target product in the obtained product is improved;

[0073] Step 5: Ultrafiltration

[0074] After the product is purified, the salt substances introduced during the purification process need to be removed and desalination treatment is performed by ultrafiltration. Before ultrafiltration, the ultrafiltration membrane needs to be rinsed with 16% NaOH solution; after rinsing, injection water is used for replacement desalination. The ultrafiltration membrane is replaced regularly during the ultrafiltration process. This process produces rinsing wastewater, ultrafiltration wastewater, and waste ultrafiltration membranes;

[0075] Step 6: Freeze-drying

[0076] A drying method that freezes water-containing materials below freezing point (-40°C, 12h) to convert water into ice, and then converts the ice into steam under a relatively high vacuum to remove it. The water vapor generated by freeze drying is captured by a low-temperature condenser and then turns into ice, which is then thawed into water to obtain a dehydrated product.

[0077] The above is a detailed description of an embodiment of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A process for synthesizing pharmaceutical nucleic acid, characterized in that: The steps include: The first step is to add the prepared raw materials into a fully automatic synthesizer and perform the synthesis operation under normal temperature and fully closed conditions. The entire synthesis cycle is 24 hours; The chain extension reaction process extends from 3'-5' direction, including four steps of deprotection, activation coupling, sulfurization / oxidation, and capping. After the four steps are completed, the next round of chain extension reaction is carried out; The deprotection reaction comprises: removing the protecting group DMT on the nucleotide with dichloroacetic acid to obtain the 5'-hydroxyl end, i.e., the nucleotide monomer, for the next coupling step; The activated coupling reaction comprises: before coupling, the 5'-hydroxyl end protected by phosphoramidite is mixed with an activating agent ethylthiotetrazolyl to obtain a nucleoside phosphorous acid activated intermediate, whose 3' end is activated, the 5'-hydroxyl is protected by DMT, and a condensation reaction occurs with a free 5'-hydroxyl in the solution; The sulfurization / oxidation reaction comprises: under the action of a sulfurizing agent, phenylacetyl disulfide, or an oxidizing agent, iodine solution, the phosphorous acid form is converted into a phosphate triester; The capping reaction comprises: terminating the subsequent reaction of the 5'-hydroxyl group with acetic anhydride; The second step is amine washing; The third step is aminolysis; Step 4: purification; Step 5: Ultrafiltration; Step 6: freeze-drying to obtain the finished product; When a synthesis reaction is carried out in a fully automatic synthesizer, a synthesis column structure (4) is supported by a synthesis plate, wherein the synthesis plate comprises an upper sealing plate (1) and a lower sealing plate (2), a distance is maintained between the upper sealing plate (1) and the lower sealing plate (2) to form an air pressure adjustment area (3), and a plurality of adjustment connection pipes (5) are provided between the upper sealing plate (1) and the lower sealing plate (2) corresponding to the synthesis column structure (4); The regulating connecting pipe (5) comprises a pipe body (52), the pipe body (52) being a hollow tubular structure with openings at both ends, the pipe walls at both ends of the pipe body (52) being seamlessly sealed and connected to an upper sealing plate (1) and a lower sealing plate (2) respectively; a feeding circular hole (11) is provided on the upper sealing plate (1) at one end corresponding to the pipe body (52), a vent pipe (51) is provided on the side wall of the pipe body (52), a flow regulating valve is provided on the vent pipe (51), a sealing retaining ring (53) is provided on the inner side wall of the pipe body (52), the inner diameter of the sealing retaining ring (53) matches the inner diameter of the feed port of the synthetic column structure (4), and a threaded area (54) matching the synthetic column structure (4) is provided on the inner wall of the pipe body (52) below the sealing retaining ring (53).

2. The process for synthesizing pharmaceutical nucleic acid according to claim 1, characterized in that: The vulcanization reaction and the oxidation reaction do not proceed simultaneously in a single chain extension reaction process, but the number of reactions is the same in a single batch production process.

3. The pharmaceutical nucleic acid synthesis process according to claim 1, characterized in that: In the activated coupling reaction, ethylthiotetrazolyl is used in excess.

4. The process for synthesizing pharmaceutical nucleic acid according to claim 1, characterized in that: The product is rinsed with diethylamine to remove the protective group cyanoethyl. The rinsing time of the diethylamine and acetonitrile mixed solution during the amine washing process is 10 minutes.

5. The process for synthesizing pharmaceutical nucleic acid according to claim 4, characterized in that: After the amine wash is completed, acetonitrile is used to clean the synthesizer and the synthesis column in the synthesizer.

6. The process for synthesizing pharmaceutical nucleic acid according to claim 4, characterized in that: Ammonolysis is to transfer the reaction product in the synthesis column together with the solid phase carrier into the ammonolysis tank, use nitrogen to pressurize 25% ammonia water to the ammonolysis tank, elute the oligonucleic acid synthesized on the solid phase support, and then press the ammonia water containing the oligonucleic acid product into the mobile tank through a nitrogen closed pipeline.

7. The process for synthesizing pharmaceutical nucleic acid according to claim 4, characterized in that: A water inlet and a water outlet are respectively installed at both ends of the synthesis column, and a valve is respectively set at both ends of the synthesis column. Valves are also set on the water inlet and the water outlet. When performing aminolysis, the valves at both ends of the synthesis column are closed, and 25% ammonia water is pressed from the storage barrel to the synthesis column by nitrogen to elute the oligonucleic acid synthesized on the solid support. Then, the ammonia water containing the oligonucleic acid product is discharged through the water outlet of the synthesis column and pressed into the mobile tank.

8. The process for synthesizing pharmaceutical nucleic acid according to claim 1, characterized in that: Purification methods include ion exchange and reverse phase chromatography.

9. The process for synthesizing pharmaceutical nucleic acid according to claim 1, characterized in that: Before ultrafiltration, the ultrafiltration membrane was rinsed with a 16% by mass NaOH solution, and after rinsing, injection water was used for displacement and desalination.

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