CN114591387A - Amine washing process after synthesis of medicinal nucleic acid - Google Patents
Amine washing process after synthesis of medicinal nucleic acid Download PDFInfo
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- 238000005406 washing Methods 0.000 title claims abstract description 87
- 150000001412 amines Chemical class 0.000 title claims abstract description 67
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 22
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 22
- 102000039446 nucleic acids Human genes 0.000 title claims description 22
- 108020004707 nucleic acids Proteins 0.000 title claims description 22
- 150000007523 nucleic acids Chemical class 0.000 title claims description 22
- 239000007788 liquid Substances 0.000 claims abstract description 69
- 239000000243 solution Substances 0.000 claims abstract description 58
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 33
- NDKBVBUGCNGSJJ-UHFFFAOYSA-M benzyltrimethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)CC1=CC=CC=C1 NDKBVBUGCNGSJJ-UHFFFAOYSA-M 0.000 claims abstract description 27
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 239000003054 catalyst Substances 0.000 claims abstract description 11
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 36
- 239000012528 membrane Substances 0.000 claims description 27
- 239000002253 acid Substances 0.000 claims description 26
- 238000000909 electrodialysis Methods 0.000 claims description 26
- 239000003513 alkali Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 24
- 230000008569 process Effects 0.000 claims description 21
- 239000000872 buffer Substances 0.000 claims description 19
- -1 benzyl trimethyl ammonium halide salt Chemical class 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 230000005684 electric field Effects 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 6
- UUZYBYIOAZTMGC-UHFFFAOYSA-M benzyl(trimethyl)azanium;bromide Chemical compound [Br-].C[N+](C)(C)CC1=CC=CC=C1 UUZYBYIOAZTMGC-UHFFFAOYSA-M 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- ORWKVZNEPHTCQE-UHFFFAOYSA-N acetic formic anhydride Chemical compound CC(=O)OC=O ORWKVZNEPHTCQE-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000013078 crystal Substances 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 238000001668 nucleic acid synthesis Methods 0.000 abstract description 4
- 239000000463 material Substances 0.000 description 11
- 229940079593 drug Drugs 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 239000002699 waste material Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 229910052736 halogen Inorganic materials 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000004073 vulcanization Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- YOUGRGFIHBUKRS-UHFFFAOYSA-N benzyl(trimethyl)azanium Chemical compound C[N+](C)(C)CC1=CC=CC=C1 YOUGRGFIHBUKRS-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000003014 ion exchange membrane Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010815 organic waste Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/0234—Nitrogen-, phosphorus-, arsenic- or antimony-containing compounds
- B01J31/0235—Nitrogen containing compounds
- B01J31/0239—Quaternary ammonium compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses an amine washing process after medicinal nucleic acid synthesis, which comprises the following steps: step 1: preparing an amine washing solution: mixing diethylamine, acetonitrile and benzyl trimethyl ammonium hydroxide according to the mass ratio of 48-82: 150-200: 2-4, adding the mixture into a mixing container to obtain amine washing liquor; step 2: washing the synthesis column with buffer solution with pH of 5.0, and controlling flow rate at 20 ml/min; and step 3: washing with amine washing solution, controlling the flow rate at 50-65ml/min, and detecting the amine washing solution in real time; the invention takes benzyl trimethyl ammonium hydroxide as a catalyst, and can effectively improve the reaction efficiency between diethylamine and a protective group cyanoethyl in a product; by improving the amine washing liquid, the amine washing time can be effectively shortened.
Description
Technical Field
The invention relates to the technical field of medicinal nucleic acid, in particular to an amine washing process after medicinal nucleic acid synthesis.
Background
The medicinal nucleic acid is widely applied to the development of small nucleic acid drugs and the development of novel vaccine adjuvants. The development of a drug can be roughly divided into three stages, namely: small molecule drugs, protein antibody drugs, nucleic acid drugs. In recent years, the third generation of drugs based on nucleic acid therapy is developed rapidly, and the nucleic acid drugs are characterized in that the drugs can be directly combined with pathogenic genes and prevented from translating, so that the production of pathogenic proteins is prevented, and the nucleic acid drugs can be accurately guided to directly reach targets and are easy to industrially amplify and produce;
in the prior art, the medicinal nucleic acid is fully-automatically synthesized by using a fully-automatic synthesizer at normal temperature in a fully-closed environment, and the whole synthesis period is 24 hours; all the prepared raw materials are conveyed into a synthesizer through a pipeline, and the synthesizer is provided with synthesis columns of 350mm, 600mm and 800 mm. The chain extension reaction process is extended from the 3 '-5' direction, and mainly comprises deprotection, activation coupling, vulcanization/oxidation (the vulcanization and oxidation reactions are not carried out simultaneously in the single chain extension reaction process, the reaction times are the same in the single batch production process), and 4-step reaction of a cap, after the four-step reaction is completed, the product is washed by 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 mixed solution of diethylamine and acetonitrile in the amine washing process is about 10min, and the problems of longer washing time and influence on the overall efficiency of medicinal nucleic acid synthesis exist.
Disclosure of Invention
The present invention aims to solve the problems of the background art, and provides an amine washing process after the synthesis of medicinal nucleic acid.
The purpose of the invention can be realized by the following technical scheme:
the amine washing process after the synthesis of the medicinal nucleic acid comprises the following steps:
step 1: preparing an amine washing solution: mixing diethylamine, acetonitrile and benzyl trimethyl ammonium hydroxide according to the mass ratio of 48-82: 150-200: 2-4, adding the mixture into a mixing container to obtain amine washing liquor;
step 2: washing the synthesis column with buffer solution with pH of 5.0, and controlling flow rate at 20 ml/min;
and step 3: washing with amine washing solution, controlling the flow rate at 50-65ml/min, detecting the amine washing solution in real time, and observing the washing progress.
As a further scheme of the invention: the buffer solution with pH of 5.0 is prepared from 0.01mol/L potassium dihydrogen phosphate-dipotassium hydrogen phosphate according to the mass ratio of 1: 8 proportion is dissolved by purified water and the pH value is adjusted to 5.0.
As a further scheme of the invention: the washing progress detection method comprises the following steps: the method comprises the following steps:
placing 2 drops of washing liquid into a conical flask, adding 10mL of formic acid-acetic anhydride acylating agent and 30mL of chloroform-glacial acetic acid solvent with the mass fraction of 10%, standing for 10min, adding 1-2 drops of crystal violet-glacial acetic acid indicator, titrating the solution by using 0.1mol/L perchloric acid-glacial acetic acid standard solution, and changing the color of the solution from violet to blue, namely completely eluting the protective group cyanoethyl in the product by using amine washing liquid.
As a further scheme of the invention: the preparation process of the catalyst benzyltrimethylammonium hydroxide comprises the following steps:
s1: introducing the aqueous solution of benzyl trimethyl ammonium halide salt into a material liquid chamber, introducing deionized water into an acid liquid chamber and an alkali liquid chamber respectively, and introducing a sulfuric acid solution into an electrode liquid chamber and a buffer chamber respectively;
s2: applying direct current electric fields on the cathode chamber and the anode chamber, controlling the temperature in the electrodialysis membrane module to be kept in a preset temperature range, and performing a bipolar membrane electrodialysis process;
s3: and after the bipolar membrane electrodialysis process is finished, recovering the hydrohalic acid from the acid liquor chamber, and recovering the benzyltrimethyl ammonium hydroxide from the alkali liquor chamber.
As a further scheme of the invention: 2L of aqueous solution of benzyltrimethylammonium bromide with the mass fraction of 16-18% is fed into a feed liquid chamber, 2L of sulfuric acid solution with the mass fraction of 2-4% is used as feed materials of an anode chamber and a cathode chamber, and the feed materials are fed into the anode chamber and the cathode chamber.
As a further scheme of the invention: 2L of deionized water is respectively introduced into the acid liquid chamber and the alkali liquid chamber, and 2L of sulfuric acid solution with the mass fraction of 2-4% is introduced into the buffer chamber.
As a further scheme of the invention: in the bipolar membrane electrodialysis process, the current density of the applied direct current electric field is 150-600A/m2。
As a further scheme of the invention: the temperature in the electrodialysis membrane module is kept between 20 and 35 ℃.
As a further scheme of the invention: the liquid flow rates in the buffer chamber, the acid liquid chamber, the feed liquid chamber and the alkali liquid chamber are 200-2000L/h.
The invention has the beneficial effects that:
according to the invention, benzyl trimethyl ammonium hydroxide is added into an amine washing solution of diethylamine and acetonitrile as a catalyst, and the product and a solid phase carrier in a synthesizer are subjected to amine washing, wherein the amine washing adopts diethylamine to wash the product, so that the diethylamine reacts with a protective group cyanoethyl to achieve the purpose of removing the protective group cyanoethyl, and a waste liquid generated by washing is connected to a waste liquid storage tank through a pipeline;
the benzyl trimethyl ammonium hydroxide is used as a catalyst, so that the reaction efficiency between diethylamine and a protective group cyanoethyl in a product can be effectively improved; therefore, the amine washing time can be effectively shortened by improving the amine washing liquid;
electrolyzing benzyl trimethyl ammonium halide salt by using a bipolar membrane electrodialysis assembly to obtain halogen ions and benzyl trimethyl ammonium cations; meanwhile, water dissociation is carried out by using a bipolar membrane to generate hydrogen ions and hydroxyl ions; under the action of an external electric field, an ion exchange membrane is utilized to combine the obtained benzyl trimethyl ammonium cation with hydroxide ions to generate a benzyl trimethyl ammonium hydroxide solution.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention relates to an amine washing process after medicinal nucleic acid synthesis, which comprises the following steps:
step 1: preparing an amine washing solution: mixing diethylamine, acetonitrile and benzyl trimethyl ammonium hydroxide according to a mass ratio of 48: 150: 2, adding the mixture into a mixing container, and stirring for 2min to obtain an amine washing solution;
wherein, the diethylamine reacts with the protection group cyanoethyl in the product to achieve the purpose of removing the protection group cyanoethyl; acetonitrile is used as a solvent in amine washing liquor, and benzyl trimethyl ammonium hydroxide is used as a catalyst, so that the reaction efficiency between diethylamine and a protective group cyanoethyl in a product can be effectively improved; therefore, the amine washing liquid is improved, so that the amine washing time can be effectively shortened;
step 2: washing the synthesis column with buffer solution with pH of 5.0, and controlling flow rate at 20 ml/min;
wherein the buffer solution with pH of 5.0 is prepared from 0.01mol/L potassium dihydrogen phosphate-dipotassium hydrogen phosphate according to the mass ratio of 1: 8 proportion is dissolved by purified water and the pH value is adjusted to 5.0;
and step 3: washing with amine washing liquid, controlling the flow rate at 50ml/min, detecting the amine washing liquid in real time, and observing the washing progress;
the washing progress detection method comprises the following steps: the method comprises the following steps:
placing 2 drops of flushing liquid into a 100mL dry conical flask, adding 10mL formic acid-acetic anhydride acylating agent and 30mL chloroform-glacial acetic acid solvent with the mass fraction of 10%, standing for 10min, adding 1-2 drops of crystal violet-glacial acetic acid indicator, titrating the solution by using 0.1mol/L perchloric acid-glacial acetic acid standard solution, and changing the color of the solution from violet to blue, namely completely eluting the protective group cyanoethyl in the product by using amine washing liquid;
according to the invention, the product and the solid phase carrier in the synthesizer are subjected to amine washing, and the product is washed by diethylamine, so that the diethylamine reacts with the protective group cyanoethyl to achieve the purpose of removing the protective group cyanoethyl, and waste liquid generated by washing is connected to a waste liquid storage tank through a pipeline;
adopt acetonitrile to wash the synthesizer after the amine is washed to accomplish, get rid of the impurity in the synthesizer, synthetic post in the synthesizer washs to get rid of the residue in the synthetic post, the cleaning process is produced by organic waste liquid, carries to the waste liquid storage tank through airtight pipeline and keeps in.
Example 2
Compared with the embodiment 1, the difference is that: the amine washing process after the synthesis of the medicinal nucleic acid comprises the following steps:
step 1: preparing an amine washing solution: mixing diethylamine, acetonitrile and benzyl trimethyl ammonium hydroxide according to a mass ratio of 65: 180: 3, adding the mixture into a mixing container, and stirring for 3min to obtain amine washing liquor;
step 2: washing the synthesis column with buffer solution with pH of 5.0, and controlling flow rate at 20 ml/min;
and 3, step 3: washing with amine washing solution, controlling the flow rate at 65ml/min, detecting the amine washing solution in real time, and observing the washing progress.
Example 3
Compared with the embodiment 1, the difference is that: the amine washing process after the synthesis of the medicinal nucleic acid comprises the following steps:
step 1: preparation of amine wash liquor: mixing diethylamine, acetonitrile and benzyltrimethylammonium hydroxide according to a mass ratio of 82: 200: 4, adding the mixture into a mixing container, and stirring for 4min to obtain amine washing liquor;
step 2: washing the synthesis column with buffer solution with pH of 5.0, and controlling flow rate at 20 ml/min;
and step 3: and (4) washing with an amine washing solution, controlling the flow rate at 65ml/min, detecting the amine washing solution in real time, and observing the washing progress.
Example 4
The preparation process of the catalyst benzyltrimethylammonium hydroxide comprises the following steps:
s1: introducing the aqueous solution of benzyl trimethyl ammonium halide salt into a material liquid chamber, introducing deionized water into an acid liquid chamber and an alkali liquid chamber respectively, and introducing a sulfuric acid solution into an electrode liquid chamber and a buffer chamber respectively;
wherein 2L of aqueous solution of benzyltrimethylammonium bromide with the mass fraction of 16% is fed into a feed liquid chamber, 2L of sulfuric acid solution with the mass fraction of 2% is used as feed materials of an anode chamber and a cathode chamber, and the feed materials are fed into the anode chamber and the cathode chamber; respectively introducing 2L of deionized water into the acid liquid chamber and the alkali liquid chamber, and introducing 2L of sulfuric acid solution with the mass fraction of 2% into the buffer chamber;
s2: applying direct current electric fields on the cathode chamber and the anode chamber, controlling the temperature in the electrodialysis membrane module to be kept in a preset temperature range, and performing a bipolar membrane electrodialysis process;
s3: after the bipolar membrane electrodialysis process is finished, recovering hydrohalic acid from the acid liquor chamber, and recovering benzyl trimethyl ammonium hydroxide from the alkali liquor chamber;
in the bipolar membrane electrodialysis process, the applied DC electric field has a current density of 150A/m2The temperature in the electrodialysis membrane module is kept at 20 ℃, and the liquid flow rate in the buffer chamber, the acid liquid chamber, the feed liquid chamber and the alkali liquid chamber is 200L/h;
the mass fraction of the sulfuric acid solution respectively introduced into the polar liquid chamber and the buffer chamber is 1%; the mass fraction of the benzyltrimethyl ammonium hydroxide solution recovered from the alkali liquor chamber is 10 percent, the recovery rate is more than 95 percent, and the concentration of impurity halogen ions in the solution is less than 70 ppm.
Example 5
The preparation process of the catalyst benzyltrimethylammonium hydroxide comprises the following steps:
s1: introducing the aqueous solution of benzyl trimethyl ammonium halide salt into a material liquid chamber, introducing deionized water into an acid liquid chamber and an alkali liquid chamber respectively, and introducing a sulfuric acid solution into an electrode liquid chamber and a buffer chamber respectively;
wherein 2L of aqueous solution of benzyltrimethylammonium bromide with the mass fraction of 17% is fed into a feed liquid chamber, 2L of sulfuric acid solution with the mass fraction of 3% is used as feed materials of an anode chamber and a cathode chamber, and the feed materials are fed into the anode chamber and the cathode chamber; respectively introducing 2L of deionized water into the acid liquor chamber and the alkali liquor chamber, and introducing 2L of sulfuric acid solution with the mass fraction of 3% into the buffer chamber;
s2: applying direct current electric fields on the cathode chamber and the anode chamber, controlling the temperature in the electrodialysis membrane module to be kept in a preset temperature range, and performing a bipolar membrane electrodialysis process;
s3: after the bipolar membrane electrodialysis process is finished, recovering hydrohalic acid from the acid liquor chamber, and recovering benzyl trimethyl ammonium hydroxide from the alkali liquor chamber;
in the bipolar membrane electrodialysis process, the applied DC electric field has a current density of 350A/m2The temperature in the electrodialysis membrane module is kept at 25 ℃, and the liquid flow rate in the buffer chamber, the acid liquid chamber, the feed liquid chamber and the alkali liquid chamber is 1100L/h;
the mass fraction of the sulfuric acid solution introduced into the anolyte chamber and the buffer chamber is 2%. The mass fraction of the benzyltrimethyl ammonium hydroxide solution recovered from the alkali liquor chamber is 15 percent, the recovery rate is more than 95 percent, and the concentration of impurity halogen ions in the solution is less than 70 ppm.
Example 6
The preparation process of the catalyst benzyltrimethyl ammonium hydroxide comprises the following steps:
s1: introducing the aqueous solution of benzyl trimethyl ammonium halide salt into a material liquid chamber, introducing deionized water into an acid liquid chamber and an alkali liquid chamber respectively, and introducing a sulfuric acid solution into an electrode liquid chamber and a buffer chamber respectively;
wherein 2L of aqueous solution of benzyltrimethylammonium bromide with the mass fraction of 18 percent is fed into a feed liquid chamber, 2L of sulfuric acid solution with the mass fraction of 4 percent is used as feed materials of an anode chamber and a cathode chamber and is fed into the anode chamber and the cathode chamber; respectively introducing 2L of deionized water into the acid liquid chamber and the alkali liquid chamber, and introducing 2L of sulfuric acid solution with the mass fraction of 4% into the buffer chamber;
s2: applying direct current electric fields on the cathode chamber and the anode chamber, controlling the temperature in the electrodialysis membrane module to be kept in a preset temperature range, and performing a bipolar membrane electrodialysis process;
s3: after the bipolar membrane electrodialysis process is finished, recovering hydrohalic acid from the acid liquor chamber, and recovering benzyl trimethyl ammonium hydroxide from the alkali liquor chamber;
in the bipolar membrane electrodialysis process, the applied DC electric field has a current density of 600A/m2In electrodialysis membrane moduleThe temperature of the buffer chamber, the acid liquid chamber, the feed liquid chamber and the alkali liquid chamber is kept at 35 ℃, and the liquid flow rate in the buffer chamber, the acid liquid chamber, the feed liquid chamber and the alkali liquid chamber is 2000L/h;
the mass fraction of the sulfuric acid solution introduced into the polar liquid chamber and the buffer chamber is 3 percent; the mass fraction of the benzyltrimethyl ammonium hydroxide solution recovered from the alkali liquor chamber is 20 percent, the recovery rate is more than 95 percent, and the concentration of impurity halogen ions in the solution is less than 70 ppm.
Comparative example 1
Comparative example is the amine wash process of example 1 without the addition of the catalyst benzyltrimethylammonium hydroxide,
the amine wash processes of examples 1-3 and comparative example 1 were tested for performance, and the results are shown in the table below; the time spent by the amine washing of the product and the solid phase carrier in the synthesizer by different amine washing solutions is tested by the washing progress detection method in example 1; the test results are shown in the following table:
from the above table, it can be seen that the reaction efficiency between diethylamine and the protecting group cyanoethyl group in the product can be effectively improved by adding benzyltrimethylammonium hydroxide as a catalyst in the amine washing solution of diethylamine and acetonitrile, and it can be seen in particular that the amine washing solution in examples 1-3 has the amine washing time of 4.6-5.9min for the product and the solid phase carrier in the synthesizer; while the amine wash of comparative example 1 without the addition of benzyltrimethylammonium hydroxide used an amine wash time of 13.5min, which was much greater than the amine wash times of examples 1-3.
While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (9)
1. The amine washing process after the synthesis of the medicinal nucleic acid is characterized by comprising the following steps of:
step 1: preparing an amine washing solution: mixing diethylamine, acetonitrile and benzyl trimethyl ammonium hydroxide according to the mass ratio of 48-82: 150-200: 2-4, adding the mixture into a mixing container to obtain amine washing liquor;
step 2: washing the synthesis column with buffer solution with pH of 5.0, and controlling flow rate at 20 ml/min;
and step 3: washing with amine washing solution, controlling the flow rate at 50-65ml/min, detecting the amine washing solution in real time, and observing the washing progress.
2. The process of amine washing after synthesis of a medicinal nucleic acid according to claim 1, wherein the buffer solution having a pH of 5.0 is prepared from 0.01mol/L potassium dihydrogen phosphate-dipotassium hydrogen phosphate in a mass ratio of 1: 8 proportion is dissolved by purified water and the pH value is adjusted to 5.0.
3. The amine washing process after the synthesis of the medicinal nucleic acid according to claim 1, wherein the washing progress detection method comprises the following steps: the method comprises the following steps:
placing 2 drops of washing liquid into a conical flask, adding 10mL of formic acid-acetic anhydride acylating agent and 30mL of chloroform-glacial acetic acid solvent with the mass fraction of 10%, standing for 10min, adding 1-2 drops of crystal violet-glacial acetic acid indicator, titrating the solution by using 0.1mol/L perchloric acid-glacial acetic acid standard solution, and changing the color of the solution from violet to blue, namely completely eluting the protective group cyanoethyl in the product by using amine washing liquid.
4. The amine washing process after the synthesis of the medicinal nucleic acid according to claim 1, wherein the preparation process of the catalyst benzyltrimethylammonium hydroxide comprises the following steps:
s1: introducing the aqueous solution of benzyl trimethyl ammonium halide salt into a feed liquid chamber, introducing deionized water into an acid liquid chamber and an alkali liquid chamber respectively, and introducing a sulfuric acid solution into a polar liquid chamber and a buffer chamber respectively;
s2: applying direct current electric fields on the cathode chamber and the anode chamber, controlling the temperature in the electrodialysis membrane module to be kept in a preset temperature range, and carrying out a bipolar membrane electrodialysis process;
s3: and after the bipolar membrane electrodialysis process is finished, recovering the hydrohalic acid from the acid liquor chamber, and recovering the benzyltrimethyl ammonium hydroxide from the alkali liquor chamber.
5. The process of claim 4, wherein 2L of aqueous benzyltrimethylammonium bromide solution with a mass fraction of 16-18% is fed into the feed chamber, and 2L of sulfuric acid solution with a mass fraction of 2-4% is fed into the anode chamber and the cathode chamber.
6. The amine washing process after the synthesis of medicinal nucleic acid according to claim 4, wherein 2L of deionized water is introduced into the acid solution chamber and the alkali solution chamber, respectively, and 2L of sulfuric acid solution with a mass fraction of 2-4% is introduced into the buffer chamber.
7. The amine washing process after the synthesis of the medicinal nucleic acid as claimed in claim 4, wherein the current density of the applied DC electric field during the bipolar membrane electrodialysis process is 150-600A/m2。
8. The amine washing process after synthesis of a pharmaceutical nucleic acid according to claim 7, wherein the temperature in the electrodialysis membrane module is maintained at 20-35 ℃.
9. The amine washing process after the synthesis of medicinal nucleic acid as claimed in claim 7, wherein the liquid flow rate in the buffer chamber, the acid solution chamber, the solution chamber and the alkali solution chamber is 200-2000L/h.
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