CN117362372A - Ammonolysis deprotection process for nucleic acid production - Google Patents
Ammonolysis deprotection process for nucleic acid production Download PDFInfo
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- CN117362372A CN117362372A CN202311307288.2A CN202311307288A CN117362372A CN 117362372 A CN117362372 A CN 117362372A CN 202311307288 A CN202311307288 A CN 202311307288A CN 117362372 A CN117362372 A CN 117362372A
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- nucleic acid
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- ammonolysis
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 67
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 62
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 62
- 238000005915 ammonolysis reaction Methods 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000008569 process Effects 0.000 title claims abstract description 21
- 238000010511 deprotection reaction Methods 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 239000007790 solid phase Substances 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 33
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 18
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 14
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 12
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 239000012298 atmosphere Substances 0.000 claims abstract description 4
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 239000001913 cellulose Substances 0.000 claims description 16
- 229920002678 cellulose Polymers 0.000 claims description 16
- 238000007254 oxidation reaction Methods 0.000 claims description 13
- 125000006239 protecting group Chemical group 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- 230000003647 oxidation Effects 0.000 claims description 9
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 8
- 229920002472 Starch Polymers 0.000 claims description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- 239000004005 microsphere Substances 0.000 claims description 8
- 239000008107 starch Substances 0.000 claims description 8
- 235000019698 starch Nutrition 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004202 carbamide Substances 0.000 claims description 6
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 6
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 claims description 6
- BFXIKLCIZHOAAZ-UHFFFAOYSA-N methyltrimethoxysilane Chemical compound CO[Si](C)(OC)OC BFXIKLCIZHOAAZ-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229920002261 Corn starch Polymers 0.000 claims description 4
- 239000008120 corn starch Substances 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 229960005215 dichloroacetic acid Drugs 0.000 claims description 3
- 150000008300 phosphoramidites Chemical class 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 2
- 240000003183 Manihot esculenta Species 0.000 claims description 2
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims description 2
- 229920001592 potato starch Polymers 0.000 claims description 2
- 239000012351 deprotecting agent Substances 0.000 claims 1
- 238000001668 nucleic acid synthesis Methods 0.000 abstract description 4
- 230000000052 comparative effect Effects 0.000 description 10
- 230000002194 synthesizing effect Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000008782 xin-kang Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000007098 aminolysis reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006355 external stress Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 229920000548 poly(silane) polymer Polymers 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses an ammonolysis protection process for nucleic acid production, and belongs to the technical field of nucleic acid synthesis. The process for producing the ammonolysis and deprotection of the nucleic acid comprises the following steps: firstly, preparing a solid phase carrier; preparing a solid phase carrier containing nucleic acid by using the prepared solid phase carrier as a raw material; and secondly, carrying out ammonolysis reaction and purifying treatment to obtain the nucleic acid. Wherein the ammonolysis reaction comprises the following steps: adding concentrated ammonia water and sodium hydroxide solution into dioxane, and uniformly stirring to obtain ammonolysis solution; the method comprises the steps of (1) placing a solid phase carrier containing nucleic acid into an ammonolysis solution for pre-reaction, adjusting pH for continuous reaction, placing the solid phase carrier into a high-pressure container, performing pressurized reaction in an ammonia atmosphere, filtering and separating, and washing with acetonitrile solution to obtain the ammonolyzed nucleic acid; purifying to obtain nucleic acid. The ammonolysis reaction adopted in the invention can efficiently carry out deprotection reaction on the nucleic acid, and the obtained nucleic acid has higher integrity and purity.
Description
Technical Field
The invention belongs to the technical field of nucleic acid synthesis, and particularly relates to an ammonolysis protection process for nucleic acid production.
Background
Nucleic acid molecules are used as carriers of biological genetic information in nature, are the most important biological information molecules, have unique chemical or structural characteristics and are main objects of molecular biological research. In the prior art, a nucleic acid reaction solution is synthesized on controllable microporous glass beads (CPG) by a phosphoramidite method, then the reaction solution is subjected to a series of deprotection, condensation, sealing and oxidation steps by a synthesis column, the cyclic operation is performed, and finally, a target nucleic acid sequence is dissociated from a solid phase carrier by ammonolysis, and a nucleic acid finished product is obtained by purification.
In the prior art, when saturated ammonia water is used for ammonolysis, a long period of time is usually required, and as the reaction time increases, the ammonia water solution is easy to volatilize, so that the operation is complex, and the whole time is long; in the microwave ammonolysis, the alkali is easy to consume in the reaction, which leads to OH in the reaction system - The ion concentration decreases, so that the reaction cannot proceed any further.
In CN111704644B, an ammonolysis solution and an ammonolysis method are disclosed, and the method uses an organic base with a low boiling point instead of ammonia water to perform an experiment, but when an organic amine such as triethylamine, diethylamine, isopropylamine and the like is used in the experiment, the reaction solution is liable to have a jelly shape, and the problems cannot be avoided in any way of increasing the feed ratio or selecting a dropping mode.
Disclosure of Invention
The invention aims to provide a nucleic acid production ammonolysis deprotection process for solving the problems of low ammonolysis rate and low yield.
The aim of the invention can be achieved by the following technical scheme:
a nucleic acid production ammonolysis deprotection process, comprising the steps of:
first, preparing a solid phase carrier:
adding urea, cetyl trimethyl ammonium chloride and methyltrimethoxysilane into an acetic acid solution, and stirring to obtain a silica gel solution; adding starch, cellulose and lithium chloride/dimethylacetamide solution into a silica gel solution, continuously stirring, adding into absolute ethyl alcohol to obtain cellulose microspheres, washing, and vacuum drying until the water content is lower than 8% to obtain a solid phase carrier; preparing a solid phase carrier containing nucleic acid by using the prepared solid phase carrier as a raw material;
second, ammonolysis reaction:
adding concentrated ammonia water and sodium hydroxide solution into dioxane, and uniformly stirring to obtain ammonolysis solution; the method comprises the steps of (1) placing a solid phase carrier containing nucleic acid into an ammonolysis solution for pre-reaction, adjusting pH for continuous reaction, placing the solid phase carrier into a container, performing pressurized reaction in an ammonia atmosphere, filtering and separating, and washing with acetonitrile solution to obtain the ammonolyzed nucleic acid; purifying to obtain nucleic acid.
Preferably, the ratio of urea, cetyltrimethylammonium chloride, methyltrimethoxysilane and acetic acid solution is 3g:0.6g:4-6mL:10mL; the dosage ratio of starch, cellulose, lithium chloride/dimethylacetamide solution and silica gel solution was 0.2g:0.8g:15mL:20mL; wherein the concentration of lithium chloride in the lithium chloride/dimethylacetamide solution is 8%.
Preferably, the ammonia solution is concentrated: the volume ratio of the sodium hydroxide solution to the dioxane is 3.5:1.5:9.5; wherein the concentration of the sodium hydroxide solution is 1M.
Preferably, the starch is one or two or more of corn starch, tapioca starch and potato starch, which are mixed in any proportion.
Preferably, the pH is adjusted to 7.5-9.5.
Preferably, the pressurization reaction is carried out to 960-970KPa.
Preferably, the preparation method of the solid phase carrier containing the nucleic acid is a solid phase phosphoramidite method, and the specific steps are as follows:
deprotection reaction is carried out on the protecting groups on the surface of the solid phase carrier by adopting a deprotection agent; coupling reaction is carried out on the nucleic acid fragment with the protecting group and the hydroxyl to obtain a conjugate; carrying out oxidation reaction on the conjugate to obtain an oxidation product; and (3) carrying out capping reaction on the oxidation product to obtain the solid phase carrier with the nucleic acid.
Preferably, the deprotection agent is one or both of dichloroacetic acid and trichloroacetic acid mixed in any ratio.
The invention has the beneficial effects that:
in the ammonolysis process, firstly, a stable and mild protecting group is removed from ammonolysis liquid, and then an unstable protecting group is removed in an ammonia atmosphere, so that the protecting group can be used forThe nucleic acid production efficiency can be improved by completely removing the nucleic acid; the dioxane is added as a solvent, so that the introduction of water is reduced, and the interference of the water on the synthesis of nucleic acid is reduced; in order to provide sufficient OH in time - Ensure that the ammonolysis reaction can be smoothly carried out, and the reaction rate is further improved by properly adjusting the pH. The ammonia hydrolysis deprotection process for nucleic acid production provided by the invention has the advantages of simple preparation process, low-cost ammonia water/sodium hydroxide solution/dioxane system for removing protecting groups, high nucleic acid integrity and high purity, and provides an economic and efficient method for the nucleic acid production process.
The solid phase carrier prepared by the invention, namely the cellulose microsphere, has smooth surface, honeycomb-shaped inside and more porous gaps, and has higher loading capacity compared with the traditional solid phase synthesis carrier; the cellulose reacts with the silane to form a closely combined cellulose-polysilane network structure, so that the impact of external stress can be relieved, the deformation is enough, the stress is transferred, and meanwhile, the cellulose network is used for bridging, so that the flexibility of the cellulose microsphere is improved. The surface of the cellulose microsphere contains a large number of hydroxyl groups, on one hand, the cellulose microsphere is highly hydrophobic, the residual moisture on the surface can be thoroughly removed by leaching with an anhydrous solvent, the interference of the moisture on the synthesis of nucleic acid is reduced, the surface active sites are relatively few, the error sequence is not easy to generate in the process of synthesizing the nucleic acid, and the purity of the obtained product is high; on the other hand, when the ammonolysis reaction is carried out, the protecting group is easy to remove, the ammonolysis reaction rate is accelerated, and the nucleic acid synthesis efficiency is improved.
The solid phase carrier prepared by the invention has a large number of modifiable hydroxyl groups on the surface, can be further functionalized according to the needs, and expands the application range of the solid phase carrier, so that the nucleic acid synthesis technology field has wider application prospect.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Preparing a solid phase carrier:
3g of urea, 0.6g of hexadecyl trimethyl ammonium chloride and 4mL of methyltrimethoxysilane are added into 10mL of acetic acid solution (the mass fraction is 17 percent) and stirred uniformly to obtain a silica gel solution; adding 0.2g of corn starch, 0.8g of cellulose (purity is 99%, hubei Xinkang pharmaceutical chemical Co., ltd.) and 15mL of lithium chloride/dimethylacetamide solution (concentration of lithium chloride is 8%) into 20mL of silica gel solution, continuously stirring until uniform, dripping into absolute ethyl alcohol to obtain modified cellulose microspheres, washing 3 times by absolute ethyl alcohol, and vacuum drying until the water content is 7%, thus obtaining the solid phase carrier.
Synthesizing nucleic acid: removing protecting groups on the surface of the solid phase carrier by using dichloroacetic acid to expose hydroxyl; then, the nucleic acid fragment with the protecting group and the hydroxyl are subjected to coupling reaction to obtain a conjugate; carrying out oxidation reaction on the conjugate to obtain an oxidation product; capping reaction is carried out on the oxidation product, and a solid phase carrier with 220bp nucleic acid is obtained through synthesis;
ammonolysis reaction: adding 3.5mL of concentrated ammonia water (with the concentration of 26%) and 1.5mL of sodium hydroxide solution into 9.5mL of dioxane, and uniformly stirring to obtain an ammonolysis solution; then placing the solid phase carrier containing the nucleic acid into an ammonolysis solution for pre-reaction for 20min, adjusting the pH value to 8.5, continuing the reaction for 10min, placing the solid phase carrier into a high-pressure container for continuing the reaction, introducing ammonia gas, continuing the reaction for 5min under 965KPa, filtering and separating, and washing an acetonitrile solution (the mass fraction is 80%) for 3 times; obtaining the ammonolyzed nucleic acid; and (3) performing a purification reaction by a magnetic bead method to obtain the nucleic acid.
Example 2
Preparing a solid phase carrier:
3g of urea, 0.6g of hexadecyl trimethyl ammonium chloride and 5mL of methyltrimethoxysilane are added into 10mL of acetic acid solution (the mass fraction is 17 percent) and stirred uniformly to obtain a silica gel solution; adding 0.2g of corn starch, 0.8g of cellulose (purity is 99%, hubei Xinkang pharmaceutical chemical Co., ltd.) and 15mL of lithium chloride/dimethylacetamide solution (concentration of lithium chloride is 8%) into 20mL of silica gel solution, continuously stirring until uniform, dripping into absolute ethyl alcohol to obtain modified cellulose microspheres, washing 3 times by absolute ethyl alcohol, and vacuum drying until the water content is 7%, thus obtaining the solid phase carrier.
Synthesizing nucleic acid: removing protecting groups on the surface of the solid phase carrier by trichloroacetic acid to expose hydroxyl; then, the nucleic acid fragment with the protecting group and the hydroxyl are subjected to coupling reaction to obtain a conjugate; carrying out oxidation reaction on the conjugate to obtain an oxidation product; capping reaction is carried out on the oxidation product, and a solid phase carrier with 350bp nucleic acid is obtained through synthesis;
ammonolysis reaction: adding 3.5mL of concentrated ammonia water (with the concentration of 26%) and 1.5mL of sodium hydroxide solution into 9.5mL of dioxane, and uniformly stirring to obtain an ammonolysis solution; then placing the solid phase carrier containing the nucleic acid into an ammonolysis solution for pre-reaction for 25min, adjusting the pH value to 8.5, continuing the reaction for 10min, placing the solid phase carrier into a high-pressure container for continuing the reaction, introducing ammonia gas, continuing the reaction for 5min under 967KPa, filtering and separating, and washing an acetonitrile solution (the mass fraction is 80%) for 3 times to obtain the ammonolysis-treated nucleic acid; and (3) performing a purification reaction by a magnetic bead method to obtain the nucleic acid.
Example 3
Synthesizing to obtain a solid phase carrier with 480bp nucleic acid;
ammonolysis reaction: adding 3.5mL of concentrated ammonia water (with the concentration of 26%) and 1.5mL of sodium hydroxide solution into 9.5mL of dioxane, and uniformly stirring to obtain an ammonolysis solution; then placing the solid phase carrier containing the nucleic acid into an ammonolysis solution for pre-reaction for 30min, adjusting the pH value to 8.5, continuing the reaction for 10min, placing the solid phase carrier into a high-pressure container for continuing the reaction, introducing ammonia gas, continuing the reaction for 10min under 963KPa, filtering and separating, and washing the acetonitrile solution for 3 times to obtain the ammonolysis-treated nucleic acid; performing a purification reaction by a magnetic bead method to obtain nucleic acid; the other raw materials and steps were the same as in example 1.
Comparative example 1
In the step of synthesizing nucleic acid, CPG solid phase carrier (load is 35 mu mmol/g) is adopted to obtain nucleic acid; the other raw materials and steps were the same as in example 1.
Comparative example 2
In the ammonolysis reaction step, concentrated ammonia water (the concentration is 28%) is used for ammonolysis reaction to obtain nucleic acid; the other raw materials and steps were the same as in example 1.
Comparative example 3
CPG solid phase carrier (load is 35 mu mmol/g) is adopted in the step of synthesizing nucleic acid, and concentrated ammonia water (concentration is 26%) is adopted in the step of ammonolysis reaction to obtain nucleic acid; the other raw materials and steps were the same as in example 1.
Performance tests were performed on examples 1-3 and comparative examples 1-3:
statistical analysis of the length of aminolysis in examples and comparative examples purified nucleic acids were tested using fluorescent quantitative PCR techniques; the analysis results are shown in table 1:
TABLE 1
| Project | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Ammonolysis duration (min) | 35 | 40 | 50 | 45 | 78 | 93 |
| Yield (%) | 76 | 75 | 72 | 64 | 74 | 61 |
| Purity (%) | 95 | 93 | 92 | 78 | 86 | 75 |
As can be seen from Table 1, examples 1-3 have short reaction times and higher yields and purities. From the results of the yield and purity of the nucleic acid shown in comparative example 1, the solid phase carrier prepared by the present invention can effectively increase the yield of the nucleic acid; the ammonolysis time in comparative examples 2 and 3 is longer, and the ammonolysis and deprotection process for producing nucleic acid provided by the invention has the advantages of high purity of nucleic acid, good yield and high reaction time.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A process for producing ammonolysis protection from nucleic acid, comprising the steps of:
first, preparing a solid phase carrier:
adding urea, cetyl trimethyl ammonium chloride and methyltrimethoxysilane into an acetic acid solution, and stirring to obtain a silica gel solution; adding starch, cellulose and lithium chloride/dimethylacetamide solution into a silica gel solution, continuously stirring, adding into absolute ethyl alcohol to obtain cellulose microspheres, washing, and vacuum drying until the water content is lower than 8% to obtain a solid phase carrier; preparing a solid phase carrier containing nucleic acid by using the prepared solid phase carrier as a raw material;
second, ammonolysis reaction:
adding concentrated ammonia water and sodium hydroxide solution into dioxane, and uniformly stirring to obtain ammonolysis solution; the method comprises the steps of (1) placing a solid phase carrier containing nucleic acid into an ammonolysis solution for pre-reaction, adjusting pH for continuous reaction, placing the solid phase carrier into a container, performing pressurized reaction in an ammonia atmosphere, filtering and separating, and washing with acetonitrile solution to obtain the ammonolyzed nucleic acid; purifying to obtain nucleic acid.
2. The process for the production of ammonia according to claim 1, wherein the ratio of urea, cetyltrimethylammonium chloride, methyltrimethoxysilane and acetic acid solution is 3g:0.6g:4-6mL:10mL; the dosage ratio of starch, cellulose, lithium chloride/dimethylacetamide solution and silica gel solution was 0.2g:0.8g:15mL:20mL; wherein the concentration of lithium chloride in the lithium chloride/dimethylacetamide solution is 8%.
3. The process for producing ammonia according to claim 1, wherein the ammonia solution is concentrated ammonia water: the volume ratio of the sodium hydroxide solution to the dioxane is 3.5:1.5:9.5; wherein the concentration of the sodium hydroxide solution is 1M.
4. The process for producing ammonia according to claim 1, wherein the starch is one or two or more of corn starch, tapioca starch and potato starch.
5. The process for producing ammonia according to claim 1, wherein the pH is adjusted to 7.5 to 9.5.
6. The process for producing ammonia according to claim 1, wherein the pressurizing reaction is performed to 960-970KPa.
7. The process for producing ammonia according to claim 1, wherein the preparation method of the solid phase carrier containing nucleic acid is a solid phase phosphoramidite method, and the specific steps are as follows:
deprotection reaction is carried out on the protecting groups on the surface of the solid phase carrier by adopting a deprotection agent; coupling reaction is carried out on the nucleic acid fragment with the protecting group and the hydroxyl to obtain a conjugate; carrying out oxidation reaction on the conjugate to obtain an oxidation product; and (3) carrying out capping reaction on the oxidation product to obtain the solid phase carrier with the nucleic acid.
8. The process for producing ammonia according to claim 7, wherein the deprotecting agent is one or both of dichloroacetic acid and trichloroacetic acid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311307288.2A CN117362372A (en) | 2023-10-10 | 2023-10-10 | Ammonolysis deprotection process for nucleic acid production |
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| CN202311307288.2A CN117362372A (en) | 2023-10-10 | 2023-10-10 | Ammonolysis deprotection process for nucleic acid production |
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