CN117327236A - Synthesis method of high GC content primer - Google Patents
Synthesis method of high GC content primer Download PDFInfo
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- CN117327236A CN117327236A CN202311203020.4A CN202311203020A CN117327236A CN 117327236 A CN117327236 A CN 117327236A CN 202311203020 A CN202311203020 A CN 202311203020A CN 117327236 A CN117327236 A CN 117327236A
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- silane coupling
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- 238000001308 synthesis method Methods 0.000 title abstract description 5
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 claims abstract description 21
- 239000007790 solid phase Substances 0.000 claims abstract description 21
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000011737 fluorine Substances 0.000 claims abstract description 10
- 229910052731 fluorine Inorganic materials 0.000 claims abstract description 10
- 239000000178 monomer Substances 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 125000006239 protecting group Chemical group 0.000 claims abstract description 5
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 4
- 238000006482 condensation reaction Methods 0.000 claims abstract description 3
- 239000007800 oxidant agent Substances 0.000 claims abstract description 3
- 230000001590 oxidative effect Effects 0.000 claims abstract description 3
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 0.000 claims abstract description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 238000003756 stirring Methods 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 239000002131 composite material Substances 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 25
- 238000001035 drying Methods 0.000 claims description 24
- 239000006087 Silane Coupling Agent Substances 0.000 claims description 16
- 150000004756 silanes Chemical class 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 14
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 238000000227 grinding Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000007873 sieving Methods 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 230000002194 synthesizing effect Effects 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 6
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 6
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 6
- LFRDHGNFBLIJIY-UHFFFAOYSA-N trimethoxy(prop-2-enyl)silane Chemical compound CO[Si](OC)(OC)CC=C LFRDHGNFBLIJIY-UHFFFAOYSA-N 0.000 claims description 5
- FMQPBWHSNCRVQJ-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-yl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OC(C(F)(F)F)C(F)(F)F FMQPBWHSNCRVQJ-UHFFFAOYSA-N 0.000 claims description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- NKSJNEHGWDZZQF-UHFFFAOYSA-N ethenyl(trimethoxy)silane Chemical compound CO[Si](OC)(OC)C=C NKSJNEHGWDZZQF-UHFFFAOYSA-N 0.000 claims description 3
- DEQJNIVTRAWAMD-UHFFFAOYSA-N 1,1,2,4,4,4-hexafluorobutyl prop-2-enoate Chemical compound FC(F)(F)CC(F)C(F)(F)OC(=O)C=C DEQJNIVTRAWAMD-UHFFFAOYSA-N 0.000 claims description 2
- ZNJXRXXJPIFFAO-UHFFFAOYSA-N 2,2,3,3,4,4,5,5-octafluoropentyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(F)(F)C(F)(F)C(F)(F)C(F)F ZNJXRXXJPIFFAO-UHFFFAOYSA-N 0.000 claims description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 12
- 238000003786 synthesis reaction Methods 0.000 abstract description 12
- 230000002209 hydrophobic effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 8
- 229910021641 deionized water Inorganic materials 0.000 description 8
- 239000011148 porous material Substances 0.000 description 8
- 238000009777 vacuum freeze-drying Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 238000010907 mechanical stirring Methods 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 229910004298 SiO 2 Inorganic materials 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000004438 BET method Methods 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F292/00—Macromolecular compounds obtained by polymerising monomers on to inorganic materials
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/06—Oxidation
-
- 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)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a synthesis method of a primer with high GC content, which belongs to the technical field of biological molecular science and comprises the following steps: step one, reacting nucleotides which are pre-connected on a modified solid phase carrier CPG and are protected by active groups with trichloroacetic acid, and removing a protecting group DMT of a 5 '-hydroxyl group of the nucleotides to obtain a free 5' -hydroxyl group; step two, condensation reaction; step three, reacting with a cap; step four, oxidation reaction: oxidizing the phosphorous acid with an oxidizing agent to form a phosphotriester; the modified solid phase carrier is obtained by modifying the di-oxidation of chitosan oligosaccharide and fluorine-containing monomer; the modified solid phase carrier used in the invention has better hydrophobic property and controllability, effectively improves the synthesis efficiency of the primer and reduces the production cost.
Description
Technical Field
The invention belongs to the technical field of biological molecular science, and particularly relates to a method for synthesizing a primer with high GC content.
Background
With the development of molecular biology techniques, nucleic acid drug development, diagnosis of genetic diseases and infectious diseases, and gene research have progressed rapidly, so that synthesis of a large number of oligonucleotides is required to support the above research. The current common synthesis method is a solid-phase phosphoramidite method, most of the solid-phase carriers of the synthetic primers used in the method are powdery controllable microporous glass beads (CPG), and the CPG is used as an inorganic silica-based carrier, has the advantages of easy surface derivatization, higher reaction activity, no swelling and the like, and is a solid-phase synthesis carrier with very excellent performance.
However, as the surface of the nucleic acid is rich in hydroxyl groups and has strong hydrophilicity, the protecting group of the nucleic acid is easy to fall off in the synthesis process, and the synthesis efficiency and quality of the nucleic acid are affected to a certain extent; in the synthesis process, CPG needs a large filling amount, and is easy to react with reagents insufficiently, so that the synthesis efficiency is low, the amount of synthesized target primers is small, and the cost is high; in addition, when CPG carrier is used, the amount of the powder (generally 4 mg) is generally fixed, resulting in about 5OD of the synthesized primer, while the primer generally required in the experiment only needs 0.1-2OD, which causes waste to a certain extent, so that CPG is used as solid phase synthesis carrier and has great limitation in the application field of primer synthesis.
Disclosure of Invention
The invention aims to provide a synthesis method of a primer with high GC content, which aims to solve the problem of low synthesis efficiency of CPG carrier.
The aim of the invention can be achieved by the following technical scheme:
a method for synthesizing a primer with high GC content comprises the following steps:
step one, reacting nucleotides which are pre-connected on a modified solid phase carrier and are protected by active groups with trichloroacetic acid, and removing a protecting group DMT of a 5 '-hydroxyl group of the nucleotides to obtain a free 5' -hydroxyl group;
step two, condensation reaction;
step three, reacting with a cap;
step four, oxidation reaction: oxidizing the phosphorous acid with an oxidizing agent to form a phosphotriester;
wherein the modified solid phase carrier is prepared by the following steps:
firstly, adding chitosan oligosaccharide into acetic acid solution, stirring until the chitosan oligosaccharide is completely dissolved, adding tetraethyl orthosilicate, stirring for 12-15h, standing for 2-3h, washing with water, freeze-drying, grinding and sieving to obtain a composite material;
secondly, adding the composite material into distilled water for ultrasonic dispersion, dripping an unsaturated silane coupling agent into the dispersion liquid, adjusting the pH value to 9-11 by using strong ammonia water, heating to 80 ℃, stirring and reacting for 24 hours, and drying to obtain the unsaturated silane coupling agent modified composite material;
thirdly, adding the unsaturated silane coupling agent modified composite material, sodium dodecyl sulfate, potassium carbonate and fluorine-containing monomer into distilled water, introducing nitrogen, heating to 65 ℃ and stirring for 1-2h, adding potassium persulfate for reaction for 6h, adding magnesium sulfate, filtering, washing with water, and drying to obtain the modified solid phase carrier.
Preferably, the dosage ratio of chitosan oligosaccharide, acetic acid solution and tetraethyl orthosilicate is 0.25-1g:50mL:5mL.
Preferably, the composite, distilled water and unsaturated silane coupling agent are used in a ratio of 1.5g:50mL:0.03mol.
Preferably, the unsaturated silane coupling agent comprises one of allyltrimethoxysilane and vinyltrimethoxysilane.
Preferably, the dosage ratio of the unsaturated silane coupling agent modified composite material, sodium dodecyl sulfate, potassium carbonate, fluorine-containing monomer, distilled water and potassium persulfate is 1g:0.16g:0.125g:9g:40mL:0.1g.
Preferably, the fluorine-containing monomer is one or more of hexafluorobutyl acrylate, hexafluoroisopropyl methacrylate, octafluoropentyl acrylate, hexafluoroisopropyl methacrylate and octafluoropentyl methacrylate.
The invention has the beneficial effects that:
the invention relates to a method for synthesizing a primer with high GC content, which relates to a self-made modified solid phase carrier in the synthesis process, wherein the self-made modified solid phase carrier SiO 2 After grafting reaction with fluorine-containing monomer, siO with excellent hydrophobic performance is obtained 2 Meanwhile, the content of chitosan oligosaccharide can be changed, so that the purpose of controlling the pore size of the solid phase carrier is achieved, and the solid phase carrier with the corresponding pore size can be selected according to the amount to be synthesized in the primer synthesis process, so that the waste caused by the product in the synthesis process is reduced; compared with the existing solid phase carrier, the solid phase carrier prepared by the invention has better hydrophobicity and controllable pore size, can effectively reduce the falling-off of protecting groups, and simultaneously reduces the waste of products, thereby improving the efficiency of primer synthesis.
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
Adding 0.25g of chitosan oligosaccharide (with the viscosity less than 200 mPa.s) into 50mL of acetic acid solution (99.5% of analytical grade), stirring until the chitosan oligosaccharide is completely dissolved, adding 5mL of tetraethyl orthosilicate, setting the stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing with deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material;
adding 1.5g of the composite material into 50mL of distilled water, performing strong ultrasonic and mechanical stirring, dripping 0.045mol of allyltrimethoxysilane into the dispersion liquid, adjusting the pH to 9-11 with concentrated ammonia water, heating to 80 ℃, stirring for reaction for 24 hours, and drying the mixed liquid in a drying oven at 65 ℃ to obtain the unsaturated silane coupling agent modified composite material;
thirdly, 1g of modified nano SiO 2 Adding 0.16g of dodecyl sulfuric acid, 0.125g of potassium carbonate and 9 fluorine-containing monomers into 40mL of water, introducing nitrogen, heating to 65 ℃ and stirring for 1-2h; and adding 0.1g of potassium persulfate into the reaction system, performing polymerization reaction for 6 hours, adding magnesium sulfate, filtering, washing with water, and then putting into a drying oven for drying for 24 hours to obtain the modified solid-phase carrier.
Example 2
Adding 0.5g of chitosan oligosaccharide (with the viscosity less than 200 mPa.s) into 50mL of acetic acid solution (99.5% of analytical grade), stirring until the chitosan oligosaccharide is completely dissolved, adding 5mL of tetraethyl orthosilicate, setting the stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing with deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material;
adding 1.5g of the composite material into 50mL of distilled water, performing strong ultrasonic and mechanical stirring, dripping 0.045mol of vinyltrimethoxysilane into the dispersion liquid, adjusting the pH to 9-11 with concentrated ammonia water, heating to 80 ℃, stirring for reaction for 24 hours, and drying the mixed liquid in a drying oven at 65 ℃ to obtain the unsaturated silane coupling agent modified composite material; the rest of the procedure is the same as in example 1.
Example 3
Adding 0.75g of chitosan oligosaccharide (with the viscosity less than 200 mPa.s) into 50mL of acetic acid solution (99.5% of analytical grade), stirring until the chitosan oligosaccharide is completely dissolved, adding 5mL of tetraethyl orthosilicate, setting the stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing with deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material; the rest of the procedure is the same as in example 1.
Example 4
Firstly, adding 1g of chitosan oligosaccharide (with the viscosity less than 200 mPa.s) into 50mL of acetic acid solution (99.5% of analytically pure), stirring until the chitosan oligosaccharide is completely dissolved, adding 5mL of tetraethyl orthosilicate, setting the stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing with deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material; the rest of the procedure is the same as in example 1.
Example 5
Firstly, adding 1.25g of chitosan oligosaccharide (with the viscosity less than 200 mPa.s) into 50mL of acetic acid solution (99.5% of analytical grade), stirring until the chitosan oligosaccharide is completely dissolved, adding 5mL of tetraethyl orthosilicate, setting the stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material; the rest of the procedure is the same as in example 1.
Example 6
Firstly, adding 1.5g of chitosan oligosaccharide (with the viscosity less than 200 mPa.s) into 50mL of acetic acid solution (99.5% of analytical grade), stirring until the chitosan oligosaccharide is completely dissolved, adding 5mL of tetraethyl orthosilicate, setting the stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material; the rest of the procedure is the same as in example 1.
Comparative example 1
Adding 5mL of tetraethyl orthosilicate into 50mL of acetic acid solution (99.5% of analytically pure), setting stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing with deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material;
adding 1.5g of the composite material into 50mL of distilled water, performing strong ultrasonic and mechanical stirring, dripping 0.045mol of allyltrimethoxysilane into the dispersion liquid, adjusting the pH to 9-11 with concentrated ammonia water, heating to 80 ℃, stirring for reaction for 24 hours, and placing the mixed liquid into a drying oven at 65 ℃ for drying and grinding to obtain the unsaturated silane coupling agent modified composite material;
thirdly, 1g of modified nano SiO 2 Adding 0.16g of dodecyl sulfuric acid, 0.125g of potassium carbonate and 9 fluorine-containing monomers into 40mL of water, introducing nitrogen gas, and heating to 65Stirring for 1-2h at the temperature; and adding 0.1g of potassium persulfate into the reaction system, performing polymerization reaction for 6 hours, adding magnesium sulfate, filtering, washing with water, and then putting into a drying oven for drying for 24 hours to obtain the modified solid-phase carrier.
Comparative example 2
Adding 5mL of tetraethyl orthosilicate into 50mL of acetic acid solution (99.5% of analytically pure), setting stirring speed to 900r/min, stirring for 12-15h, standing for 2-3h, washing with deionized water for three times, drying in a vacuum freeze drying box at-25 ℃, grinding, and sieving with a 300-mesh sieve to obtain a composite material;
and secondly, adding 1.5g of the composite material into 50mL of distilled water, dropwise adding 0.045mol of allyltrimethoxysilane into the dispersion liquid after strong ultrasonic and mechanical stirring, adjusting the pH to 9-11 by using concentrated ammonia water, heating to 80 ℃, stirring and reacting for 24 hours, and placing the mixed liquid into a drying oven at 65 ℃ for drying and grinding to obtain the unsaturated silane coupling agent modified composite material.
Performance tests were performed on examples 1-6 and comparative examples 1-2:
wettability test: pressing the powder into a wafer by a tablet press, removing 5-10 mu L of water by a precise needle tube, measuring the angle formed by water drops on the surface of the wafer by using a JC2000D contact angle measuring instrument, and repeatedly testing to obtain the average value of the contact angles of 10 liquid drops as the contact angle of water on the surface of the solid;
specific surface area measurement: full automatic specific surface and pore analyzer model Tristar3020 was used, N 2 Performing adsorption and desorption at the temperature of liquid nitrogen of-196 ℃ as an adsorption medium, and calculating by a BET method from isotherm data to obtain the aperture, the pore surface area and the specific surface area of a sample; the test results are shown in table 1:
TABLE 1
| Project | Contact angle (°) | Specific surface area (m) 2 /g) | Pore volume (cm) 3 /g) |
| Example 1 | 110.14 | 300 | 0.92 |
| Example 2 | 111.21 | 347 | 0.98 |
| Example 3 | 108.06 | 453 | 1.07 |
| Example 4 | 110.56 | 598 | 1.43 |
| Example 5 | 113.12 | 565 | 1.36 |
| Example 6 | 110.52 | 554 | 1.31 |
| Comparative example 1 | 105.85 | 189 | 0.91 |
| Comparative example 2 | 65.74 | 177 | 0.86 |
As can be seen from table 1, the contact angles of examples 1 to 6 and comparative example 1 are both higher than comparative example 2, compared to comparative example 2, indicating that the self-made solid phase synthesis support has better hydrophobic properties; meanwhile, as shown in the table, the examples 1-6 have higher specific surface area and pore volume, and according to the examples 1-6, the content of the chitosan oligosaccharide is different, so that the pore volume and specific surface area of the synthetic material are different, and the self-made solid phase synthetic carrier in the invention has the controllability which is not available in the existing solid phase synthetic carriers, so that the synthetic efficiency is improved, and the waste of products is avoided.
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 (6)
1. A method for synthesizing a primer with high GC content, which is characterized by comprising the following steps:
step one, reacting nucleotides which are pre-connected on a modified solid phase carrier and are protected by active groups with trichloroacetic acid, and removing a protecting group DMT of a 5 '-hydroxyl group of the nucleotides to obtain a free 5' -hydroxyl group;
step two, condensation reaction;
step three, reacting with a cap;
step four, oxidation reaction: oxidizing the phosphorous acid with an oxidizing agent to form a phosphotriester;
wherein the modified solid phase carrier is prepared by the following steps:
firstly, adding chitosan oligosaccharide into acetic acid solution, stirring until the chitosan oligosaccharide is completely dissolved, adding tetraethyl orthosilicate, stirring for 12-15h, standing for 2-3h, washing with water, freeze-drying, grinding and sieving to obtain a composite material;
secondly, adding the composite material into distilled water for ultrasonic dispersion, dripping an unsaturated silane coupling agent into the dispersion liquid, adjusting the pH value to 9-11 by using strong ammonia water, heating to 80 ℃, stirring and reacting for 24 hours, and drying to obtain the unsaturated silane coupling agent modified composite material;
thirdly, adding the unsaturated silane coupling agent modified composite material, sodium dodecyl sulfate, potassium carbonate and fluorine-containing monomer into distilled water, introducing nitrogen, heating to 65 ℃ and stirring for 1-2h, adding potassium persulfate for reaction for 6h, adding magnesium sulfate, filtering, washing with water, and drying to obtain the modified solid phase carrier.
2. The method for synthesizing a primer having a high GC content according to claim 1, wherein the ratio of chitosan oligosaccharide, acetic acid solution and tetraethyl orthosilicate is 0.25-1 g/50 mL/5 mL.
3. The method for synthesizing a primer having a high GC content according to claim 1, wherein the amount ratio of the composite material, distilled water and the unsaturated silane coupling agent is 1.5 g/50 mL/0.03 mol.
4. The method for synthesizing a primer having a high GC content according to claim 1, wherein the unsaturated silane coupling agent comprises one of allyltrimethoxysilane and vinyltrimethoxysilane.
5. The method for synthesizing the primer with high GC content according to claim 1, wherein the amount ratio of the unsaturated silane coupling agent modified composite material, sodium dodecyl sulfate, potassium carbonate, fluorine-containing monomer, distilled water and potassium persulfate is 1g:0.16g:0.125g:9g:40mL:0.1g.
6. The method for synthesizing the primer with high GC content according to claim 1, wherein the fluorine-containing monomer is one or more of hexafluorobutyl acrylate, hexafluoroisopropyl methacrylate, octafluoropentyl acrylate, hexafluoroisopropyl methacrylate and octafluoropentyl methacrylate.
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