CN117801298A - Solid phase carrier for primer synthesis and preparation method thereof - Google Patents
Solid phase carrier for primer synthesis and preparation method thereof Download PDFInfo
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- CN117801298A CN117801298A CN202311614739.7A CN202311614739A CN117801298A CN 117801298 A CN117801298 A CN 117801298A CN 202311614739 A CN202311614739 A CN 202311614739A CN 117801298 A CN117801298 A CN 117801298A
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- 239000007790 solid phase Substances 0.000 title claims abstract description 23
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 238000001308 synthesis method Methods 0.000 title description 2
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000004005 microsphere Substances 0.000 claims abstract description 35
- 239000004793 Polystyrene Substances 0.000 claims abstract description 30
- 229920002223 polystyrene Polymers 0.000 claims abstract description 30
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000003756 stirring Methods 0.000 claims abstract description 27
- 229920000587 hyperbranched polymer Polymers 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 18
- 239000004593 Epoxy Substances 0.000 claims abstract description 17
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910000077 silane Inorganic materials 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000008961 swelling Effects 0.000 claims abstract description 11
- 238000007265 chloromethylation reaction Methods 0.000 claims abstract description 8
- 238000004821 distillation Methods 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 3
- 125000003700 epoxy group Chemical group 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- MUUOUUYKIVSIAR-UHFFFAOYSA-N 2-but-3-enyloxirane Chemical compound C=CCCC1CO1 MUUOUUYKIVSIAR-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 5
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 4
- RRSXICBKOPODSP-UHFFFAOYSA-N 1,4-bis(chloromethoxy)butane Chemical compound ClCOCCCCOCCl RRSXICBKOPODSP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000005457 ice water Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 230000006837 decompression Effects 0.000 claims description 3
- 239000002841 Lewis acid Substances 0.000 claims description 2
- 150000007517 lewis acids Chemical class 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims 6
- 238000011068 loading method Methods 0.000 abstract description 6
- 229920000642 polymer Polymers 0.000 abstract description 6
- 238000010923 batch production Methods 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 7
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 229910021627 Tin(IV) chloride Inorganic materials 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 239000005289 controlled pore glass Substances 0.000 description 3
- 239000010954 inorganic particle Substances 0.000 description 3
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 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 2
- 238000009835 boiling Methods 0.000 description 2
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000001668 nucleic acid synthesis Methods 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
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- 230000007423 decrease Effects 0.000 description 1
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- Silicon Polymers (AREA)
Abstract
The invention discloses a solid phase carrier for primer synthesis and a preparation method thereof, belonging to the technical field of polymer microspheres, wherein the preparation method comprises the following steps: firstly, adding double-end epoxy silane and diethylenetriamine into isopropanol, stirring for reaction, and performing reduced pressure distillation after the reaction is finished to obtain an amino-terminated hyperbranched polymer; and secondly, adding chloromethylation polystyrene microspheres into toluene for swelling, adding amino-terminated hyperbranched polymer for stirring reaction, extracting with ethanol after the reaction is finished, and drying in vacuum to obtain the solid phase carrier. According to the method, the amino-terminated hyperbranched polymer is grafted on the surface of the polystyrene microsphere to form the solid-phase carrier, so that the solid-phase carrier is controllable, high in loading capacity, good in universality and strong in stability, and can be directly used for large-scale and batch production by utilizing the conventional industrial equipment, and the reproducibility is strong.
Description
Technical Field
The invention belongs to the technical field of polymer microspheres, and particularly relates to a solid phase carrier for primer synthesis and a preparation method thereof.
Background
Primers are typically two oligonucleotide sequences that are synthesized artificially, one primer being complementary to one DNA template strand at one end of the target region and the other primer being complementary to the other DNA template strand at the other end of the target region, and function as a starting point for nucleotide polymerization, from the 3' end of which a nucleic acid polymerase can begin to synthesize a new nucleic acid strand. In the solid phase synthesis of primer oligonucleotides, inorganic particles such as CPG (controlled pore glass) or silica gel are generally adopted as solid phase carriers, and are used as inorganic silica-based carriers, the surfaces of the inorganic particles are easy to chemically derivatize, but the surfaces of the inorganic particles are rich in hydroxyl groups, the moisture in the carriers is more difficult to remove by washing with an anhydrous solvent (acetonitrile), the coupling efficiency is difficult to be influenced, wrong sequences are generated, the hydroxyl groups are easy to fall off in the synthesis process after being protected, and the synthesis efficiency of the oligonucleotides in the next step is easy to be influenced. In addition, the loading capacity is limited by the loading capacity in the synthesizer, so that the loading capacity is smaller, the synthesized sequences in unit time are less, and the higher market demand cannot be met.
Disclosure of Invention
The invention aims to provide a solid phase carrier for primer synthesis and a preparation method thereof, which are used for solving the problem of small solid phase carrier load.
The aim of the invention can be achieved by the following technical scheme:
a method for preparing a solid phase carrier for primer synthesis, comprising the following steps:
firstly, adding double-end epoxy silane and diethylenetriamine into isopropanol, stirring for reaction, and performing reduced pressure distillation after the reaction is finished to obtain an amino-terminated hyperbranched polymer;
and secondly, adding chloromethylation polystyrene microspheres into toluene for swelling, adding amino-terminated hyperbranched polymer for stirring reaction, extracting with ethanol after the reaction is finished, and drying in vacuum to obtain the solid phase carrier.
Further, the dosage mass ratio of the chloromethylation polystyrene microsphere to the amino-terminated hyperbranched polymer is 3:20-21.
Further, the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxy silane is 3-3.2:1, the dosage of the isopropanol accounts for 30 percent of the total weight of the raw materials.
Further, the temperature of the stirring reaction in the first step is 80 ℃, and the reaction time is 3-3.5h.
Further, the temperature of the stirring reaction in the second step is 70 ℃, and the reaction time is 20-24 hours.
Further, the double-ended epoxy silane is prepared by the steps of:
adding 1, 2-epoxy-5-hexene and chloroplatinic acid/isopropanol solution into toluene, stirring and reacting for 40min, dropwise adding hydrogen-containing double seal heads, wherein the dosage molar ratio of the hydrogen-containing double seal heads to the 1, 2-epoxy-5-hexene is 1:2, the addition amount of chloroplatinic acid is 50ppm; after the dripping is finished, the temperature is raised to 100 ℃ and the mixture is stirred for reaction for 12 hours, and after the reaction is finished, the solvent is removed by decompression concentration, so that the double-end epoxy silane is obtained.
Further, the chloromethylated polystyrene microsphere is prepared by the following steps:
5g of polystyrene microsphere (polystyrene/divinylbenzene polymer microsphere (PS-DVB microsphere), 10-50 μm and SiAnruixi biotechnology Co., ltd.) and 100mL of solvent (dichloromethane) are swelled, 12g-13g of 1, 4-bis (chloromethoxy) -butane is added after swelling, stirring is carried out under the ice water bath condition, then 4g of Lewis acid (stannic chloride) is dropwise added in batches, the temperature of a reaction system is controlled to be not more than 5 ℃ in the dropwise adding process, after the dropwise adding is finished, stirring reaction is carried out for 10-12h at room temperature (20-30 ℃), after the reaction is finished, 1mol/L of hydrochloric acid aqueous solution, dioxane and deionized water are sequentially used for washing until filtrate is free of chloride ions, and the chloromethylated polystyrene microsphere is obtained after 60 ℃ vacuum drying.
A solid phase carrier for primer synthesis is prepared by the preparation method.
The invention has the beneficial effects that:
the invention provides a preparation method of a solid phase carrier for primer synthesis, which is characterized in that amino-terminated hyperbranched polymer is grafted on the surface of polystyrene microsphere to form the solid phase carrier, and the solid phase carrier is controllable, high in loading capacity, good in universality, strong in stability, capable of directly utilizing the conventional industrial equipment to carry out large-scale and batch production, and strong in reproducibility.
According to the invention, on one hand, the branched structure of the amino-terminated hyperbranched polymer is utilized, the end groups of the outer shell of the polystyrene microsphere grafted hyperbranched polymer are modified into active sites, the carrying capacity of the carrier is improved, the purity of the primer nucleic acid synthesis is ensured, and the efficiency of further optimizing the nucleic acid synthesis is optimized.
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
The present example provides a double-ended epoxy silane prepared by the steps of:
1g of chloroplatinic acid is added into a brown reagent bottle containing 50mL of isopropanol, and after being stirred uniformly, the solution is treated for 25min in an ultrasonic oscillator of a water bath at 50 ℃ to obtain the catalyst chloroplatinic acid/isopropanol solution.
Adding 1, 2-epoxy-5-hexene and chloroplatinic acid/isopropanol solution into toluene, stirring and reacting for 40min, dropwise adding hydrogen-containing double seal heads, wherein the dosage molar ratio of the hydrogen-containing double seal heads to the 1, 2-epoxy-5-hexene is 1:2, the addition amount of chloroplatinic acid is 50ppm; after the dripping is finished, the temperature is raised to 100 ℃ and the mixture is stirred for reaction for 12 hours, and after the reaction is finished, the solvent is removed by decompression concentration, so that the double-end epoxy silane is obtained.
Example 2
The present example provides a chloromethylated polystyrene microsphere prepared by the steps of:
5g of polystyrene microsphere (polystyrene/divinylbenzene polymer microsphere (PS-DVB microsphere), 10-50 μm and 100mL of dichloromethane are swelled, 12g of 1, 4-bis (chloromethoxy) -butane is added after swelling, stirring is carried out under the ice water bath condition, then 4g of stannic chloride is dropwise added in batches, the temperature of a reaction system is controlled to be not more than 5 ℃ in the dropwise adding process, after the dropwise adding is finished, stirring is carried out at 20-30 ℃ for 10 hours, after the reaction is finished, 1mol/L of hydrochloric acid aqueous solution, dioxane and deionized water are sequentially used for washing until filtrate is free of chloride ions, and the chloromethylated polystyrene microsphere (the chloride content is about 18%) is obtained after vacuum drying at 60 ℃.
Example 3
The present example provides a chloromethylated polystyrene microsphere prepared by the steps of:
5g of polystyrene microsphere (polystyrene/divinylbenzene polymer microsphere (PS-DVB microsphere), 10-50 μm and SiAnruixi biotechnology Co., ltd.) are added into 100mL of methylene dichloride to swell, 13g of 1, 4-bis (chloromethoxy) -butane is added after swelling, stirring is carried out under the ice water bath condition, then 4g of stannic chloride is dropwise added in batches, the temperature of a reaction system is controlled to be not more than 5 ℃ in the dropwise adding process, after the dropwise adding is finished, stirring is carried out at 20-30 ℃ for 12h, after the reaction is finished, 1mol/L of hydrochloric acid aqueous solution, dioxane and deionized water are sequentially used for washing until the filtrate is free of chloride ions, and the chloromethylated polystyrene microsphere (the chloride content is about 18%) is obtained after vacuum drying at 60 ℃.
Example 4
The embodiment provides a preparation method of a solid phase carrier for primer synthesis, which comprises the following steps:
adding double-end epoxy silane and diethylenetriamine prepared according to the method in example 1 into isopropanol, stirring at 80 ℃ for reaction for 3.5 hours, and removing low-boiling-point substances such as isopropanol by reduced pressure distillation after the reaction is finished to obtain an amino-terminated hyperbranched polymer; the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxy silane is 3:1, the dosage of the isopropanol accounts for 30 percent of the total weight of the raw materials;
adding chloromethylated polystyrene microspheres into toluene for swelling, adding amino-terminated hyperbranched polymer, stirring for 20 hours at the temperature of 70 ℃, extracting with ethanol after the reaction is finished, and vacuum drying at the temperature of 60 ℃ to obtain the solid phase carrier. The dosage mass ratio of the chloromethylation polystyrene microsphere to the amino-terminated hyperbranched polymer is 3:20.
example 5
Adding double-end epoxy silane and diethylenetriamine prepared according to the method in example 1 into isopropanol, stirring at 80 ℃ for reaction for 3 hours, and removing low-boiling substances such as isopropanol and the like through reduced pressure distillation after the reaction is finished to obtain an amino-terminated hyperbranched polymer; the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxy silane is 3:1, the dosage of the isopropanol accounts for 30 percent of the total weight of the raw materials;
adding chloromethylated polystyrene microspheres into toluene for swelling, adding amino-terminated hyperbranched polymer, stirring for 24 hours at the temperature of 70 ℃, extracting with ethanol after the reaction is finished, and vacuum drying at the temperature of 60 ℃ to obtain the solid phase carrier. The dosage mass ratio of the chloromethylation polystyrene microsphere to the amino-terminated hyperbranched polymer is 3:20.
example 6
Adding double-end epoxy silane and diethylenetriamine prepared according to the method in example 1 into isopropanol, stirring at 80 ℃ for reaction for 3 hours, and removing low-boiling substances such as isopropanol and the like through reduced pressure distillation after the reaction is finished to obtain an amino-terminated hyperbranched polymer; the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxy silane is 3:1, the dosage of the isopropanol accounts for 30 percent of the total weight of the raw materials;
adding chloromethylated polystyrene microspheres into toluene for swelling, adding amino-terminated hyperbranched polymer, stirring for 24 hours at the temperature of 70 ℃, extracting with ethanol after the reaction is finished, and vacuum drying at the temperature of 60 ℃ to obtain the solid phase carrier. The dosage mass ratio of the chloromethylation polystyrene microsphere to the amino-terminated hyperbranched polymer is 3:21.
comparative example 1
In this comparative example, the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxysilane was 2:1.
comparative example 2
In this comparative example, the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxysilane was 4:1.
comparative example 3
Compared with the example 5, the dosage mass ratio of the chloromethylation polystyrene microsphere to the amino-terminated hyperbranched polymer is 3:15.
comparative example 4
The CPG vector is commercialized.
Test example:
synthesizing a nucleic acid sequence 5'-AGCUA-3'; 30mg of the vectors of example 4 to example 6 and comparative examples 1 to 4 were weighed, and packed in a column synthesizer to synthesize nucleic acids. And (3) carrying out shear deprotection after synthesis: 2mol/L ammonia in methanol is added, the reaction is carried out for 1h at 20 ℃, the product is cut from the carrier, 32wt% ammonia water is added, and the reaction is carried out for 4h at 55 ℃ to remove the protecting group. The supernatant was collected, concentrated in vacuo at 65℃and 200. Mu.L of TE buffer (pH=7.5) was added thereto, and the mixture was allowed to stand at room temperature for 1 hour to dissolve the supernatant sufficiently, and the insoluble matter was discarded from the supernatant and placed in a fresh 2mLEP tube.
Desalting and purifying: to the nucleic acid to be purified, 1/10 of the nucleic acid solution volume (20 μl) of sodium acetate (3 mol/LpH =5.2) was added and thoroughly mixed: adding 220 μl ethanol, mixing thoroughly, standing at-20deg.C for 20min, centrifuging for 10min, removing supernatant, adding 1mL70% ethanol, centrifuging for 2min, removing supernatant, opening at room temperature, volatilizing to dryness, and adding 100mL buffer for dissolving (4deg.C overnight). The nucleic acid product obtained was diluted 100 times and quantified with NanoDrop2000, and the concentration was recorded spectrophotometrically. The results are shown in table 1 below:
TABLE 1
| Project | Example 4 | Example 5 | Example 6 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| Concentration (ng/. Mu.L) | 257.1 | 269.6 | 267.9 | 225.0 | 236.3 | 185.9 | 197.2 |
From the test results, the self-made carrier has high load, and from the results of comparative examples 1 and 2, it is known that adjusting the raw material proportion of the treated amino-terminated hyperbranched polymer reduces the branching degree of the polymer, and that too small an amount of epoxy groups in the double-ended epoxy silane can hardly form a branched structure, and too much an amount of epoxy groups in the double-ended epoxy silane can cause the reduction of the content of amino-terminated groups, which can affect the load. From the results of comparative example 3, it is seen that decreasing the content of amino-terminated hyperbranched polymer in the carrier decreases the loading.
The product was dissolved in 20% acetonitrile in water and tested by reverse phase high performance liquid chromatography (RP-HPLC), and the purity of the samples of the examples and comparative examples were greater than 90%. The self-made carrier can obtain the product with the purity similar to that of the commercial products.
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 method for preparing a solid phase carrier for primer synthesis, which is characterized by comprising the following steps:
firstly, adding double-end epoxy silane and diethylenetriamine into isopropanol, stirring for reaction, and performing reduced pressure distillation after the reaction is finished to obtain an amino-terminated hyperbranched polymer;
and secondly, adding chloromethylation polystyrene microspheres into toluene for swelling, adding amino-terminated hyperbranched polymer for stirring reaction, extracting with ethanol after the reaction is finished, and drying in vacuum to obtain the solid phase carrier.
2. The method for preparing a solid phase carrier for primer synthesis according to claim 1, wherein the dosage mass ratio of chloromethylated polystyrene microsphere to amino-terminated hyperbranched polymer is 3:20-21.
3. The method for preparing a solid support for primer synthesis according to claim 1, wherein the molar ratio of epoxy groups to diethylenetriamine in the double-ended epoxysilane is 3-3.2:1, the dosage of the isopropanol accounts for 30 percent of the total weight of the raw materials.
4. The method for preparing a solid support for primer synthesis according to claim 1, wherein the temperature of the stirring reaction in the first step is 80℃and the reaction time is 3 to 3.5 hours.
5. The method for preparing a solid support for primer synthesis according to claim 1, wherein the temperature of the stirring reaction in the second step is 70℃and the reaction time is 20 to 24 hours.
6. The method for preparing a solid support for primer synthesis according to claim 1, wherein the double-ended epoxysilane is prepared by the steps of:
adding 1, 2-epoxy-5-hexene and chloroplatinic acid/isopropanol solution into toluene, stirring and reacting for 40min, dropwise adding hydrogen-containing double seal heads, wherein the dosage molar ratio of the hydrogen-containing double seal heads to the 1, 2-epoxy-5-hexene is 1:2, the addition amount of chloroplatinic acid is 50ppm; after the dripping is finished, the temperature is raised to 100 ℃ and the mixture is stirred for reaction for 12 hours, and after the reaction is finished, the solvent is removed by decompression concentration, so that the double-end epoxy silane is obtained.
7. The method for preparing a solid support for primer synthesis according to claim 1, wherein the chloromethylated polystyrene microspheres are prepared by the steps of:
adding polystyrene microspheres into a solvent for swelling, adding 1, 4-bis (chloromethoxy) -butane after swelling, stirring under ice water bath conditions, then dropwise adding Lewis acid in batches, controlling the temperature of a reaction system to be not more than 5 ℃ in the dropwise adding process, stirring at room temperature for reaction after the dropwise adding is finished, washing with 1mol/L hydrochloric acid aqueous solution, dioxane and deionized water in sequence until filtrate is free of chloride ions after the reaction is finished, and vacuum drying to obtain chloromethylated polystyrene microspheres.
8. A solid support for primer synthesis prepared by the method of any one of claims 1 to 7.
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