Solid support for primer synthesis and method for its preparation

Abstract

The application discloses a solid phase carrier for primer synthesis and a preparation method thereof, and belongs to the technical field of polymer microspheres, and the preparation method comprises the following steps: in the first step, a double-end epoxy silane and diethylenetriamine are added into isopropyl alcohol, and stirring reaction is carried out; after the reaction is completed, vacuum distillation is carried out to obtain an end-amino hyperbranched polymer; in the second step, chloromethylated polystyrene microspheres are added into toluene to be swelled, then the end-amino hyperbranched polymer is added to carry out stirring reaction, after the reaction is completed, ethanol extraction is carried out, and vacuum drying is carried out to obtain the solid phase carrier. According to the method, the end-amino hyperbranched polymer is grafted on the surface of the polystyrene microspheres to form the solid phase carrier, the method is controllable, the loading capacity is high, the universality is good, the stability is strong, the existing industrial conventional equipment can be directly used for large-scale and batch production, and the reproducibility is strong.

Description

Technical Field

[0001] This invention belongs to the field of polymer microsphere technology, specifically relating to a solid-phase support for primer synthesis and its preparation method. Background Technology

[0002] Primers are typically two artificially synthesized oligonucleotide sequences. One primer is complementary to one DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region. Their function is to serve as the initiation point for nucleotide polymerization, allowing nucleic acid polymerase to synthesize new nucleic acid chains starting from their 3' ends. In solid-phase synthesis of primer oligonucleotides, inorganic particles such as CPG (converterless glass) or silica gel are generally used as solid-phase supports. While inorganic silica-based supports are easily chemically derivatized, their surface is rich in hydroxyl groups, making it difficult to remove moisture using anhydrous solvents (acetonitrile). This difficulty in dehydration affects coupling efficiency, generates incorrect sequences, and the protected hydroxyl groups are prone to detachment during synthesis, potentially affecting the efficiency of subsequent oligonucleotide synthesis. Furthermore, limitations in the packing capacity of the synthesizer result in a small loading capacity, leading to fewer sequences synthesized per unit time, which cannot meet higher market demands. Summary of the Invention

[0003] The purpose of this invention is to provide a solid-phase support for primer synthesis and its preparation method, so as to solve the problem of low loading capacity of solid-phase supports.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] A method for preparing a solid-phase support for primer synthesis includes the following steps:

[0006] Step 1: Add di-terminated epoxy silane and diethylenetriamine to isopropanol, stir and react. After the reaction is complete, distill under reduced pressure to obtain the amino-terminated hyperbranched polymer.

[0007] The second step involves adding chloromethylated polystyrene microspheres to toluene to swell, then adding terminal amino hyperbranched polymers and stirring to react. After the reaction is complete, the mixture is extracted with ethanol and dried under vacuum to obtain a solid support.

[0008] Furthermore, the mass ratio of chloromethylated polystyrene microspheres to amino-terminated hyperbranched polymers is 3:20-21.

[0009] Furthermore, the molar ratio of epoxy group to diethylenetriamine in the di-terminated epoxy silane is 3-3.2:1, and the amount of isopropanol accounts for 30% of the total weight of the raw materials.

[0010] Furthermore, the temperature of the stirring reaction in the first step is 80℃, and the reaction time is 3-3.5h.

[0011] Furthermore, in the second step, the stirring reaction temperature is 70℃, and the reaction time is 20-24h.

[0012] Furthermore, the di-terminated epoxy silane is prepared by the following steps:

[0013] 1,2-Epoxy-5-hexene and chloroplatinic acid / isopropanol solution were added to toluene and stirred for 40 min. Hydrogen-containing double-ended heads were then added dropwise, with a molar ratio of 1:2 between the hydrogen-containing double-ended heads and 1,2-epoxy-5-hexene, and 50 ppm of chloroplatinic acid was added. After the addition was complete, the temperature was raised to 100 °C and the reaction was stirred for 12 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a double-terminated epoxy silane.

[0014] Furthermore, the chloromethylated polystyrene microspheres are prepared by the following steps:

[0015] 5g of polystyrene microspheres (polystyrene / divinylbenzene polymer microspheres (PS-DVB microspheres), 10-50μm, Xi'an Ruixi Biotechnology Co., Ltd.) were swollen in 100mL of solvent (dichloromethane). After swelling, 12g-13g of 1,4-di(chloromethoxy)-butane was added, and the mixture was stirred in an ice-water bath. Then, 4g of Lewis acid (tin tetrachloride) was added dropwise in batches. During the dropwise addition, the temperature of the reaction system was controlled not to exceed 5℃. After the dropwise addition was completed, the mixture was stirred at room temperature (20-30℃) for 10-12h. After the reaction was completed, the mixture was washed successively with 1mol / L hydrochloric acid aqueous solution, dioxane, and deionized water until the filtrate was free of chloride ions. After vacuum drying at 60℃, chloromethylated polystyrene microspheres were obtained.

[0016] A solid-phase support for primer synthesis is prepared by the above-described method.

[0017] The beneficial effects of this invention are:

[0018] This invention provides a method for preparing a solid-phase support for primer synthesis. The method forms a solid-phase support by grafting terminal amino hyperbranched polymers onto the surface of polystyrene microspheres. The solid-phase support is controllable, has a high loading capacity, good versatility, strong stability, and can be directly produced on a large scale and in batches using existing industrial conventional equipment. It also has strong reproducibility.

[0019] In this invention, on the one hand, the branched structure of the terminal amino hyperbranched polymer is utilized to modify the terminal groups of the hyperbranched polymer shell into active sites by grafting them onto polystyrene microspheres, thereby increasing the carrier's loading capacity and ensuring the purity of primer nucleic acid synthesis, thus optimizing the efficiency of nucleic acid synthesis. On the other hand, the terminal amino hyperbranched polymer is a hyperbranched organosilicon. The polystyrene carrier surface is hydrophobic. After treatment with the terminal amino hyperbranched polymer, the active sites after amino derivatization are increased, and the hyperbranched polymer forms a hydrophobic cavity. Rinsing with anhydrous organic solvent can completely remove residual moisture from the surface, reducing the interference of moisture on nucleic acid primer synthesis. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1

[0022] This embodiment provides a doubly epoxy-terminated silane, prepared through the following steps:

[0023] Add 1g of chloroplatinic acid to a brown reagent bottle containing 50mL of isopropanol, stir well, and then treat in an ultrasonic oscillator at 50℃ for 25min to obtain the catalyst chloroplatinic acid / isopropanol solution.

[0024] 1,2-Epoxy-5-hexene and chloroplatinic acid / isopropanol solution were added to toluene and stirred for 40 min. Hydrogen-containing double-ended heads were then added dropwise, with a molar ratio of 1:2 between the hydrogen-containing double-ended heads and 1,2-epoxy-5-hexene, and 50 ppm of chloroplatinic acid was added. After the addition was complete, the temperature was raised to 100 °C and the reaction was stirred for 12 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a double-terminated epoxy silane.

[0025] Example 2

[0026] This embodiment provides chloromethylated polystyrene microspheres, which are prepared through the following steps:

[0027] 5g of polystyrene microspheres (polystyrene / divinylbenzene polymer microspheres (PS-DVB microspheres), 10-50μm, Xi'an Ruixi Biotechnology Co., Ltd.) were swollen in 100mL of dichloromethane. After swelling, 12g of 1,4-di(chloromethoxy)-butane was added, and the mixture was stirred in an ice-water bath. Then, 4g of tin tetrachloride was added dropwise in batches, and the temperature of the reaction system was controlled not to exceed 5℃ during the dropwise addition. After the dropwise addition was completed, the mixture was stirred at 20-30℃ for 10h. After the reaction was completed, the mixture was washed successively with 1mol / L hydrochloric acid aqueous solution, dioxane, and deionized water until the filtrate was free of chloride ions. After vacuum drying at 60℃, chloromethylated polystyrene microspheres (chlorine content of approximately 18%) were obtained.

[0028] Example 3

[0029] This embodiment provides chloromethylated polystyrene microspheres, which are prepared through the following steps:

[0030] 5g of polystyrene microspheres (polystyrene / divinylbenzene polymer microspheres (PS-DVB microspheres), 10-50μm, Xi'an Ruixi Biotechnology Co., Ltd.) were added to 100mL of dichloromethane to swell. After swelling, 13g of 1,4-di(chloromethoxy)-butane was added, and the mixture was stirred in an ice-water bath. Then, 4g of tin tetrachloride was added dropwise in batches, and the temperature of the reaction system was controlled not to exceed 5℃ during the dropwise addition. After the dropwise addition was completed, the mixture was stirred at 20-30℃ for 12h. After the reaction was completed, the mixture was washed successively with 1mol / L hydrochloric acid aqueous solution, dioxane, and deionized water until the filtrate was free of chloride ions. After vacuum drying at 60℃, chloromethylated polystyrene microspheres (chlorine content of approximately 18%) were obtained.

[0031] Example 4

[0032] This embodiment provides a method for preparing a solid-phase support for primer synthesis, comprising the following steps:

[0033] The di-terminated epoxy silane and diethylenetriamine prepared according to the method in Example 1 were added to isopropanol and stirred at 80°C for 3.5 h. After the reaction was completed, isopropanol and other low-boiling substances were removed by vacuum distillation to obtain the terminal amino hyperbranched polymer. The molar ratio of epoxy groups to diethylenetriamine in the di-terminated epoxy silane was 3:1, and the amount of isopropanol accounted for 30% of the total weight of the raw materials.

[0034] Chloromethylated polystyrene microspheres were swollen in toluene, and then an amino-terminated hyperbranched polymer was added. The mixture was stirred at 70°C for 20 h. After the reaction was complete, the mixture was extracted with ethanol and dried under vacuum at 60°C to obtain a solid support. The mass ratio of chloromethylated polystyrene microspheres to amino-terminated hyperbranched polymer was 3:20.

[0035] Example 5

[0036] The di-terminated epoxy silane and diethylenetriamine prepared according to the method in Example 1 were added to isopropanol and stirred at 80°C for 3 hours. After the reaction was completed, isopropanol and other low-boiling substances were removed by vacuum distillation to obtain the terminal amino hyperbranched polymer. The molar ratio of epoxy group to diethylenetriamine in the di-terminated epoxy silane was 3:1, and the amount of isopropanol accounted for 30% of the total weight of the raw materials.

[0037] Chloromethylated polystyrene microspheres were swollen in toluene, and then an amino-terminated hyperbranched polymer was added. The mixture was stirred at 70°C for 24 hours. After the reaction was completed, the mixture was extracted with ethanol and dried under vacuum at 60°C to obtain a solid support. The mass ratio of chloromethylated polystyrene microspheres to amino-terminated hyperbranched polymer was 3:20.

[0038] Example 6

[0039] The di-terminated epoxy silane and diethylenetriamine prepared according to the method in Example 1 were added to isopropanol and stirred at 80°C for 3 hours. After the reaction was completed, isopropanol and other low-boiling substances were removed by vacuum distillation to obtain the terminal amino hyperbranched polymer. The molar ratio of epoxy group to diethylenetriamine in the di-terminated epoxy silane was 3:1, and the amount of isopropanol accounted for 30% of the total weight of the raw materials.

[0040] Chloromethylated polystyrene microspheres were swollen in toluene, and then an amino-terminated hyperbranched polymer was added. The mixture was stirred at 70°C for 24 hours. After the reaction was completed, the mixture was extracted with ethanol and dried under vacuum at 60°C to obtain a solid support. The mass ratio of chloromethylated polystyrene microspheres to amino-terminated hyperbranched polymer was 3:21.

[0041] Comparative Example 1

[0042] Compared with Example 5, this comparative example uses a 2:1 molar ratio of epoxy groups and diethylenetriamine in a di-terminated epoxy silane.

[0043] Comparative Example 2

[0044] Compared with Example 5, this comparative example uses a 4:1 molar ratio of epoxy groups and diethylenetriamine in a di-terminated epoxy silane.

[0045] Comparative Example 3

[0046] Compared with Example 5, the comparative example uses a mass ratio of chloromethylated polystyrene microspheres to amino-terminated hyperbranched polymers of 3:15.

[0047] Comparative Example 4

[0048] This is a commercially available CPG carrier.

[0049] Test example:

[0050] The nucleic acid sequence 5′-AGCUA-3′ was synthesized. 30 mg of the vectors from Examples 4-6 and Comparative Examples 1-4 were weighed and filled into a column synthesizer for nucleic acid synthesis. After synthesis, deprotection was performed by cleavage: a methanol solution of 2 mol / L ammonia was added, and the reaction was carried out at 20°C for 1 h. The product was then cleaved from the vector, and 32 wt% ammonia was added, followed by reaction at 55°C for 4 h to remove the protecting group. The supernatant was collected, concentrated under vacuum at 65°C, and 200 μL of TE buffer (pH = 7.5) was added. The mixture was allowed to stand at room temperature for 1 h to fully dissolve, and the insoluble matter was discarded from the supernatant. The solution was then transferred to a new 2 mL EP tube.

[0051] Desalting and purification: Add 1 / 10 volume (20 μL) of sodium acetate (3 mol / L, pH 5.2) to the nucleic acid to be purified and mix thoroughly. Add 220 μL of ethanol, mix thoroughly, incubate at -20℃ for 20 min, centrifuge for 10 min, remove the supernatant, add 1 mL of 70% ethanol, centrifuge for 2 min, remove the supernatant, leave exposed at room temperature to evaporate to dryness, then dissolve in 100 mL of LTE buffer (4℃, overnight). Dilute the obtained nucleic acid product 100-fold and quantify using NanoDrop2000. Record the concentration using spectrophotometry. The results are shown in Table 1 below.

[0052] Table 1

[0053] project Example 4 Example 5 Example 6 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Concentration (ng / μL) 257.1 269.6 267.9 225.0 236.3 185.9 197.2

[0054] The test results show that the self-made carrier has a high loading capacity. The results of Comparative Examples 1 and 2 indicate that adjusting the proportion of the terminal amino hyperbranched polymer raw materials reduces the degree of branching of the polymer. Insufficient epoxy groups in the di-epoxy silane make it difficult to form branched structures, while excessive epoxy groups reduce the content of terminal amino groups, both affecting the loading capacity. The results of Comparative Example 3 show that reducing the content of the terminal amino hyperbranched polymer in the carrier reduces the loading capacity.

[0055] The product was dissolved in a 20% acetonitrile aqueous solution and analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The purity of the samples in the examples and comparative examples was greater than 90%. The self-made carrier yielded products with purity similar to commercially available products.

[0056] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0057] Although embodiments of the 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 to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a solid-phase support for primer synthesis, characterized in that, Includes the following steps: Step 1: Add di-terminated epoxy siloxane and diethylenetriamine to isopropanol, stir and react. After the reaction is complete, distill under reduced pressure to obtain the terminal amino hyperbranched polymer. The second step involves adding chloromethylated polystyrene microspheres to toluene to swell, then adding terminal amino hyperbranched polymers and stirring to react. After the reaction is complete, the mixture is extracted with ethanol and dried under vacuum to obtain a solid support. The doubly epoxy-terminated siloxane is prepared by the following steps: 1,2-Epoxy-5-hexene and chloroplatinic acid / isopropanol solution were added to toluene and stirred for 40 min. Hydrogen-containing double-ended heads were then added dropwise, with a molar ratio of 1:2 between the hydrogen-containing double-ended heads and 1,2-epoxy-5-hexene, and 50 ppm of chloroplatinic acid was added. After the addition was complete, the temperature was raised to 100 °C and the reaction was stirred for 12 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a double-terminated epoxysiloxane.

2. The method for preparing a solid-phase support for primer synthesis according to claim 1, characterized in that, The mass ratio of chloromethylated polystyrene microspheres to amino-terminated hyperbranched polymers is 3:20-21.

3. The method for preparing a solid-phase support for primer synthesis according to claim 1, characterized in that, The molar ratio of epoxy groups and diethylenetriamine in the di-terminated epoxy siloxane is 3-3.2:1, and the amount of isopropanol accounts for 30% of the total weight of the raw materials.

4. The method for preparing a solid-phase support for primer synthesis according to claim 1, characterized in that, The temperature of the stirring reaction in the first step is 80℃, and the reaction time is 3-3.5h.

5. The method for preparing a solid-phase support for primer synthesis according to claim 1, characterized in that, In the second step, the stirring reaction temperature is 70℃, and the reaction time is 20-24h.

6. The method for preparing a solid-phase support for primer synthesis according to claim 1, characterized in that, The chloromethylated polystyrene microspheres were prepared by the following steps: Polystyrene microspheres were added to a solvent to swell, and then 1,4-di(chloromethoxy)-butane was added. The mixture was stirred under ice-water bath conditions, and then Lewis acid was added dropwise in batches. During the dropwise addition, the temperature of the reaction system was controlled not to exceed 5°C. After the dropwise addition was completed, the reaction was stirred at room temperature. After the reaction was completed, the mixture was washed successively with 1 mol / L hydrochloric acid aqueous solution, dioxane, and deionized water until the filtrate was free of chloride ions. After vacuum drying, chloromethylated polystyrene microspheres were obtained.

7. A solid-phase support for primer synthesis, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.