A styrene / nitrogen-containing heterocyclic copolymer, its preparation method and application

Abstract

This invention belongs to the field of oligonucleotide synthesis, specifically relating to a styrene / nitrogen-containing heterocyclic copolymer, its preparation method, and its application. The modified styrene / nitrogen-containing heterocyclic copolymer has the structure shown in formula (2). The key to this invention is the introduction of a nitrogen-containing heterocycle into the polystyrene molecular chain, which effectively increases the effective loading of nucleotides. Furthermore, compared to introducing a nitrogen-containing heterocyclic compound alone into the oligonucleotide synthesis system, it significantly improves the synthesis efficiency and purity of nucleic acids. Formula (2)

Description

Technical Field

[0001] This invention belongs to the field of oligonucleotide synthesis, specifically relating to a styrene / nitrogen-containing heterocyclic copolymer, its preparation method, and its application. Background Technology

[0002] Oligonucleotides are short-chain nucleic acids composed of multiple nucleotide monomers linked by phosphodiester bonds, and they have wide applications in gene research, drug development, and molecular diagnostics. With the development of molecular biology techniques, nucleic acid drug development, diagnosis of genetic and infectious diseases, and gene research are progressing rapidly, thus requiring the synthesis of large quantities of oligonucleotides to support these studies. The commonly used synthetic method for oligonucleotides is the solid-phase phosphorous amide method (solid-phase synthesis). In solid-phase synthesis reactions, the synthetic support plays a crucial role; controllable microporous glass (CPG) and polystyrene (PS) are two commonly used types of supports.

[0003] Controllable porous glass (CPG) is made of silica, a rigid, non-expanding solid support with advantages such as good mechanical properties, non-swelling, and controllable pore size. It also possesses definite pore size and surface chemical properties, making it ideal for solid-phase oligonucleotide synthesis. Functionalized controllable porous glass (CPG) is a key solid support in oligonucleotide synthesis, allowing nucleosides to be attached to the CPG surface, followed by chain extension via phosphoramidite chemistry. This functionalization can be used to synthesize oligonucleotides with various modifications and applications, and offers the following advantages: (1) Highly efficient oligonucleotide synthesis: Functionalized CPG supports allow for efficient and reproducible synthesis of oligonucleotides using standard phosphoramidite chemistry methods; (2) Wide range of applications: It can synthesize oligonucleotides with various modifications, making functionalized CPG supports suitable for a wide range of applications, including drug development, diagnostics, and research. Furthermore, CPG spheres contain many irregular channels with pore size stability. Due to spatial constraints, larger pore sizes are suitable for long-fragment synthesis, while smaller pore sizes are suitable for short-fragment synthesis. CPGs with different pore sizes can be used to synthesize oligonucleotide products with different loading capacities: (1) CPG products with pore sizes of 500 Å / 600 Å are suitable for the synthesis of oligonucleotides <35 mers, such as therapeutic oligonucleotides, with a synthesis loading capacity of up to 100 μmol / g; (2) CPG products with pore sizes of 1000 Å are suitable for the synthesis of oligonucleotides >35 mers or highly modified oligonucleotides; (3) CPG products with pore sizes of 2000 Å / 3000 Å are suitable for the synthesis of oligonucleotides on a scale of 80 mers or more, with a synthesis loading capacity of typically 10~20 μmol / g. However, because it is difficult to achieve high loading capacities (<100 μmol / g) with CPG, and because CPG is easily corroded by chemical reagents, thus contaminating the synthesized products, the application scope of CPG as a carrier for large-scale oligonucleotide synthesis is greatly limited.

[0004] Polystyrene (PS) supports are commonly used in solid-phase organic synthesis. They consist of small beads with diameters ranging from 20 to 150 μm. Monodisperse polystyrene microspheres are cross-linked using a cross-linking agent, typically divinylbenzene, with a cross-linking degree between 1% and 2%. This low-cross-linking resin is widely used for the synthesis of small nucleotides with fewer than 30 base units. Within this cross-linking range, the resin exhibits excellent swelling properties in DMF and DCM, forming a three-dimensional network structure that allows reactant molecules to move freely within the resin. While polystyrene (PS) supports offer the advantage of high loading capacity, making them suitable for large-scale oligonucleotide synthesis, they also suffer from drawbacks such as high swelling (3-6 times expansion), high solvent consumption, and the limitation to short-chain synthesis. A solid-phase support that is insoluble in various solvents but capable of swelling is a fundamental structural element. Furthermore, research indicates that using polystyrene (PS) as a support does not improve the synthesis efficiency of oligonucleotides and makes it difficult to obtain high-purity oligonucleotides. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings of existing controllable microporous glass carriers, such as low loading capacity and low synthesis efficiency and purity when synthesizing oligonucleotides using polystyrene carriers. Instead, this invention provides a styrene / nitrogen-containing heterocyclic copolymer with high loading capacity, which can improve the synthesis efficiency and purity when used as a carrier to synthesize oligonucleotides.

[0006] A second objective of this invention is to provide a modified styrene / nitrogen-containing heterocyclic copolymer.

[0007] A third objective of this invention is to provide a method for preparing the above-mentioned styrene / nitrogen-containing heterocyclic copolymer.

[0008] A fourth objective of this invention is to provide a method for preparing the above-mentioned modified styrene / nitrogen-containing heterocyclic copolymer.

[0009] The fifth objective of this invention is to provide the above-mentioned styrene / nitrogen-containing heterocyclic copolymers and / or modified styrene / nitrogen-containing heterocyclic copolymers as oligonucleotide synthesis carriers.

[0010] The styrene / nitrogen-containing heterocyclic copolymer provided by this invention has the structure shown in formula (1): Equation (1) In formula (1), A, B and Y are N, C, S or O independently; R1 is a single bond or a C1~C5 alkylene group; X1 is -NH2 or -OH; m and n represent the degree of polymerization of styrene-derived structural units and nitrogen-containing heterocyclic structural units, respectively, m:n=(1~9):1; * represents the bond end.

[0011] The modified styrene / nitrogen-containing heterocyclic copolymer provided by this invention has the structure shown in formula (2): Equation (2) In formula (2), A, B and Y are N, C, S or O independently; R1 is a single bond or a C1~C5 alkylene group; X2 is -NH- or -O-; m and n represent the degree of polymerization of styrene-derived structural units and nitrogen-containing heterocyclic structural units, respectively, m:n=(1~9):1; Z is a linking group that can undergo coupling reaction with nucleoside phosphoramidite; * represents the bond terminator.

[0012] The method for preparing styrene / nitrogen-containing heterocyclic copolymers provided by this invention includes the following steps: S1: Halogenated styrene and vinyl nitrile are subjected to free radical polymerization under inert gas protection to obtain a halogenated styrene / vinyl nitrile copolymer; S2: The halogenated styrene / vinyl nitrile copolymer is substituted with sodium azide to obtain styrene-azide / nitrogen-containing heterocyclic copolymer; S3: The styrene / nitrogen-containing heterocyclic copolymer is reduced to obtain the styrene / nitrogen-containing heterocyclic copolymer.

[0013] The method for preparing the modified styrene / nitrogen-containing heterocyclic copolymer provided by the present invention includes condensing a styrene / nitrogen-containing heterocyclic copolymer having the structure shown in formula (1) with a linker to attach a linker group capable of coupling with nucleoside phosphoramidite to the X1 position of the styrene / nitrogen-containing heterocyclic copolymer.

[0014] The key to this invention lies in introducing nitrogen-containing heterocycles into the polystyrene molecular chain. Compared to introducing nitrogen-containing heterocyclic compounds alone into the oligonucleotide synthesis system, this significantly improves the synthesis efficiency and purity of nucleic acids. The reason for this is presumably that the nitrogen-containing heterocycle can react with phosphonium amide monomers to form an activated intermediate. Introducing the nitrogen-containing heterocycle into the polystyrene molecular chain accelerates the formation of this activated intermediate and reduces the formation of interfering substances, thereby significantly accelerating the reaction and improving its efficiency and purity. Furthermore, the styrene / nitrogen-containing heterocyclic copolymer and modified styrene / nitrogen-containing heterocyclic copolymer provided by this invention are not only corrosion-resistant but also prevent the expansion of traditional polymer carriers during heating / chain extension and from being affected by solvents. This avoids the problem of low loading capacity due to expansion, and effectively utilizes the high loading capacity of the resin, greatly improving the effective loading of nucleotides and possessing a higher nucleotide monomer loading capacity. Detailed Implementation

[0015] The styrene / nitrogen-containing heterocyclic copolymer provided by this invention has the structure shown in formula (1): Equation (1) In formula (1), A, B, and Y are independently N, C, S, or O; R1 is a single bond or a C1~C5 alkylene group; X1 is -NH2 or -OH; m and n represent the degree of polymerization of the styrene-derived structural unit and the nitrogen-containing heterocyclic structural unit, respectively, m:n=(1~9):1; * represents the bond terminator. The styrene-derived structural unit refers to the structural unit containing a benzene ring in formula (1), that is, the structural unit shown in formula (A); the nitrogen-containing heterocyclic structural unit refers to the structural unit containing a nitrogen heterocycle in formula (1), that is, the structural unit shown in formula (B). Preferably, A is N and B and Y are independently N or C, or A is C and B and Y are independently N or C. In this case, the nitrogen-containing heterocycle has N or C elements at at least two specific positions, which is more conducive to further improving the efficiency and purity of nucleic acid synthesis. C1-C5 alkylene groups can be listed as methylene, ethylene, n-propylene, isopropylene, n-butylene, secondary butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, tert-pentylene, or neopentylene. The ratio of m to n is (1-9):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or any value between them. It should be noted that m and n are calculated based on the amount of material fed.

[0016] Formula (A) Formula (B) In this invention, formula (1) above is only used to represent the content of each structure and each structural unit contained in the styrene / nitrogen-containing heterocyclic copolymer, and cannot represent the arrangement relationship between the structural units. The styrene / nitrogen-containing heterocyclic copolymer can be a random copolymer, a block copolymer, or an alternating copolymer, preferably a random copolymer. In addition, the number average molecular weight of the styrene / nitrogen-containing heterocyclic copolymer is preferably 5,000 to 20,000, such as 5,000, 8,000, 10,000, 12,000, 15,000, 18,000, 20,000, or any value between them.

[0017] The modified styrene / nitrogen-containing heterocyclic copolymer provided by this invention has the structure shown in formula (2): Equation (2) In formula (2), A, B, and Y are independently N, C, S, or O; R1 is a single bond or a C1~C5 alkylene group; X2 is -NH- or -O-; m and n represent the degree of polymerization of the styrene-derived structural unit and the nitrogen-containing heterocyclic structural unit, respectively, m:n=(1~9):1; Z is a linking group capable of coupling with nucleoside phosphoramidite; * represents the bond terminator. The styrene-derived structural unit refers to the structural unit containing a benzene ring in formula (2), that is, the structural unit shown in formula (C); the nitrogen-containing heterocyclic structural unit refers to the structural unit containing a nitrogen heterocycle in formula (2), that is, the structural unit shown in formula (D). Preferably, A is N and B and Y are independently N or C, or A is C and B and Y are independently N or C. In this case, the nitrogen-containing heterocycle has N or C elements at at least two specific positions, which is more conducive to further improving the efficiency and purity of nucleic acid synthesis. C1-C5 alkylene groups can be listed as methylene, ethylene, n-propylene, isopropylene, n-butylene, secondary butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, tert-pentylene, or neopentylene. The ratio of m to n is (1-9):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or any value between them. It should be noted that m and n are calculated based on the amount of material fed.

[0018] Formula (C) Equation (D) In one specific embodiment, Z is derived from at least one of the following linkers (Unylinker), and it should be noted that the term "derived from" refers to the residue obtained after the linker reacts with group X1 in the styrene / nitrogen-containing heterocyclic copolymer.

[0019] ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; In this group, R is H, a halogen, or a C1-C5 alkoxy group, and R' is a group derived from adenine (A), guanine (G), cytosine (C), or thymine (T). Specific examples of the C1-C5 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, or isobutoxy. The structures of adenine (A), guanine (G), cytosine (C), or thymine (T) are shown below: .

[0020] In the above linkers, DMT represents a protecting group, which can be dimethoxytriphenylmethyl, p-methoxytriphenylmethyl, triphenylmethyl, etc. The carboxyl group in the linker undergoes a condensation reaction with the amino / hydroxyl group in the styrene / nitrogen-containing heterocyclic copolymer to form a linker group capable of coupling with the nucleoside phosphoramidite. When the linker contains the protecting group DMT, deprotection is required during oligonucleotide synthesis; when the linker does not contain the protecting group DMT, deprotection is not required during oligonucleotide synthesis.

[0021] In this invention, formula (2) above is only used to represent the content of each structure and each structural unit contained in the modified styrene / nitrogen-containing heterocyclic copolymer, and cannot represent the arrangement relationship between the structural units. The modified styrene / nitrogen-containing heterocyclic copolymer can be a random copolymer, a block copolymer, or an alternating copolymer, preferably a random copolymer. In addition, the number average molecular weight of the modified styrene / nitrogen-containing heterocyclic copolymer is preferably 5,000 to 20,000, such as 5,000, 8,000, 10,000, 12,000, 15,000, 18,000, 20,000, or any value between them.

[0022] The method for preparing styrene / nitrogen-containing heterocyclic copolymers provided by this invention includes the following steps: S1: Halogenated styrene and vinyl nitrile are subjected to free radical polymerization under inert gas protection to obtain a halogenated styrene / vinyl nitrile copolymer; S2: The halogenated styrene / vinyl nitrile copolymer is substituted with sodium azide to obtain styrene-azide / nitrogen-containing heterocyclic copolymer; S3: The styrene / nitrogen-containing heterocyclic copolymer is reduced to obtain the styrene / nitrogen-containing heterocyclic copolymer.

[0023] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, in step S1, the molar ratio of the halostyrene to acrylonitrile is preferably (0.5~9):1, such as 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or any value between them. The conditions of the free radical polymerization reaction preferably include a temperature of 70ºC~80ºC, such as 70ºC, 72ºC, 74ºC, 76ºC, 78ºC, 80ºC or any value between them; and a time of 10h~48h, such as 10h, 12h, 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h or any value between them. The free radical polymerization reaction is preferably carried out in solution polymerization. The solvent used can be any existing inert liquid medium that does not interact with the reactants and reaction products, as those skilled in the art will know, and will not be elaborated here. Furthermore, the free radical polymerization reaction is usually carried out under an inert gas atmosphere. The inert gas can be, for example, at least one of nitrogen, argon, helium, etc.

[0024] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, in step S1, the type of initiator used in the free radical polymerization reaction is not particularly limited, and can be selected from at least one of azo initiators, peroxide initiators, and redox initiators. Specific examples of azo initiators include, but are not limited to, at least one of: dimethyl azobisisobutyrate, azobisisobutyramidine hydrochloride, azodicarbonamide, azobisisopropylimidazoline hydrochloride, azoisobutylcyanoformamide, azodicyclohexylformonitrile, azodicyanovalerate, azobisisopropylimidazoline, azobisisobutyronitrile, azobisisovalerate, and azobisisoheptanenitrile. Specific examples of peroxide initiators include, but are not limited to, at least one of: hydrogen peroxide, ammonium persulfate, sodium persulfate, potassium persulfate, benzoyl peroxide, and benzoyl tert-butyl peroxide. Specific examples of the redox initiator include, but are not limited to, at least one of the following: sulfate-sulfite, persulfate-thiourea, persulfate-organic salt, and ammonium persulfate-aliphatic amine. The sulfate-sulfite may be selected from at least one of sodium sulfate-sodium sulfite, potassium sulfate-potassium sulfite, and ammonium sulfate-ammonium sulfite. The persulfate-thiourea may be selected from at least one of sodium persulfate-thiourea, potassium persulfate-thiourea, and ammonium persulfate-thiourea. The persulfate-organic salt may be selected from at least one of sodium persulfate-potassium acetate, potassium persulfate-potassium acetate, and ammonium persulfate-ammonium acetate. The ammonium persulfate-aliphatic amine may be ammonium persulfate-N,N-tetramethylethylenediamine and / or ammonium persulfate-diethylamine.

[0025] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, in step S1, after the free radical polymerization reaction is completed, the target polymer can be separated from the polymerization product by alcohol precipitation. After alcohol precipitation, it is preferable to wash with dichloromethane and methanol sequentially. After washing, the product can also be dried. The drying conditions typically include a temperature of 30ºC to 50ºC and a time of 5h to 24h.

[0026] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, the purpose of the substitution reaction in step S2 is to convert the halogen on the halostyrene structural unit into azide, and at the same time convert the nitrile group on the vinyl nitrile structural unit into a nitrogen-containing heterocycle. The preferred mass ratio of the halostyrene / vinyl nitrile copolymer to sodium azide used in the substitution reaction is 1:(1.1~1.3), such as 1:1.1, 1:1.2, 1:1.3, or any value between them. The preferred conditions for the substitution reaction include a temperature of 70ºC to 100ºC, such as 70ºC, 75ºC, 80ºC, 85ºC, 90ºC, 95ºC, 100ºC, or any value between them; and a time of 10h to 48h, such as 10h, 12h, 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h, or any value between them. The substitution reaction is preferably carried out in the presence of ammonium chloride. The mass ratio of the ammonium chloride to the halostyrene / vinyl nitrile copolymer is preferably (0.9 to 1.1):1, such as 0.9:1, 1:1, 1.1:1, or any value between them. The substitution reaction is preferably carried out in a solvent, and the solvent can be any existing inert liquid medium that does not interact with the reactants and reaction products; this is well known to those skilled in the art and will not be elaborated upon here. The substitution reaction is typically carried out under an inert gas atmosphere. The inert gas can be, for example, at least one of nitrogen, argon, helium, etc. Furthermore, after the substitution reaction is complete, the product is preferably washed sequentially with 2-methylamide, water, and methanol. After washing, the product can be dried. Drying conditions typically include a temperature of 30ºC to 50ºC and a time of 5 h to 48 h.

[0027] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, the purpose of the reduction reaction in step S3 is to convert the azide groups on the styrene / nitrogen-containing heterocyclic copolymer into amino groups. The reduction reaction can be performed using at least one of the following methods: catalytic hydrogenation, triphenylphosphine reduction, sodium thiosulfate reduction, LAH reduction, sodium borohydride / Lewis acid reduction, or TMSCl / NaCl system reduction. When using the LAH reduction method, the preferred conditions for the reduction reaction include a temperature of 0ºC to 40ºC, such as 0ºC, 5ºC, 10ºC, 15ºC, 20ºC, 25ºC, 30ºC, 35ºC, 40ºC, or any value between these values; and a time of 10h to 48h, such as 10h, 12h, 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h, or any value between these values. The reduction reaction is typically carried out in a solvent, which can be any existing inert liquid medium that does not interact with the reactants and products. This is well-known to those skilled in the art and will not be elaborated upon here. The reduction reaction is usually carried out under an inert gas atmosphere. The inert gas can be, for example, at least one of nitrogen, argon, and helium. After the reduction reaction is complete, the resulting reduction product is preferably washed sequentially with hydrochloric acid, sodium bicarbonate solution, 2-methylamide, methanol, and tetrahydrofuran. After washing, the product can be dried. Drying conditions typically include a temperature of 30ºC to 50ºC and a time of 5 hours to 48 hours.

[0028] The method for preparing the modified styrene / nitrogen-containing heterocyclic copolymer provided by the present invention includes condensing a styrene / nitrogen-containing heterocyclic copolymer having the structure shown in formula (1) with a linker to attach a linker group capable of coupling with nucleoside phosphoramidite to the X1 position of the styrene / nitrogen-containing heterocyclic copolymer.

[0029] In the preparation process of the above-mentioned modified styrene / nitrogen-containing heterocyclic copolymer, the styrene / nitrogen-containing heterocyclic copolymer can be obtained by directly reacting amino / hydroxy styrene with a nitrogen-containing heterocyclic compound via free radical polymerization, or it can be prepared according to the above method, which will not be elaborated here.

[0030] In one specific embodiment, the method for preparing the modified styrene / nitrogen-containing heterocyclic copolymer includes the following steps: S1': A condensation reaction is carried out between a styrene / nitrogen-containing heterocyclic copolymer and a linker (Unylinker) to obtain the condensation reaction product; S2': The condensation reaction product is subjected to a capping reaction with capping agent A and capping agent B, wherein capping agent A is a mixture of acetic anhydride and tetrahydrofuran, and capping agent B is a mixture of tetrahydrofuran, triethylamine and N-methylimidazole, to obtain a modified styrene / nitrogen-containing heterocyclic copolymer.

[0031] In the preparation process of the modified styrene / nitrogen-containing heterocyclic copolymer described above, in step S1`, the condensation reaction conditions preferably include a temperature of room temperature and a time of 10h to 24h, such as 10h, 12h, 15h, 18h, 20h, 22h, 24h, or any value between them. The molar ratio of the total content of amino and hydroxyl groups in the styrene / nitrogen-containing heterocyclic copolymer to Unylinker is preferably 1:(1~1.2), such as 1:1, 1:1.05, 1:1.1, 1:1.15, 1:2, or any value between them. The condensation reaction preferably uses triethylamine as a catalyst. The preferred ratio of triethylamine to Unylinker is (0.5~1) mL:1g, such as 0.5mL:1g, 0.6mL:1g, 0.7mL:1g, 0.8mL:1g, 0.9mL:1g, 1mL:1g, or any value between them. The condensation reaction is typically carried out in a solvent. The solvent can be any existing inert liquid medium that does not interact with the reactants and products; this is well known to those skilled in the art and will not be elaborated upon here. After the condensation reaction is complete, the resulting condensation product is preferably washed with an acetonitrile / dichloromethane solution.

[0032] In the preparation process of the above-mentioned modified styrene / nitrogen-containing heterocyclic copolymer, in step S2', the capping agent A is a mixture of acetic anhydride and tetrahydrofuran, wherein the volume ratio of acetic anhydride to tetrahydrofuran is preferably (5~10):1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or any value between them. The capping agent B is a mixture of tetrahydrofuran, triethylamine and N-methylimidazole, wherein the volume ratio of tetrahydrofuran, triethylamine and N-methylimidazole is preferably (5~10):(5~10):1. Specifically, the volume ratio of tetrahydrofuran to N-methylimidazole is preferably (5~10):1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or any value between them. The preferred volume ratio of triethylamine to N-methylimidazole is (5~10):1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any value between them. The preferred dosage ratio of capping agent A to Unylinker is (50~100) mL:1g, such as 0.5 mL:1g, 0.6 mL:1g, 0.7 mL:1g, 0.8 mL:1g, 0.9 mL:1g, 1 mL:1g, or any value between them. The preferred dosage ratio of capping agent B to Unylinker is (50~100) mL:1g, such as 50 mL:1g, 60 mL:1g, 70 mL:1g, 80 mL:1g, 90 mL:1g, 100 mL:1g, or any value between them. The preferred conditions for the capping reaction include a temperature of room temperature and a time of 10h~24h. The capping reaction is typically carried out in a solvent, which can be any existing inert liquid medium that does not interact with the reactants and products. This is well-known to those skilled in the art and will not be elaborated upon here. The capping reaction is usually carried out under an inert gas atmosphere. The inert gas can be, for example, at least one of nitrogen, argon, and helium. After the capping reaction is complete, the resulting capped product is preferably washed sequentially with acetonitrile, dichloromethane, and tetrahydrofuran. After washing, the product can be dried. Drying conditions typically include a temperature of 30ºC to 50ºC and a time of 5 hours to 48 hours.

[0033] The present invention also provides the application of the styrene / nitrogen-containing heterocyclic copolymer and / or modified styrene / nitrogen-containing heterocyclic copolymer as an oligonucleotide synthesis carrier.

[0034] In this invention, the styrene / nitrogen-containing heterocyclic copolymer and / or modified styrene / nitrogen-containing heterocyclic copolymer can be used alone as an oligonucleotide synthesis carrier, or it can be used as a composite carrier after being loaded / coated onto the CPG surface. When the latter is used, the CPG can be loaded / coated after the polymer preparation is completed; or it can be loaded / coated during the polymer preparation process, and the polymer preparation can continue after the loading / coating is completed. Compared with traditional CPG carriers or PS carriers, the novel carrier of CPG coated with styrene / nitrogen-containing heterocyclic copolymer and / or modified styrene / nitrogen-containing heterocyclic copolymer has several advantages. Firstly, this polymer carrier material contains nitrogen-containing cyclic structural units. These nitrogen-containing cyclic structural units can react with phosphorus on nucleoside monomers during the synthesis of oligonucleotide monomers, forming an intermediate similar to an intramolecular reaction, which can effectively promote the synthesis of long-chain oligonucleotide monomer chains. Secondly, it can fully utilize the advantages of CPG materials, such as good mechanical properties, non-swelling, and controllable pore size. Thirdly, due to the polymer coating, the problem of CPG being easily corroded under alkaline conditions during oligonucleotide monomer synthesis can be effectively avoided. Fourthly, it can also prevent the expansion of traditional polymer carriers under heat (during chain extension) and solvent influence, thus avoiding the problem of low loading capacity due to expansion. Fifthly, the tetrazolium on this novel solid-phase carrier reacts with phosphorus on nucleoside monomers during nucleic acid synthesis, forming an intermediate similar to an intramolecular reaction, thereby improving the efficiency of nucleic acid synthesis. Finally, this novel solid-phase carrier effectively utilizes the high loading capacity of resin, greatly increasing the effective loading capacity of nucleosides. These advantages significantly increase the industrial yield of oligonucleotide monomers and further reduce the cost-effectiveness and production costs.

[0035] In one specific embodiment, the method for synthesizing the oligonucleotide includes the following steps: (1) Deprotection reaction: Under the action of a deprotecting agent, the protecting group on the linking group of the support is removed; if there is no protecting group on the support, this step can be omitted; (2) Coupling reaction: The nucleoside phosphoramide synthesis unit is coupled in the presence of a coupling agent and a support, wherein the nucleoside phosphoramide synthesis unit includes nucleoside phosphoramide monomer and / or nucleoside phosphoramide polymer, to obtain the coupling reaction product. (3) Capping reaction: The coupling reaction product is subjected to a capping reaction in the presence of a capping agent to obtain the capping reaction product; (4) Oxidation reaction: The capped reaction product is oxidized in the presence of an oxidizing agent to obtain the oxidation reaction product; (5) Cleavage and deprotection: The oxidation reaction product is cleaved and deprotected in the presence of a cleavage and deprotection agent so that the oligonucleotide chain is removed from the support to obtain oligonucleotide.

[0036] The present invention will be described in detail below through embodiments.

[0037] In the following test examples, the catalog number of adenine A is A111000; the catalog number of thymine T is T111000; the catalog number of guanine G is G111000; and the catalog number of cytosine C is C111000.

[0038] Example 1 S1': In a 100 mL three-necked round-bottom flask, add p-chlorostyrene (9.12 g, 60 mmol) and acrylonitrile (3.18 g, 60 mmol), then add 100 mL of toluene and stir to obtain the reactant. In another 50 mL round-bottom flask, add azobisisobutylene (AIBN, 0.10 g, 0.6 mmol), then add 30 mL of toluene and stir until all the solids are dissolved. Then add this solution to the above reactant. Evacuate the reactant for 1 min, then purge with argon gas. Repeat this operation two more times. Under argon protection, heat the reactant to 75ºC and stir for 42 h to obtain a viscous product.

[0039] The viscous product was cooled to 40ºC and slowly added to 210 mL of stirred methanol solution. The resulting white suspension was stirred at room temperature for 2 hours and then filtered to obtain a white product. This product was dissolved in 70 mL of dichloromethane, and this solution was slowly added to 700 mL of stirred methanol solution. The mixture was stirred for 2 hours and then filtered. The resulting filter cake was washed twice with methanol, 30 mL each time. The product was then vacuum dried at 40ºC for 12 hours to obtain 12.25 g of white halostyrene / vinyl nitrile copolymer, denoted as PACS.

[0040] 1 H NMR (DMSO-d6, ppm:0.88-2): (CH2-CH),4.85 (CH2-Cl),6.9-7.7 (Ar-H);FT-IR(cm-1): 3085-3026 (aromatic CH), 2926-2860 (aliphatic CH),2240 (CN),1600-1490 (aromatic C=C).

[0041] S2': In a 50 mL three-necked round-bottom flask, add PACS (0.54 g), NaN3 (0.65 g), ammonium chloride (0.54 g), and dimethylamide (20 mL). Evacuate the reactants for 1 min, then purge with argon gas. Repeat this process twice. Under argon protection, heat the reactants to 90ºC and continue reacting for 24 h. Cool the reaction to room temperature and filter. Wash the product twice with 10 mL of 2-methylamide each time, then twice with 15 mL of deionized water each time, and finally twice with 10 mL of methanol each time. Vacuum dry the resulting product for 24 h to obtain 6.99 g of azide styrene / nitrogen-containing heterocyclic copolymer.

[0042] According to GPC testing, the number average molecular weight of this styrene-azidopolymer / nitrogen-containing heterocyclic copolymer is 10045.

[0043] 1 H NMR (DMSO-d6, ppm): 1-1.7 (CH2-CH), 4.4 (CH2-N3), 6.8-7.7 (Ar-H). FT-IR: 3446, 3027 (aromatic CH), 2090 (azide N3), 1600-1490 (aromatic C=C).

[0044] S3`: In a 250 mL three-necked round-bottom flask, 0.52 g of styrene-azidopolymer / nitrogen-containing heterocyclic copolymer and tetrahydrofuran were added and stirred for 30 min. Then, 3 g of glass microparticles (bare spheres, particle size 150 ± 10 mesh, pore size 1000 Å) and 2.0 mL of triethylamine were added. The above reaction mixture was stirred at room temperature for 1 h under argon protection, then heated to reflux and held for 24 h. The reaction mixture was cooled to room temperature and stirred at room temperature for 24 h. The resulting reaction product was filtered and washed twice with dimethylamide (30 mL each time), then twice with deionized water (30 mL each time), and finally washed three times with tetrahydrofuran (30 mL each time). The resulting product was filtered through air for 24 h to obtain 3.43 g of light-colored styrene-azidopolymer / nitrogen-containing heterocyclic copolymer-coated CPG, denoted as Co-polymer coated CPG-N3.

[0045] S4`: In a 250 mL three-necked round-bottom flask, add Co-polymer coated CPG-N3 and 50 mL of anhydrous tetrahydrofuran. Evacuate the reactants for 1 min, then purge with argon gas. Repeat this process twice. Under argon protection, cool the reactants to 0°C. Slowly add lithium aluminum hydride (LAH 1.0 M THF, 5 mL) over 15 min using a dropping funnel. Then, stir the reactants at room temperature for 24 h. Filter the product and wash twice with 0.1 N hydrochloric acid (50 mL each time), then twice with 1.0 M sodium bicarbonate solution (50 mL each time), then once with 2-methylamide (50 mL), then twice with methanol (50 mL each time), and finally twice with tetrahydrofuran (50 mL each time). Dry the resulting product in air for 40 h to obtain aminostyrene / nitrogen-containing heterocyclic copolymer coated CPG, denoted as Co-polymer coated CPG-NH2. The amino loading of the CPG coated with the aminostyrene / nitrogen-containing heterocyclic copolymer was determined to be 235 µmol / g using the DMT-Cl method.

[0046]

[0047] S5: In a 250 mL three-necked round-bottom flask, add anhydrous acetonitrile (90 mL) and anhydrous dichloromethane (45 mL), then add Unylinker-1 (0.69 g, with the structure shown below), and start stirring. Add triethylamine (0.5 mL) and stir for 10 min until all solids are dissolved and a homogeneous solution is obtained. Finally, add Co-polymer coated CPG-NH2 (6.30 g, amino loading of 235 µmol / g) and stir at room temperature for 16 h. Insert a filter tube into the above reactants, turn on the vacuum filter, and dry the solution. Add the pre-prepared acetonitrile / dichloromethane solution (120 mL, 2:1 (v / v)), stir for 5 min, insert the filter tube into the above reactants, turn on the vacuum filter, and dry the solution. Repeat the above steps twice more. Under argon protection, capping agent B (CAPB, 60 mL, a mixture of tetrahydrofuran, triethylamine, and N-methylimidazole in a volume ratio of 8:8:1) was added to the above reactants and stirred for 5 min. Then, capping agent A (CAPA, 60 mL, a mixture of acetic anhydride and tetrahydrofuran in a volume ratio of 8:1) was added, and the reactants were stirred at room temperature for 12 h. The product was filtered through an argon-protected filtration system, then washed twice with acetonitrile (50 mL each time), once with dichloromethane (50 mL each time), and finally three times with tetrahydrofuran (50 mL each time). The product was dried under vacuum for 24 h to obtain 6.45 g of modified styrene / nitrogen-containing heterocyclic copolymer-coated CPG, denoted as Co-polymer coated CPG-Unylinker.

[0048] The nucleoside loading in the Co-polymer coated CPG-Unylinker was found to be 128 µmol / g, and the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier was 1:200.

[0049] Unylinker-1

[0050] Example 2 S1': In a 100 mL three-necked round-bottom flask, add p-chlorostyrene (8.21 g, 54 mmol) and acrylonitrile (3.18 g, 60 mmol), then add 50 mL of toluene and stir to obtain the reactant. In another 50 mL round-bottom flask, add azobisisobutylene (AIBN, 0.10 g, 0.6 mmol), then add 30 mL of toluene and stir until all the solids are dissolved. Then add this solution to the above reactant. Evacuate the reactant for 1 min, then purge with argon gas. Repeat this operation two more times. Under argon protection, heat the reactant to 70ºC and stir for 48 h to obtain a viscous product.

[0051] The viscous product was cooled to 40ºC and slowly added to 210 mL of stirred methanol solution. The resulting white suspension was stirred at room temperature for 2 hours and then filtered to obtain a white product. This product was dissolved in 70 mL of dichloromethane, and this solution was slowly added to 700 mL of stirred methanol solution. The mixture was stirred for 2 hours and then filtered. The resulting filter cake was washed twice with methanol, 30 mL each time. The product was then vacuum dried at 40ºC for 12 hours to obtain 6.57 g of white halostyrene / vinyl nitrile copolymer, denoted as PACS.

[0052] S2': In a 50 mL three-necked round-bottom flask, add PACS (0.54 g), NaN3 (0.65 g), ammonium chloride (0.49 g), and dimethylamide (20 mL). Evacuate the reactants for 1 min, then purge with argon gas. Repeat this process twice. Under argon protection, heat the reactants to 70ºC and continue reacting for 48 h. Cool the reaction to room temperature and filter. Wash the product twice with 10 mL of 2-methylamide each time, then twice with 15 mL of deionized water each time, and finally twice with 10 mL of methanol each time. Vacuum dry the resulting product for 24 h to obtain 0.57 g of azide styrene / nitrogen-containing heterocyclic copolymer.

[0053] According to GPC testing, the number average molecular weight of this styrene-azidopolymer / nitrogen-containing heterocyclic copolymer is 9125.

[0054] S3`: In a 250 mL three-necked round-bottom flask, 0.52 g of styrene-azidopolymer / nitrogen-containing heterocyclic copolymer and tetrahydrofuran were added and stirred for 30 min. Then, 3.0 g of glass microparticles (bare spheres, particle size 175±5 mesh, pore size 1000 Å) and 2.0 mL of triethylamine were added. The above reaction mixture was stirred at room temperature for 1 h under argon protection, then heated to reflux and held for 24 h. The reaction mixture was cooled to room temperature and stirred at room temperature for 24 h. The resulting reaction product was filtered and washed twice with dimethylamide (30 mL each time), then twice with deionized water (30 mL each time), and finally washed three times with tetrahydrofuran (30 mL each time). The resulting product was filtered through air for 24 h to obtain 3.43 g of light-colored styrene-azidopolymer / nitrogen-containing heterocyclic copolymer-coated CPG, denoted as Co-polymer coated CPG-N3.

[0055] S4`: In a 250 mL three-necked round-bottom flask, add Co-polymer coated CPG-N3 and 50 mL of anhydrous tetrahydrofuran. Evacuate the reactants for 1 min, then purge with argon gas. Repeat this process twice. Under argon protection, cool the reactants to 0°C. Slowly add lithium aluminum hydride (LAH 1.0 M THF, 5 mL) over 15 min using a dropping funnel. Then, stir the reactants at room temperature for 24 h. Filter the product and wash twice with 0.1 N hydrochloric acid (50 mL each time), then twice with 1.0 M sodium bicarbonate solution (50 mL each time), then once with 2-methylamide (50 mL), then twice with methanol (50 mL each time), and finally twice with tetrahydrofuran (50 mL each time). Dry the resulting product in air for 40 h to obtain aminostyrene / nitrogen-containing heterocyclic copolymer coated CPG, denoted as Co-polymer coated CPG-NH2. The amino loading of the CPG coated with the aminostyrene / nitrogen-containing heterocyclic copolymer was determined to be 242 µmol / g using the DMT-Cl method.

[0056] S5: In a 250 mL three-necked round-bottom flask, add anhydrous acetonitrile (90 mL) and anhydrous dichloromethane (45 mL), then add Unylinker-2 (0.57 g, with the structure shown below) and start stirring. Add triethylamine (0.5 mL) and stir for 10 min until all solids are dissolved and a homogeneous solution is obtained. Finally, add Co-polymer coated CPG-NH2 (3.4 g) and stir at room temperature for 10 h. Insert a filter tube into the above reaction mixture, turn on the vacuum filter, and dry the solution. Add the pre-prepared acetonitrile / dichloromethane solution (50 mL, 2:1 (v / v)), stir for 5 min, insert the filter tube into the above reaction mixture, turn on the vacuum filter, and dry the solution. Repeat the above steps twice more. Under argon protection, capping agent B (CAPB, 30 mL, a mixture of tetrahydrofuran, triethylamine, and N-methylimidazole in a volume ratio of 8:8:1) was added to the above reactants and stirred for 5 min. Then, capping agent A (CAPA, 30 mL, a mixture of acetic anhydride and tetrahydrofuran in a volume ratio of 8:1) was added, and the reactants were stirred at room temperature for 12 h. The product was filtered through an argon-protected filtration system, then washed twice with acetonitrile (50 mL each time), once with dichloromethane (50 mL each time), and finally three times with tetrahydrofuran (50 mL each time). The product was dried under vacuum for 24 h to obtain 3.85 g of modified styrene / nitrogen-containing heterocyclic copolymer-coated CPG, denoted as Co-polymer coated CPG-Unylinker.

[0057] The nucleoside loading in the Co-polymer coated CPG-Unylinker was found to be 132 µmol / g, and the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier was 1:250.

[0058] Unylinker-2 Example 3 S1': In a 100 mL three-necked round-bottom flask, add p-chlorostyrene (3.65 g, 24 mmol) and acrylonitrile (0.32 g, 6 mmol), then add 50 mL of toluene and stir to obtain the reactant. In another 50 mL round-bottom flask, add azobisisobutylene (AIBN, 0.1 g, 0.6 mmol), then add 30 mL of toluene and stir until all the solids are dissolved. Then add this solution to the above reactant. Evacuate the reactant for 1 min, then purge with argon gas. Repeat this operation two more times. Under argon protection, heat the reactant to 80ºC and stir for 10 h to obtain a viscous product.

[0059] The viscous product was cooled to 40ºC and slowly added to 210 mL of stirred methanol solution. The resulting white suspension was stirred at room temperature for 2 hours and then filtered to obtain a white product. This product was dissolved in 70 mL of dichloromethane, and this solution was slowly added to 700 mL of stirred methanol solution. The mixture was stirred for 2 hours and then filtered. The resulting filter cake was washed twice with methanol, 30 mL each time. The product was then vacuum dried at 40ºC for 12 hours to obtain 3.82 g of white halostyrene / vinyl nitrile copolymer, denoted as PACS.

[0060] S2': In a 50 mL three-necked round-bottom flask, add PACS (0.54 g), NaN3 (0.65 g), ammonium chloride (0.7 g), and dimethylamide (20 mL). Evacuate the reactants for 1 min, then purge with argon gas. Repeat this process twice. Under argon protection, heat the reactants to 100ºC and continue reacting for 10 h. Cool the reaction to room temperature and filter. Wash the product twice with 10 mL of 2-methylamide each time, then twice with 15 mL of deionized water each time, and finally twice with 10 mL of methanol each time. Vacuum dry the resulting product for 24 h to obtain 0.55 g of azide styrene / nitrogen-containing heterocyclic copolymer.

[0061] According to GPC testing, the number average molecular weight of this styrene-azidopolymer / nitrogen-containing heterocyclic copolymer is 9550.

[0062] S3`: In a 250 mL three-necked round-bottom flask, 0.52 g of styrene-azidopolymer / nitrogen-containing heterocyclic copolymer and tetrahydrofuran were added and stirred for 30 min. Then, 3.0 g of glass microparticles (bare spheres, particle size 240 ± 10 mesh, pore size 1000 Å) and 2.0 mL of triethylamine were added. The above reaction mixture was stirred at room temperature for 1 h under argon protection, then heated to reflux and held for 24 h. The reaction mixture was cooled to room temperature and stirred at room temperature for 24 h. The resulting reaction product was filtered and washed twice with dimethylamide (30 mL each time), then twice with deionized water (30 mL each time), and finally washed three times with tetrahydrofuran (30 mL each time). The resulting product was filtered through air for 24 h to obtain 3.43 g of light-colored styrene-azidopolymer / nitrogen-containing heterocyclic copolymer-coated CPG, denoted as Co-polymer coated CPG-N3.

[0063] S4`: In a 250 mL three-necked round-bottom flask, add Co-polymer coated CPG-N3 and 50 mL of anhydrous tetrahydrofuran. Evacuate the reactants for 1 min, then purge with argon gas. Repeat this process twice. Under argon protection, cool the reactants to 0°C. Slowly add lithium aluminum hydride (LAH 1.0 M THF, 5 mL) over 15 min using a dropping funnel. Then, stir the reactants at room temperature for 24 h. Filter the product and wash twice with 0.1 N hydrochloric acid (50 mL each time), then twice with 1.0 M sodium bicarbonate solution (50 mL each time), then once with 2-methylamide (50 mL), then twice with methanol (50 mL each time), and finally twice with tetrahydrofuran (50 mL each time). Dry the resulting product in air for 40 h to obtain aminostyrene / nitrogen-containing heterocyclic copolymer coated CPG, denoted as Co-polymer coated CPG-NH2. The amino loading of the CPG coated with the aminostyrene / nitrogen-containing heterocyclic copolymer was determined to be 246 µmol / g using the DMT-Cl method.

[0064] S5: In a 250 mL three-necked round-bottom flask, add anhydrous acetonitrile (90 mL) and anhydrous dichloromethane (45 mL), then add Unylinker-3 (0.26 g, with the structure shown below) and start stirring. Add triethylamine (0.5 mL) and stir for 10 min until all solids are dissolved and a homogeneous solution is obtained. Finally, add Co-polymer coated CPG-NH2 (3.3 g) and stir at room temperature for 24 h. Insert a filter tube into the above reaction mixture, turn on the vacuum filter, and dry the solution. Add the pre-prepared acetonitrile / dichloromethane solution (50 mL, 2:1 (v / v)), stir for 5 min, insert the filter tube into the above reaction mixture, turn on the vacuum filter, and dry the solution. Repeat the above steps twice more. Under argon protection, capping agent B (CAPB, 30 mL, a mixture of tetrahydrofuran, triethylamine, and N-methylimidazole in a volume ratio of 8:8:1) was added to the above reactants and stirred for 5 min. Then, capping agent A (CAPA, 30 mL, a mixture of acetic anhydride and tetrahydrofuran in a volume ratio of 8:1) was added, and the reactants were stirred at room temperature for 12 h. The product was filtered through an argon-protected filtration system, then washed twice with acetonitrile (50 mL each time), once with dichloromethane (50 mL each time), and finally three times with tetrahydrofuran (50 mL each time). The product was dried under vacuum for 24 h to obtain 3.48 g of modified styrene / nitrogen-containing heterocyclic copolymer-coated CPG, denoted as Co-polymer coated CPG-Unylinker.

[0065] The nucleoside loading in the Co-polymer coated CPG-Unylinker was found to be 142 µmol / g, and the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier was 1:220.

[0066] Unylinker-3 Comparative Example 1 A reference polymer-coated CPG was prepared according to the method of Example 1, except that in step S1', the same molar amount of chlorostyrene was used to replace the acetonitrile, and the other conditions were the same as in Example 1, resulting in polystyrene-coated CPG, denoted as polymer coated CPG-Unylinker.

[0067] The nucleoside loading in the polymer-coated CPG-Unylinker was found to be 85 µmol / g.

[0068] Comparative Example 2 The glass microparticles (Unylinker-CPG, bare spheres, particle size 150±10 mesh, 1000 Å) used in Example 1 were directly used as the carrier. The loading of this CPG was measured to be 32 µmol / g.

[0069] Comparative Example 3 The polymer-coated CPG-Unylinker was prepared according to the method of Example 1, except that the amount of styrene-azidopolymer / nitrogen-containing heterocyclic copolymer was increased in step S3' so that the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass substrate in the final Co-polymer coated CPG-Unylinker was 1:150.

[0070] Comparative Example 4 The polymer-coated CPG-Unylinker was prepared according to the method of Example 1, except that the amount of styrene-azide / nitrogen-containing heterocyclic copolymer was reduced in step S3' so that the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier in the final Co-polymer coated CPG-Unylinker was 1:400.

[0071] Test case The carriers obtained in the above embodiments and comparative examples were respectively loaded into a solid-phase synthesizer (GE AKTA OP-100, using UNICORN control software). TM7) The oligonucleotide is synthesized under the same conditions in the matching empty synthesis column. The oligonucleotide is DNA and its sequence is 5`-GATTCCTAGGAGGTGATTCCTAGGAGGTGATTCCTAGGAGGT-3`, that is, 5`-[GATTCCTAGGAGGT]3-3` (abbreviated as I). During synthesis, the target oligonucleotide sequence is input into the control software and carried out at room temperature according to steps (1)-(5). All the following steps are carried out under argon protection. The specific process is as follows: (1) Deprotection: A toluene solution of deprotecting agent was added to the synthesis column to remove the protecting matrix on the linker in the support. The concentration of the deprotecting agent in the toluene solution was 5 wt%. The deprotecting agent was composed of trichloroacetic acid, dichloroacetic acid and 3,5-dinitrobenzoic acid in a weight ratio of 1:1:2. Then, acetonitrile was used for washing. (2) Coupling reaction: Add acetonitrile solution of coupling agent and acetonitrile solution of nucleoside phosphoramide synthesis unit to the synthesis column; the concentration of coupling agent in the acetonitrile solution is 0.2 mol / L, and the coupling agent is composed of 5-benzylthio-1H-tetrazole, N-methylimidazolium and 4,5-dicyanimidazolium in a weight ratio of 10:1:1; the concentration of acetonitrile solution of nucleoside phosphoramide synthesis unit is 1 mol / L, and the nucleoside phosphoramide synthesis unit contains adenine A, thymine T, guanine G and cytosine C in a molar ratio of 1:1:1:1. (3) Capping reaction: Capping agent A and an equal volume of capping agent B were added to the synthesis column. Capping agent A was a mixture of acetic anhydride and tetrahydrofuran in a volume ratio of 10%:90%, and capping agent B was a mixture of tetrahydrofuran, triethylamine and N-methylimidazole in a volume ratio of 80%:10%:10%. (4) Oxidation reaction: Add a tetrahydrofuran solution of iodine to the synthesis column, wherein the concentration of iodine is 0.05 mol / L; (5) Ligation and deprotection: After the synthesis is completed, the solid support in the synthesis column is removed, and 30wt% ammonia water is added to it. The column is treated at 60℃ for 2 hours, and then the synthesis column is eluted with deionized water to obtain oligonucleotides.

[0072] The purity of oligonucleotides was determined by ultraviolet-visible spectrophotometry (UV-Vis) and high-performance liquid chromatography, and the results are shown in Table 1.

[0073] FLP (OD's A260) was determined using ultraviolet-visible spectrophotometry (UV-Vis). FLP (OD's A260) = A 260nm / A 320nm , where A 260nm A represents the absorbance of the oligonucleotide solution at its characteristic peak of 260 nm. 320nmThe absorbance of the oligonucleotide solution at the characteristic peak of 320 nm is represented. A higher FLP (OD's A260) indicates a higher oligonucleotide concentration, which supports the conclusion that the oligonucleotide synthesis efficiency is higher; conversely, a lower FLP indicates a lower oligonucleotide concentration, which supports the conclusion that the oligonucleotide synthesis efficiency is lower. The results are shown in Table 1.

[0074] Table 1

[0075] As can be seen from the data in Table 1, the polymer-loaded glass support provided by this invention has a loading capacity of over 128 µmol / g, enabling large-scale synthesis of oligonucleotide supports and thus improving the synthesis efficiency of oligonucleotides. Furthermore, the purity of the oligonucleotides obtained from the polymer-loaded glass support provided by this invention is over 82.2%. A comparison between Example 1 and Comparative Example 1 shows that when no nitrogen-containing heterocyclic compound is introduced into the polymer, the loading capacity of the resulting support is relatively small, and its use in oligonucleotide synthesis cannot effectively improve the purity of the oligonucleotides. A comparison between Example 1 and Comparative Example 2 shows that it is difficult to achieve a high loading capacity with CPG, and its use in oligonucleotide synthesis cannot effectively improve the purity of the oligonucleotides. A comparison between Example 1 and Comparative Examples 3 and 4 shows that when the ratio of the copolymer thickness to the average pore size of the glass support in the polymer-loaded glass support is outside the range of this invention, either the loading capacity is too small or the purity of the obtained oligonucleotides is too low. Furthermore, when the polymer-loaded glass carrier provided by this invention is used to synthesize long-chain oligonucleotides with 42 bases, its FLP (OD's A260) can reach more than 27648, which means it has high efficiency.

[0076] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A styrene / nitrogen-containing heterocyclic copolymer, characterized in that, The styrene / nitrogen-containing heterocyclic copolymer has the structure shown in formula (1): Equation (1) In formula (1), A, B and Y are N, C, S or O independently; R1 is a single bond or a C1~C5 alkylene group; X1 is -NH2 or -OH; m and n represent the degree of polymerization of styrene-derived structural units and nitrogen-containing heterocyclic structural units, respectively, m:n=(1~9):1; * represents the bond end.

2. The styrene / nitrogen-containing heterocyclic copolymer according to claim 1, characterized in that, The styrene / nitrogen-containing heterocyclic copolymer is a random copolymer; the number average molecular weight of the styrene / nitrogen-containing heterocyclic copolymer is 5000~20000.

3. The styrene / nitrogen-containing heterocyclic copolymer according to claim 1, characterized in that, A is N, and B and Y are independently N or C; or, A is C, and B and Y are independently N or C.

4. A modified styrene / nitrogen-containing heterocyclic copolymer, characterized in that, The modified styrene / nitrogen-containing heterocyclic copolymer has the structure shown in formula (2): Equation (2) In formula (2), A, B and Y are N, C, S or O independently; R1 is a single bond or a C1~C5 alkylene group; X2 is -NH- or -O-; m and n represent the degree of polymerization of styrene-derived structural units and nitrogen-containing heterocyclic structural units, respectively, m:n=(1~9):1; Z is a linking group that can undergo coupling reaction with nucleoside phosphoramidite; * represents the bond terminator.

5. The modified styrene / nitrogen-containing heterocyclic copolymer according to claim 4, characterized in that, The modified styrene / nitrogen-containing heterocyclic copolymer is a random copolymer; the number-average molecular weight of the modified styrene / nitrogen-containing heterocyclic copolymer is 5000~20000.

6. The modified styrene / nitrogen-containing heterocyclic copolymer according to claim 4, characterized in that, A is N, and B and Y are independently N or C; or, A is C, and B and Y are independently N or C.

7. The modified styrene / nitrogen-containing heterocyclic copolymer according to claim 4, characterized in that, Z is derived from at least one of the following connectors: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； In this group, R is H, halogen, or C1-C5 alkoxy group, and R' is a group derived from adenine, guanine, cytosine, or thymine.

8. A method for preparing the styrene / nitrogen-containing heterocyclic copolymer according to any one of claims 1 to 3, characterized in that, The method includes the following steps: S1: Halogenated styrene and vinyl nitrile are subjected to free radical polymerization under inert gas protection to obtain a halogenated styrene / vinyl nitrile copolymer; S2: The halogenated styrene / vinyl nitrile copolymer is substituted with sodium azide to obtain styrene-azide / nitrogen-containing heterocyclic copolymer; S3: The styrene / nitrogen-containing heterocyclic copolymer is reduced to obtain the styrene / nitrogen-containing heterocyclic copolymer.

9. The method for preparing the styrene / nitrogen-containing heterocyclic copolymer according to claim 8, characterized in that, In step S1, the molar ratio of the halostyrene to the acrylonitrile is (0.5~9):1; the conditions for the free radical polymerization reaction include a temperature of 70ºC~80ºC and a time of 10h~48h. In step S2, the mass ratio of the halostyrene / vinyl nitrile copolymer to sodium azide is 1:(1.1~1.3); the conditions for the substitution reaction include a temperature of 70ºC~100ºC and a time of 10h~48h; the substitution reaction is carried out in the presence of ammonium chloride, and the mass ratio of ammonium chloride to the halostyrene / vinyl nitrile copolymer is (0.9~1.1):1; In step S3, the conditions for the reduction reaction include a temperature of 0ºC to 40ºC and a time of 10h to 48h.

10. A method for preparing the modified styrene / nitrogen-containing heterocyclic copolymer according to any one of claims 4 to 7, characterized in that, The method includes a condensation reaction of a styrene / nitrogen-containing heterocyclic copolymer having the structure shown in formula (1) with a linker to attach a linker group capable of coupling with nucleoside phosphoramidide to the X1 position of the styrene / nitrogen-containing heterocyclic copolymer.

11. The use of the styrene / nitrogen-containing heterocyclic copolymer according to any one of claims 1 to 3 and / or the modified styrene / nitrogen-containing heterocyclic copolymer according to any one of claims 4 to 7 as a carrier for oligonucleotide synthesis.

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