Glass carrier loaded with polymer, preparation method and application thereof, and synthesis method of oligonucleotide

Abstract

The invention belongs to the field of oligonucleotide synthesis, and particularly relates to a polymer-loaded glass carrier, a preparation method and application thereof, and an oligonucleotide synthesis method. The polymer-loaded glass carrier comprises a glass carrier and a modified styrene / nitrogen-containing heterocyclic ring copolymer coating the surface of the glass carrier, the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic ring copolymer to the average pore size of the glass carrier is 1: (200-250), and the modified styrene / nitrogen-containing heterocyclic ring copolymer has a structure as shown in a formula (1). The preparation method is characterized in that a nitrogen-containing heterocyclic ring is introduced into a polystyrene molecular chain and coupled to CPG, and meanwhile, the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic ring copolymer to the average pore size of CPG is strictly controlled, so that the effective load of nucleotide can be effectively improved; meanwhile, compared with the method that a nitrogen-containing heterocyclic compound is independently introduced into an oligonucleotide synthesis system, the synthesis efficiency and purity of nucleic acid can be remarkably improved. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of oligonucleotide synthesis, and particularly relates to a glass carrier loaded with a polymer, a preparation method and application thereof, and a method for synthesizing oligonucleotides. Background Art

[0002] Oligonucleotides are short-chain nucleic acids composed of multiple nucleotide monomers connected by phosphodiester bonds. They are widely used in genetic research, drug development, molecular diagnosis and other fields. With the development of molecular biology technology, the development of nucleic acid drugs, diagnosis of genetic diseases and infectious diseases, and genetic research have developed rapidly, so a large number of oligonucleotides need to be synthesized to support the above research. The commonly used method for synthesizing oligonucleotides is the solid-phase phosphoramidite method (abbreviated as solid-phase synthesis). In the solid-phase synthesis reaction, the synthetic carrier plays an important role. Controlled pore glass (CPG) and polystyrene (PS) are two commonly used types of carriers.

[0003] Controlled pore glass (CPG) is made of silica, a rigid, non-swelling solid support with advantages such as good mechanical properties, no swelling, and controllable pore size. It also has defined pore size and surface chemical properties, making it very suitable for solid-phase oligonucleotide synthesis. Functionalized controlled pore glass (CPG) is a key solid support in oligonucleotide synthesis. Nucleosides can be attached to the CPG surface and then chain extended by phosphoramidite chemistry. This functionalization can be used to synthesize oligonucleotides with various modifications and applications, and has the following advantages: (1) Efficient oligonucleotide synthesis: Functionalized CPG carriers allow efficient and reproducible synthesis of oligonucleotides using standard phosphoramidite chemistry; (2) Wide range of uses: The ability to synthesize oligonucleotides with various modifications makes functionalized CPG carriers suitable for a wide range of applications, including drug development, diagnosis, and research. In addition, there are many irregular pores inside the CPG spheres, and they have pore size stability. Due to spatial reasons, large pores are suitable for the synthesis of long fragments, while small pores are suitable for the synthesis of short fragments. CPGs with different pore sizes can be used to synthesize oligonucleotide products with different loadings: (1) CPG products with a large pore size are suitable for the synthesis of oligonucleotides <35mers. For example, the synthetic loading capacity of therapeutic oligonucleotides can be as high as 100 μmol / g; (2) CPG products with large pore size are suitable for the synthesis of oligonucleotides >35mers or highly modified oligonucleotides; (3) CPG products with a large pore size are suitable for oligonucleotide synthesis at scales exceeding 80mers, with a typical loading capacity of 10-20 μmol / g. However, since CPG is difficult to achieve high loading capacities (<100 μmol / g) and is susceptible to corrosion by chemical reagents, which can contaminate the synthesized product, its application as a carrier for large-scale oligonucleotide synthesis is greatly limited.

[0004] Polystyrene (PS) supports are also commonly used in solid-phase organic synthesis. They consist of small beads with diameters ranging from 20 to 150 μm. Monodisperse polystyrene microspheres are crosslinked using a crosslinker, typically divinylbenzene, with a crosslinking degree of 1% to 2%. This low-crosslinked resin is widely used for the synthesis of small nucleotides with fewer than 30 base units. Within this crosslinking 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. Polystyrene (PS) supports have the advantage of a high loading capacity (350 μmol / g), making them suitable for large-scale oligonucleotide synthesis. However, PS supports have disadvantages such as high swelling capacity (3-6 times the swelling), high solvent consumption, and the limited ability to synthesize short chains. Solid-phase supports, which are insoluble but swellable in various solvents, are their fundamental structural element.

[0005] Studies have shown that controlled pore glass (CPG) and polystyrene (PS) cannot improve the efficiency of oligonucleotide synthesis. Furthermore, the oligonucleotide synthesis process requires the use of chemical reagents such as deprotecting agents, coupling agents, and capping agents, all of which can cause reactions, and incomplete capping can produce impurities. High-purity oligonucleotides cannot be obtained using controlled pore glass (CPG) and polystyrene (PS). Summary of the Invention

[0006] The first purpose of the present invention is to overcome the defects of low loading capacity of existing controllable microporous glass carriers and low synthesis efficiency and low purity when synthesizing oligonucleotides using polystyrene carriers, and to provide a glass carrier loaded with a polymer with a high loading capacity, which can improve the synthesis efficiency and purity when using it as a carrier to synthesize oligonucleotides.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned polymer-loaded glass carrier.

[0008] The third object of the present invention is to provide the use of the above-mentioned glass carrier loaded with polymer as a carrier for oligonucleotide synthesis.

[0009] A fourth object of the present invention is to provide a method for synthesizing an oligonucleotide.

[0010] The polymer-loaded glass carrier provided by the present invention comprises a glass carrier and a modified styrene / nitrogen-containing heterocyclic copolymer coated on the surface of the glass carrier, wherein the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier is 1:(200-250), and the modified styrene / nitrogen-containing heterocyclic copolymer has a structure shown in formula (1):

[0011]

[0012] 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; X2 is -NH- or -O-; m and n represent the degree of polymerization of the styrene structural unit and the nitrogen-containing heterocyclic structural unit, respectively, and m:n = (1-9):1; Z is a linking group capable of undergoing a coupling reaction with the nucleoside phosphoramidite; and * represents a bond end.

[0013] The preparation method of the polymer-loaded glass carrier provided by the present invention comprises the following steps:

[0014] S1: coating the styrene / nitrogen-containing heterocyclic copolymer having the structure shown in formula (2) on the surface of a glass carrier to obtain a glass carrier loaded with the styrene / nitrogen-containing heterocyclic copolymer;

[0015] S2: When X3 is -N3, the glass support loaded with styrene / nitrogen-containing heterocyclic copolymer is subjected to a reduction reaction to obtain a glass support loaded with aminostyrene / nitrogen-containing heterocyclic copolymer; when X3 is -OH, the glass support loaded with styrene / nitrogen-containing heterocyclic copolymer is a glass support loaded with hydroxystyrene / nitrogen-containing heterocyclic copolymer, and step S3 is directly performed;

[0016] S3: reacting the glass support loaded with the aminostyrene / nitrogen-containing heterocyclic copolymer and / or the glass support loaded with the hydroxystyrene / nitrogen-containing heterocyclic copolymer with a linker to bond a linking group capable of undergoing a coupling reaction with nucleoside phosphoramidites to the amino position of the aminostyrene / nitrogen-containing heterocyclic copolymer and / or the hydroxy position of the hydroxystyrene / nitrogen-containing heterocyclic copolymer, thereby obtaining the polymer-loaded glass support;

[0017]

[0018] In formula (4), A, B and Y are independently N, C, S or O; R1 is a single bond or a C1-C5 alkylene group; X3 is -N3 or -OH; m and n represent the degree of polymerization of the styrene structural unit and the nitrogen-containing heterocyclic structural unit, respectively, and m:n = (1-9):1; * represents the bond end.

[0019] The oligonucleotide synthesis method provided by the present invention comprises sequentially subjecting a nucleoside phosphoramidite synthesis unit to a coupling reaction, a capping reaction, and an oxidation reaction using the above-mentioned polymer-loaded glass carrier as a solid phase carrier, and then cutting the oligonucleotide chain from the solid phase carrier to obtain the oligonucleotide.

[0020] The key to the present invention lies in introducing a nitrogen-containing heterocycle onto a polystyrene molecular chain to obtain a modified styrene / nitrogen-containing heterocycle copolymer, which is then coupled to CPG. Meanwhile, the ratio of the thickness of the modified styrene / nitrogen-containing heterocycle copolymer to the average pore size of the CPG is strictly controlled. This significantly improves the efficiency and purity of nucleic acid synthesis compared to introducing the nitrogen-containing heterocycle alone into the oligonucleotide synthesis system. This is presumably because the nitrogen-containing heterocycle reacts with the phosphinamide monomer to form an activated intermediate. Introducing the nitrogen-containing heterocycle onto the polystyrene molecular chain accelerates the formation of this activated intermediate and reduces the generation of interfering substances, thereby significantly accelerating the reaction and improving its efficiency and purity. Strictly controlling the ratio of the thickness of the modified styrene / nitrogen-containing heterocycle copolymer to the average pore size of the CPG not only ensures that the CPG surface is coated with the modified styrene / nitrogen-containing heterocycle copolymer, increasing the loading capacity, but also exposes some of the original pores on the CPG surface, endowing it with the ability to synthesize long oligonucleotides. Furthermore, the polymer-loaded glass carrier provided by the present invention prevents expansion of conventional polymer carriers during heating / chain extension and expansion due to solvents, thereby avoiding the problem of low loading capacity due to expansion. At the same time, it effectively utilizes the high loading capacity of the resin, greatly increasing the effective loading capacity of nucleotides and providing a higher nucleotide monomer loading capacity. Furthermore, because the CPG in the polymer-loaded glass carrier provided by the present invention is coated with resin, it can avoid corrosion by chemical reagents and affect product quality. DETAILED DESCRIPTION

[0021] The glass carrier loaded with a polymer provided by the present invention includes a glass carrier and a modified styrene / nitrogen-containing heterocyclic copolymer coated on the surface of the glass carrier. The ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier is 1:(200-250). If the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier is lower than the lower limit, the load capacity will be reduced; if the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier is higher than the upper limit, the pores will be blocked or a complete nucleotide long chain of the required number of bases will not be obtained. Specifically, the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier can be 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250 or any value therebetween.

[0022] In the polymer-loaded glass support provided by the present invention, the mass ratio of the glass support to the modified styrene / nitrogen-containing heterocyclic copolymer is preferably 1:(0.1-1), such as 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, or any value therebetween. When the mass ratio of the glass support to the modified styrene / nitrogen-containing heterocyclic copolymer is controlled at 1:(0.1-1), the resulting polymer-loaded glass support has the largest loading capacity, which is more conducive to improving the efficiency and purity of oligonucleotide synthesis.

[0023] In the polymer-loaded glass carrier provided by the present invention, the modified styrene / nitrogen-containing heterocyclic copolymer has a structure shown in formula (1):

[0024]

[0025] 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; X2 is -NH- or -O-; m and n represent the degree of polymerization of the styrene structural unit and the nitrogen-containing heterocyclic structural unit, respectively, with m:n = (1-9):1; Z is a linking group capable of undergoing a coupling reaction with the nucleoside phosphoramidite; and * represents a bonding end. Examples of the C1-C5 alkylene group include methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, tert-pentylene, and 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 therebetween.

[0026] In a preferred embodiment, A is N, and B and Y are independently N or C. In this case, the nitrogen-containing heterocycle is N at at least two specific positions, forming a completely conjugated structure, which is more conducive to further improvement of nucleic acid synthesis efficiency and purity.

[0027] In one embodiment, Z is derived from at least one of the following unylinkers:

[0028]

[0029]

[0030]

[0031] Wherein, R is H, halogen, or a C1-C5 alkoxy group, and Rˋ is a group derived from adenine (A), guanine (G), cytosine (C), or thymine (T). Specific examples of the C1-C5 alkoxy group 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:

[0032]

[0033] In the above linkers, DMT represents a protecting group, which can be dimethoxytrityl, p-methoxytrityl, or trityl. The carboxyl groups in these linkers undergo a condensation reaction with the amino / hydroxyl groups in the styrene / nitrogen-containing heterocyclic copolymer to form a linker capable of coupling with the nucleoside phosphoramidite. When the linker contains the protecting group DMT, deprotection is required prior to oligonucleotide synthesis. When the linker does not contain the protecting group DMT, deprotection is not required prior to oligonucleotide synthesis.

[0034] In the polymer-loaded glass carrier provided by the present invention, the modified styrene / nitrogen-containing heterocyclic copolymer can be a random copolymer, a block copolymer, or an alternating copolymer, preferably a random copolymer. Furthermore, 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 therebetween.

[0035] In the glass carrier loaded with polymer provided by the present invention, the particle size of the glass carrier is preferably 120 mesh to 200 mesh, such as 120 mesh, 140 mesh, 160 mesh, 180 mesh, 200 mesh or any value therebetween. The average pore size of the glass carrier is preferably like or any value in between.

[0036] The preparation method of the polymer-loaded glass carrier provided by the present invention comprises the following steps:

[0037] S1: coating the styrene / nitrogen-containing heterocyclic copolymer having the structure shown in formula (2) on the surface of a glass carrier to obtain a glass carrier loaded with the styrene / nitrogen-containing heterocyclic copolymer;

[0038] S2: When X3 is -N3, the glass support loaded with styrene / nitrogen-containing heterocyclic copolymer is subjected to a reduction reaction to obtain a glass support loaded with aminostyrene / nitrogen-containing heterocyclic copolymer; when X3 is -OH, the glass support loaded with styrene / nitrogen-containing heterocyclic copolymer is a glass support loaded with hydroxystyrene / nitrogen-containing heterocyclic copolymer, and step S3 is directly performed;

[0039] S3: reacting the glass support loaded with the aminostyrene / nitrogen-containing heterocyclic copolymer and / or the glass support loaded with the hydroxystyrene / nitrogen-containing heterocyclic copolymer with a linker to bond a linking group capable of undergoing a coupling reaction with nucleoside phosphoramidites to the amino position of the aminostyrene / nitrogen-containing heterocyclic copolymer and / or the hydroxy position of the hydroxystyrene / nitrogen-containing heterocyclic copolymer, thereby obtaining the polymer-loaded glass support;

[0040]

[0041] In formula (4), A, B, and Y are independently N, C, S, or O; R1 is a single bond or a C1-C5 alkylene group; X3 is -N3 or -OH; m and n represent the degree of polymerization of the styrene structural unit and the nitrogen-containing heterocyclic structural unit, respectively, with m:n = (1-9):1; and * represents the bond end. Preferably, A is N and B and Y are independently N or C. In this case, the nitrogen-containing heterocyclic ring is a nitrogen element at at least two specific positions, forming a completely conjugated structure, which is more conducive to further improving the efficiency and purity of nucleic acid synthesis. Examples of C1-C5 alkylene groups include methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-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 therebetween.

[0042] In the preparation process of the above-mentioned polymer-loaded glass carrier, when the styrene / nitrogen-containing heterocyclic copolymer is a hydroxystyrene / nitrogen-containing heterocyclic copolymer, it can be obtained by free radical polymerization of hydroxystyrene and a nitrogen-containing heterocyclic compound. When the styrene / nitrogen-containing heterocyclic copolymer is an azidostyrene / nitrogen-containing heterocyclic copolymer, it can be obtained by free radical polymerization of azidostyrene and a nitrogen-containing heterocyclic compound, or it can be prepared according to the following method: S11: subjecting a halogenated styrene to a free radical polymerization reaction with vinyl nitrile under the protection of an inert gas to obtain a halogenated styrene / vinyl nitrile copolymer; S21: subjecting the halogenated styrene / vinyl nitrile copolymer to a substitution reaction with sodium azide to obtain an azidostyrene / nitrogen-containing heterocyclic copolymer.

[0043] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, in step S11, the molar ratio of the halogenated styrene to the vinyl nitrile 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 therebetween. The conditions of the free radical polymerization reaction preferably include a temperature of 70°C to 80°C, such as 70°C, 72°C, 74°C, 76°C, 78°C, 80°C or any value therebetween; 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 therebetween. The free radical polymerization reaction is preferably carried out using solution polymerization. The solvent used can be any of various existing inert liquid media that do not interact with the reactants and reaction products. This is well known to those skilled in the art and is not described in detail here. In addition, the free radical polymerization reaction is usually carried out under the protection of an inert gas. The inert gas can be, for example, at least one of nitrogen, argon, helium, etc.

[0044] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, in step S11, the type of initiator used in the free radical polymerization reaction is not particularly limited, and can be selected from at least one of an azo initiator, a peroxide initiator, and a redox initiator. Specific examples of the azo initiator include, but are not limited to, at least one of dimethyl azobisisobutyrate, azobisisobutyramidine hydrochloride, azodicarbonamide, azobisisopropylimidazoline hydrochloride, azoisobutylcyanamide, azobiscyclohexylcarbonitrile, azobiscyanovaleric acid, azobisisopropylimidazoline, azobisisobutyronitrile, azobisisovaleronitrile, and azobisisoheptylonitrile. Specific examples of the peroxide initiator include, but are not limited to, at least one of hydrogen peroxide, ammonium persulfate, sodium persulfate, potassium persulfate, benzoyl peroxide, and tert-butyl benzoyl peroxide. Specific examples of the redox initiator include, but are not limited to, at least one of sulfate-sulfite, persulfate-thiourea, persulfate-organic salt, and ammonium persulfate-fatty 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-fatty amine may be ammonium persulfate-N,N-tetramethylethylenediamine and / or ammonium persulfate-diethylamine.

[0045] In the above-mentioned preparation process of the styrene / nitrogen-containing heterocyclic copolymer, in step S11, after the free radical polymerization reaction is completed, the target polymer can be separated from the polymer product by alcohol precipitation. After the alcohol precipitation is completed, the product is preferably washed with dichloromethane and then methanol. After washing, the product can also be dried. The drying conditions generally include a temperature of 30°C to 50°C and a drying time of 5 hours to 24 hours.

[0046] In the preparation process of the above-mentioned styrene / nitrogen-containing heterocyclic copolymer, in step S21, the purpose of the substitution reaction is to convert the halogen on the halogenated styrene structural unit into azide, and at the same time convert the nitrile group on the vinyl nitrile structural unit into a nitrogen-containing heterocyclic ring. The mass ratio of the halogenated styrene / vinyl nitrile copolymer to sodium azide used in the substitution reaction is preferably 1: (1.1 to 1.3), such as 1: 1.1, 1: 1.2, 1: 1.3 or any value therebetween. The conditions of the substitution reaction preferably include a temperature of 70° C. to 100° C., such as 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C. or any value therebetween; and a time of 10 h to 48 h, such as 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, 25 h, 28 h, 30 h, 32 h, 35 h, 38 h, 40 h, 45 h, 48 h or any value therebetween. The substitution reaction is preferably carried out in the presence of ammonium chloride. The mass ratio of the ammonium chloride to the halogenated styrene / vinyl nitrile copolymer is preferably (0.9 to 1.1):1, such as 0.9:1, 1:1, 1.1:1 or any value therebetween. The substitution reaction is preferably carried out in a solvent, and the solvent used can be any existing inert liquid medium that cannot interact with the reactants and reaction products. Those skilled in the art are aware of this and will not elaborate on it here. The substitution reaction is usually carried out under the protection of an inert gas. The inert gas can be, for example, at least one of nitrogen, argon, helium, etc. In addition, after the substitution reaction is completed, it is preferably washed with 2-methylamide, water and methanol in sequence. After washing, the product can also be dried. The drying conditions generally include a temperature of 30°C to 50°C and a time of 5h to 48h.

[0047] In a specific embodiment, in the preparation process of the above-mentioned glass carrier loaded with a polymer, in step S1, the method of coating the styrene / nitrogen-containing heterocyclic copolymer on the surface of the glass carrier includes subjecting the styrene / nitrogen-containing heterocyclic copolymer and the glass carrier to a reflux reaction and a room temperature reaction in the presence of triethylamine and an organic solvent, followed by filtration, and the resulting solid product is optionally washed and impurity-removed to obtain a glass carrier loaded with styrene / nitrogen-containing heterocyclic copolymer. Wherein, the mass ratio of the glass beads to the styrene / nitrogen-containing heterocyclic copolymer is preferably 1: (0.1-1), specifically 1: 0.1, 1: 0.2, 1: 0.3, 1: 0.4, 1: 0.5, 1: 0.6, 1: 0.7, 1: 0.8, 1: 0.9, 1: 1 or any value therebetween. The amount ratio of the triethylamine to the styrene / nitrogen-containing heterocyclic copolymer is preferably (2-6) mL: 1 g. The organic solvent is preferably tetrahydrofuran. The reflux reaction time is preferably 10h to 48h, such as 10h, 12h, 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h or any value therebetween. The room temperature reaction time is preferably 10h to 48h, such as 10h, 12h, 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h or any value therebetween. The washing method preferably comprises washing with dimethylamide, deionized water and tetrahydrofuran in sequence.

[0048] In the preparation process of the polymer-loaded glass support, the purpose of the reduction reaction in step S2 is to convert the azide groups on the azidostyrene / nitrogen-containing heterocyclic copolymer into amino groups. The reduction reaction can be carried out, for example, by at least one of catalytic hydrogenation, triphenylphosphine reduction, sodium thiosulfate reduction, LAH reduction, sodium borohydride / Lewis acid reduction, TMSCl / NaCl system reduction, etc. When the LAH reduction method is used, the reduction reaction conditions preferably include a temperature of 0°C to 40°C, such as 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or any value therebetween; and a time of 10 hours to 48 hours, such as 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 25 hours, 28 hours, 30 hours, 32 hours, 35 hours, 38 hours, 40 hours, 45 hours, 48 hours, or any value therebetween. The reduction reaction is usually carried out in a solvent, and the solvent used can be various existing inert liquid media that cannot interact with the reactants and reaction products. Those skilled in the art are aware of this and will not be elaborated here. The reduction reaction is usually carried out under the protection of an inert gas. The inert gas can be, for example, at least one of nitrogen, argon, helium, etc. After the reduction reaction is completed, the obtained reduction product is preferably washed with hydrochloric acid, baking soda solution, 2-methylamide, methanol, and tetrahydrofuran in sequence. After washing, the product can also be dried. The drying conditions generally include a temperature of 30°C to 50°C and a time of 5h to 48h.

[0049] In a specific implementation, step S3 specifically includes the following steps:

[0050] S21: condensing the styrene / nitrogen-containing heterocyclic copolymer with a linker to obtain a condensation reaction product;

[0051] S22: subjecting the condensation reaction product 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.

[0052] In the preparation process of the glass carrier loaded with the polymer, in step S21, the conditions of the condensation reaction preferably include a temperature of room temperature; a time of 10h to 24h, such as 10h, 12h, 15h, 18h, 20h, 22h, 24h or any value therebetween. 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 to 1.2), such as 1: 1, 1: 1.05, 1: 1.1, 1: 1.15, 1: 2 or any value therebetween. The condensation reaction is preferably catalyzed by triethylamine. The amount ratio of triethylamine to Unylinker is preferably (0.5 to 1) 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 therebetween. The condensation reaction is usually carried out in a solvent. The solvent used can be any of various existing inert liquid media that do not interact with the reactants and reaction products. This is well known to those skilled in the art and will not be described in detail here. After the condensation reaction is completed, the resulting condensation product is preferably washed with an acetonitrile / dichloromethane solution.

[0053] In the preparation process of the glass carrier loaded with a polymer, in step S22, the capping agent A is a mixture of acetic anhydride and tetrahydrofuran, wherein the volume ratio of acetic anhydride and tetrahydrofuran is preferably (5 to 10): 1, such as 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or any value therebetween. 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 to 10): (5 to 10): 1. Specifically, the volume ratio of tetrahydrofuran and N-methylimidazole is preferably (5 to 10): 1, such as 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or any value therebetween. The volume ratio of triethylamine and N-methylimidazole is preferably (5-10):1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or any value therebetween. The amount ratio of the capping agent A to Unylinker is preferably (50-100) mL:1 g, such as 0.5 mL:1 g, 0.6 mL:1 g, 0.7 mL:1 g, 0.8 mL:1 g, 0.9 mL:1 g, 1 mL:1 g or any value therebetween. The amount ratio of the capping agent B to Unylinker is preferably (50-100) mL:1 g, such as 0.5 mL:1 g, 0.6 mL:1 g, 0.7 mL:1 g, 0.8 mL:1 g, 0.9 mL:1 g, 1 mL:1 g or any value therebetween. The conditions for the capping reaction preferably include room temperature; time of 10 h to 24 h. The capping reaction is usually carried out in a solvent, and the solvent used can be various existing inert liquid media that cannot interact with the reactants and reaction products. Those skilled in the art are aware of this and will not go into details here. The capping reaction is usually carried out under the protection of an inert gas. The inert gas can be, for example, at least one of nitrogen, argon, helium, etc. After the capping reaction is completed, the obtained capped product is preferably washed with acetonitrile, dichloromethane, and tetrahydrofuran in sequence. After washing, the product can also be dried. The drying conditions generally include a temperature of 30°C to 50°C and a time of 5h to 48h.

[0054] Compared with traditional CPG carriers or PS carriers, the polymer-loaded glass carrier provided by the present invention has multiple advantages. First, the polymer carrier material contains a nitrogen-containing cyclic structural unit. This nitrogen-containing cyclic structural unit can react with the phosphorus on the nucleoside monomer during the synthesis of the oligonucleotide monomer to form an intermediate similar to an intramolecular reaction, which can effectively promote the synthesis of long-chain oligonucleotide monomer chains; second, it can give full play to the advantages of CPG materials such as good mechanical properties, no swelling, and controllable pore size; third, due to the polymer coating, it can effectively avoid the problem of CPG being easily corroded under alkaline conditions during the synthesis of oligonucleotide monomers; fourth, it can also prevent the traditional polymer carrier from expanding when heated (during chain extension) and under the influence of solvents, thereby avoiding the problem of small load capacity due to expansion; fifth, the tetrazolium on this new solid phase carrier will react with the phosphorus on the nucleoside monomer during the synthesis of nucleic acid to form an intermediate similar to an intramolecular reaction, thereby improving the synthesis efficiency of nucleic acid; fifth, this new solid phase carrier effectively utilizes the high load characteristics of the resin, greatly increasing the effective load of nucleosides. The above advantages greatly improve the industrial production of oligonucleotide monomers and further reduce the cost-effectiveness and production costs.

[0055] The present invention also provides the use of the polymer-loaded glass carrier as an oligonucleotide synthesis carrier.

[0056] In addition, the present invention also provides a method for synthesizing an oligonucleotide, which comprises sequentially subjecting a nucleoside phosphoramidite synthesis unit to a coupling reaction, a capping reaction, and an oxidation reaction using the above-mentioned glass carrier loaded with a polymer as a solid phase carrier, and then cutting the oligonucleotide chain from the solid phase carrier to obtain the oligonucleotide.

[0057] In one embodiment, the method for synthesizing the oligonucleotide comprises the following steps:

[0058] (1) Deprotection reaction: Under the action of a deprotecting agent, the protecting group on the carrier linking group is removed; if there is no protecting group on the carrier, this step can be omitted;

[0059] (2) Coupling reaction: subjecting a nucleoside phosphoramidite synthesis unit to a coupling reaction in the presence of a coupling agent and a carrier, wherein the nucleoside phosphoramidite synthesis unit comprises a nucleoside phosphoramidite monomer and / or a nucleoside phosphoramidite polymer, to obtain a coupling reaction product;

[0060] (3) capping reaction: subjecting the coupling reaction product to a capping reaction in the presence of a capping agent to obtain a capping reaction product;

[0061] (4) oxidation reaction: subjecting the capping reaction product to an oxidation reaction in the presence of an oxidant to obtain an oxidation reaction product;

[0062] (5) Cleavage and deprotection: The oxidation reaction product is subjected to a cleavage and deprotection reaction in the presence of a cleavage and deprotection agent to separate the oligonucleotide chain segments from the carrier to obtain an oligonucleotide.

[0063] The present invention will be described in detail below through examples.

[0064] In the following test example, the product number of adenine A is A111000; the product number of thymine T is T111000; the product number of guanine G is G111000; and the product number of cytosine C is C111000.

[0065] Example 1

[0066] S1: To 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 begin stirring to obtain the reactant. To a separate 50 mL round-bottom flask, add diisobutyl azodicarbonate (AIBN, 0.10 g, 0.6 mmol) and 30 mL of toluene. Stir until all solids have dissolved, and then add this solution to the reactant. Evacuate the reaction mixture for 1 minute, then flush with argon. Repeat this procedure two more times. Under argon, heat the reaction mixture to 75°C and stir for 42 hours to obtain a viscous product.

[0067] The viscous product was cooled to 40°C and slowly added to a stirred 210 mL 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 the solution was slowly added to a stirred 700 mL methanol solution. The stirring was continued for 2 hours and filtered. The filter cake was then washed twice with 30 mL of methanol each time. The resulting product was vacuum dried at 40°C for 12 hours to obtain 12.25 g of a white halogenated styrene / vinyl nitrile copolymer, designated as PACS.

[0068] 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).

[0069]

[0070] S2: PACS (0.54 g), NaN3 (0.65 g), ammonium chloride (0.54 g), and dimethylformamide (20 mL) were added to a 50 mL three-necked round-bottom flask. The reaction mixture was evacuated for 1 minute, then flushed with argon. This process was repeated twice. Under argon, the reaction mixture was heated to 90°C and allowed to react for 24 hours. The reaction mixture was cooled to room temperature and filtered. The product was washed twice with 2-methylformamide (10 mL each), then twice with deionized water (15 mL each), and finally twice with methanol (10 mL each). The product was then dried under vacuum for 24 hours to yield 6.99 g of styrene azide / nitrogen-containing heterocyclic copolymer.

[0071] GPC analysis showed that the number average molecular weight of the styrene azide / nitrogen-containing heterocyclic copolymer was 10045.

[0072] 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).

[0073]

[0074] S3: In a 250 mL three-necked round-bottom flask, add styrene azide / nitrogen-containing heterocyclic copolymer (0.52 g) and tetrahydrofuran, stir for 30 min, and then add 3 g of glass particles (bare spheres, particle size 150 ± 10 mesh, pore size ) and triethylamine (2.0 mL). The above reactants were stirred at room temperature for 1 h under the protection of argon, and then heated to reflux and continued for 24 h. The above reactants were cooled to room temperature and stirred at room temperature for 24 h. The resulting reaction product was filtered and washed twice with dimethyl amide, each time with 30 mL, then washed twice with deionized water, each time with 30 mL, and finally washed 3 times with tetrahydrofuran, each time with 30 mL. The resulting product was filtered through air for 24 h to obtain 3.43 g of light white azidostyrene / nitrogen-containing heterocyclic copolymer-coated CPG, recorded as Co-polymer coated CPG-N3.

[0075] S4: Co-polymer coated CPG-N3 and 50 mL of anhydrous tetrahydrofuran were added to a 250 mL three-necked round-bottom flask. The reaction mixture was evacuated for 1 minute and then flushed with argon. This process was repeated twice. Under argon protection, the reaction mixture was cooled to 0°C. Lithium aluminum hydride (LAH 1.0 M THF, 5 mL) was slowly added through a dropping funnel over 15 minutes. The reaction mixture was then stirred at room temperature for 24 hours. The product was filtered and washed with 0.1 N hydrochloric acid twice, 50 mL each time, 1.0 M baking soda solution twice, 50 mL each time, 2-methylformamide once, 50 mL each time, methanol twice, and finally tetrahydrofuran twice, 50 mL each time. The resulting product was air-dried for 40 hours to obtain aminostyrene / nitrogen-containing heterocyclic copolymer-coated CPG, designated as Co-polymer coated CPG-NH2. The aminostyrene / nitrogen-containing heterocyclic copolymer-coated CPG has an amino loading of 235 μmol / g as determined by the DMT-Cl method.

[0076]

[0077] 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) and begin stirring. Add triethylamine (0.5 mL) and stir for 10 minutes until all solids are dissolved and a homogeneous solution is obtained. Finally, add co-polymer coated CPG-NH2 (6.30 g, amino group loading 235 μmol / g) and stir at room temperature for 16 hours. Insert a filter tube into the reaction mixture, turn on the suction filter, and drain the solution. Add the prepared acetonitrile / dichloromethane solution (120 mL, 2:1 (v / v)), stir for 5 minutes, insert the filter tube into the reaction mixture, turn on the suction filter, and drain the solution. Repeat this step two more times. Under argon, capping agent B (CAPB, 60 mL, a mixture of tetrahydrofuran, triethylamine, and N-methylimidazole in an 8:8:1 volume ratio) was added to the above reactants and stirred for 5 minutes. Then, capping agent A (CAPA, 60 mL, a mixture of acetic anhydride and tetrahydrofuran in an 8:1 volume ratio) was added, and the reactants were stirred at room temperature for 12 hours. The product was filtered through a filtration apparatus under argon protection and then washed twice with acetonitrile, each with 50 mL, once with 50 mL of dichloromethane, and finally three times with tetrahydrofuran, each with 50 mL. The product was dried in a vacuum for 24 hours to obtain 6.45 g of modified styrene / nitrogen-containing heterocyclic copolymer-coated CPG, designated as Co-polymer Coated CPG-Unylinker.

[0078] After testing, the nucleoside loading capacity in the Co-polymer coated CPG-Unylinker was 128 μmol / g, and the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer in the Co-polymer coated CPG-Unylinker to the average pore size of the glass carrier was 1:200.

[0079]

[0080] Example 2

[0081] S1: To 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 begin stirring to obtain the reactant. To a separate 50 mL round-bottom flask, add diisobutyl azodicarbonate (AIBN, 0.10 g, 0.6 mmol) and 30 mL of toluene. Stir until all solids have dissolved, and then add this solution to the above reactant. Evacuate the reaction mixture for 1 minute, then flush with argon. Repeat this procedure two more times. Under argon, heat the reaction mixture to 70°C and stir for 48 hours to obtain a viscous product.

[0082] The viscous product was cooled to 40°C and slowly added to a stirred 210 mL 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 slowly added to a stirred 700 mL methanol solution. The solution was stirred for 2 hours and filtered. The filter cake was then washed twice with 30 mL of methanol each time. The resulting product was vacuum dried at 40°C for 12 hours to obtain 6.57 g of a white halogenated styrene / vinyl nitrile copolymer, designated as PACS.

[0083] S2: PACS (0.54 g), NaN3 (0.65 g), ammonium chloride (0.49 g), and dimethylformamide (20 mL) were added to a 50 mL three-necked round-bottom flask. The reaction mixture was evacuated for 1 minute, then flushed with argon. This process was repeated twice. Under argon, the reaction mixture was heated to 70°C and the reaction continued for 48 hours. The reaction mixture was cooled to room temperature and filtered. The product was washed twice with 2-methylformamide (10 mL each), then twice with deionized water (15 mL each), and finally twice with methanol (10 mL each). The product was then dried under vacuum for 24 hours to yield 0.57 g of styrene azide / nitrogen-containing heterocyclic copolymer.

[0084] GPC analysis showed that the number average molecular weight of the styrene azide / nitrogen-containing heterocyclic copolymer was 9125.

[0085] S3: In a 250 mL three-necked round-bottom flask, 0.52 g of styrene azide / nitrogen-containing heterocyclic copolymer and tetrahydrofuran were added and stirred for 30 min. Then, 3.0 g of glass particles (bare spheres, particle size 175 ± 5 mesh, pore size ) and triethylamine (2.0 mL). The above reactants were stirred at room temperature for 1 h under the protection of argon, and then heated to reflux and continued for 24 h. The above reactants were cooled to room temperature and stirred at room temperature for 24 h. The resulting reaction product was filtered and washed twice with dimethyl amide, each time with 30 mL, then washed twice with deionized water, each time with 30 mL, and finally washed 3 times with tetrahydrofuran, each time with 30 mL. The resulting product was filtered through air for 24 h to obtain 3.43 g of light white azidostyrene / nitrogen-containing heterocyclic copolymer-coated CPG, recorded as Co-polymer coated CPG-N3.

[0086] S4: Co-polymer coated CPG-N3 and 50 mL of anhydrous tetrahydrofuran were added to a 250 mL three-necked round-bottom flask. The reaction mixture was evacuated for 1 minute and then flushed with argon. This process was repeated twice. Under argon protection, the reaction mixture was cooled to 0°C. Lithium aluminum hydride (LAH 1.0 M THF, 5 mL) was slowly added through a dropping funnel over 15 minutes. The reaction mixture was then stirred at room temperature for 24 hours. The product was filtered and washed with 0.1 N hydrochloric acid twice, 50 mL each time, 1.0 M baking soda solution twice, 50 mL each time, 2-methylformamide once, 50 mL each time, methanol twice, and finally tetrahydrofuran twice, 50 mL each time. The resulting product was air-dried for 40 hours to obtain aminostyrene / nitrogen-containing heterocyclic copolymer-coated CPG, designated as Co-polymer coated CPG-NH2. The aminostyrene / nitrogen-containing heterocyclic copolymer-coated CPG has an amino loading of 242 μmol / g as measured by the DMT-Cl method.

[0087] 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, having the structure shown below) and begin stirring. Add triethylamine (0.5 mL) and stir for 10 minutes 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 hours. Insert a filter tube into the above reaction mixture, turn on the suction filter, and drain the solution. Add the prepared acetonitrile / dichloromethane solution (50 mL, 2:1 (v / v)), stir for 5 minutes, insert the filter tube into the above reaction mixture, turn on the suction filter, and drain the solution. Repeat this step two more times. 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 minutes. 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 hours. The product was filtered through a filter device under argon protection, then washed twice with acetonitrile, each time with 50 mL, then washed once with 50 mL of dichloromethane, and finally washed three times with tetrahydrofuran, each time with 50 mL. The product was dried in a vacuum for 24 hours to obtain 3.85 g of modified styrene / nitrogen-containing heterocyclic copolymer-coated CPG, recorded as Co-polymer coated CPG-Unylinker.

[0088] After testing, the nucleoside loading capacity in the Co-polymer coated CPG-Unylinker was 132 μmol / g, and the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer in the Co-polymer coated CPG-Unylinker to the average pore size of the glass carrier was 1:250.

[0089]

[0090] Example 3

[0091] S1: To 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 begin stirring to obtain the reactant. To a separate 50 mL round-bottom flask, add diisobutyl azodicarbonate (AIBN, 0.1 g, 0.6 mmol) and 30 mL of toluene. Stir until all solids have dissolved, and then add this solution to the reactant. Evacuate the reaction mixture for 1 minute, then flush with argon. Repeat this procedure two more times. Under argon, heat the reaction mixture to 80°C and stir for 10 hours to obtain a viscous product.

[0092] The viscous product was cooled to 40°C and slowly added to a stirred 210 mL 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 the solution was slowly added to a stirred 700 mL methanol solution. The stirring was continued for 2 hours and filtered. The resulting filter cake was then washed twice with 30 mL of methanol each time. The resulting product was vacuum dried at 40°C for 12 hours to obtain 3.82 g of a white halogenated styrene / vinyl nitrile copolymer, designated as PACS.

[0093] S2: PACS (0.54 g), NaN3 (0.65 g), ammonium chloride (0.7 g), and dimethylformamide (20 mL) were added to a 50 mL three-necked round-bottom flask. The reaction mixture was evacuated for 1 minute, then flushed with argon. This process was repeated twice. Under argon, the reaction mixture was heated to 100°C and the reaction continued for 10 hours. The reaction mixture was cooled to room temperature and filtered. The product was washed twice with 2-methylformamide (10 mL each), then twice with deionized water (15 mL each), and finally twice with methanol (10 mL each). The product was then dried under vacuum for 24 hours to yield 0.55 g of a styrene azide / nitrogen-containing heterocyclic copolymer.

[0094] GPC analysis showed that the number average molecular weight of the styrene azide / nitrogen-containing heterocyclic copolymer was 9550.

[0095] S3: In a 250 mL three-necked round-bottom flask, add styrene azide / nitrogen-containing heterocyclic copolymer (0.52 g) and tetrahydrofuran, stir for 30 min, and then add 3.0 g of glass particles (bare spheres, particle size 240 ± 10 mesh, pore size ) and triethylamine (2.0 mL). The above reactants were stirred at room temperature for 1 h under the protection of argon, and then heated to reflux and continued for 24 h. The above reactants were cooled to room temperature and stirred at room temperature for 24 h. The resulting reaction product was filtered and washed twice with dimethyl amide, each time with 30 mL, then washed twice with deionized water, each time with 30 mL, and finally washed 3 times with tetrahydrofuran, each time with 30 mL. The resulting product was filtered through air for 24 h to obtain 3.43 g of light white azidostyrene / nitrogen-containing heterocyclic copolymer-coated CPG, recorded as Co-polymer coated CPG-N3.

[0096] S4: Co-polymer coated CPG-N3 and 50 mL of anhydrous tetrahydrofuran were added to a 250 mL three-necked round-bottom flask. The reaction mixture was evacuated for 1 minute and then flushed with argon. This process was repeated twice. Under argon protection, the reaction mixture was cooled to 0°C. Lithium aluminum hydride (LAH 1.0 M THF, 5 mL) was slowly added through a dropping funnel over 15 minutes. The reaction mixture was then stirred at room temperature for 24 hours. The product was filtered and washed with 0.1 N hydrochloric acid twice, 50 mL each time, 1.0 M baking soda solution twice, 50 mL each time, 2-methylformamide once, 50 mL each time, methanol twice, and finally tetrahydrofuran twice, 50 mL each time. The resulting product was air-dried for 40 hours to obtain aminostyrene / nitrogen-containing heterocyclic copolymer-coated CPG, designated as Co-polymer coated CPG-NH2. The aminostyrene / nitrogen-containing heterocyclic copolymer-coated CPG was measured by DMT-Cl method to have an amino group loading of 246 μmol / g.

[0097] 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, having the structure shown below) and begin stirring. Add triethylamine (0.5 mL) and stir for 10 minutes 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 hours. Insert a filter tube into the above reaction mixture, turn on the suction filter, and drain the solution. Add the prepared acetonitrile / dichloromethane solution (50 mL, 2:1 (v / v)), stir for 5 minutes, insert the filter tube into the above reaction mixture, turn on the suction filter, and drain the solution. Repeat this step two more times. 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 minutes. 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 hours. The product was filtered through a filter device under argon protection, then washed twice with acetonitrile, each time with 50 mL, then washed once with 50 mL of dichloromethane, and finally washed three times with tetrahydrofuran, each time with 50 mL. The product was dried in a vacuum for 24 hours to obtain 3.48 g of modified styrene / nitrogen-containing heterocyclic copolymer-coated CPG, recorded as Co-polymer coated CPG-Unylinker.

[0098] After testing, the nucleoside loading capacity in the Co-polymer coated CPG-Unylinker was 142 μmol / g, and the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer in the Co-polymer coated CPG-Unylinker to the average pore size of the glass carrier was 1:220.

[0099]

[0100] Comparative Example 1

[0101] A reference polymer-coated CPG was prepared according to the method of Example 1, except that in step S1, acrylonitrile was replaced with the same molar amount of p-chlorostyrene. The other conditions were the same as in Example 1, and polystyrene-coated CPG was obtained, which was recorded as polymer coated CPG-Unylinker.

[0102] After testing, the nucleoside loading capacity in the polymer coated CPG-Unylinker was 85 μmol / g.

[0103] Comparative Example 2

[0104] The glass particles used in Example 1 (Unylinker-CPG, bare spheres, particle size 150±10 mesh, ) as a carrier. After testing, the CPG loading capacity was 32 μmol / g.

[0105] Comparative Example 3

[0106] A polymer coated CPG-Unylinker was prepared according to the method of Example 1, except that the amount of the azidostyrene / nitrogen-containing heterocyclic copolymer was increased in step S3 so that the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer in the final co-polymer coated CPG-Unylinker to the average pore size of the glass support was 1:150.

[0107] Comparative Example 4

[0108] A polymer coated CPG-Unylinker was prepared according to the method of Example 1, except that the amount of the azidostyrene / nitrogen-containing heterocyclic copolymer was reduced in step S3 so that the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer in the final co-polymer coated CPG-Unylinker to the average pore size of the glass support was 1:400.

[0109] Test Case

[0110] The carriers obtained in the above examples and comparative examples were loaded into an empty synthesis column of a solid phase synthesizer (GE AKTA OP-100) and oligonucleotides were synthesized under the same conditions. Steps (1) to (4) were carried out at room temperature. All the following steps were carried out under argon protection. The specific process is as follows:

[0111] (1) Deprotection: A deprotection agent toluene solution is added to the synthesis column to remove the protecting matrix on the linker in the support. The concentration of the deprotection agent in the toluene solution is 5 wt %. The deprotection agent is composed of trichloroacetic acid, dichloroacetic acid, and 3,5-dinitrobenzoic acid in a weight ratio of 1:1:2. The column is then washed with acetonitrile.

[0112] (2) Coupling reaction: adding a coupling agent acetonitrile solution and a nucleoside phosphoramidite synthesis unit acetonitrile solution into the synthesis column; the concentration of the coupling agent in the acetonitrile solution is 0.2 mol / L, and the coupling agent is composed of 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole in a weight ratio of 10:1:1; the concentration of the acetonitrile solution of the nucleoside phosphoramidite synthesis unit is 1 mol / L, and the nucleoside phosphoramidite synthesis unit contains adenine A, thymine T, guanine G and cytosine C in a molar ratio of 1:1:1:1;

[0113] (3) Capping reaction: capping agent A and an equal volume of capping agent B were added to the synthesis column, respectively. 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%.

[0114] (4) Oxidation reaction: Add iodine tetrahydrofuran solution to the synthesis column, where the iodine concentration is 0.05 mol / L;

[0115] (5) Cleavage and deprotection: After the synthesis is completed, the solid phase carrier in the synthesis column is removed, and 30 wt% ammonia water is added thereto, and the mixture is treated at 60°C for 2 h. The oligonucleotide is then eluted with deionized water.

[0116] The purity of the oligonucleotides was determined by UV-Vis spectrophotometry (UV-Vis). The results are shown in Table 1.

[0117] Table 1

[0118]

[0119] As can be seen from the data in Table 1, the glass carrier loaded with a polymer provided by the present invention has a loading capacity of up to 128 μmol / g or more, which can be used to synthesize oligonucleotide carriers on a large scale, thereby improving the synthesis efficiency of oligonucleotides. In addition, the chain length of the oligonucleotide obtained from the glass carrier loaded with a polymer provided by the present invention is relatively long, which can reach more than 27648, with a relatively long chain length and a purity of more than 82.2%. As can be seen from the comparison of Example 1 and Comparative Example 1, when the nitrogen-containing heterocyclic compound is not introduced into the polymer, the loading capacity of the obtained carrier is small, and when it is used for oligonucleotide synthesis, the purity of the oligonucleotide cannot be effectively improved, and the resulting oligonucleotide chain is also relatively short. As can be seen from the comparison of Example 1 and Comparative Example 2, CPG is difficult to achieve a high loading capacity, and when it is used for oligonucleotide synthesis, the purity and chain length of the oligonucleotide cannot be effectively improved. As can be seen from the comparison of Example 1 with Comparative Examples 3 and 4, when the ratio of the thickness of the copolymer in the glass carrier loaded with a polymer to the average pore size of the glass carrier is not within the scope of the present invention, either the loading capacity is too small or the purity and chain length of the resulting oligonucleotide are too short.

[0120] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A glass carrier loaded with a polymer, characterized in that: The polymer-loaded glass carrier includes a glass carrier and a modified styrene / nitrogen-containing heterocyclic copolymer coated on the surface of the glass carrier, wherein the ratio of the thickness of the modified styrene / nitrogen-containing heterocyclic copolymer to the average pore size of the glass carrier is 1:(200-250), and the modified styrene / nitrogen-containing heterocyclic copolymer has a structure shown in formula (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; X2 is -NH- or -O-; m and n represent the degree of polymerization of the styrene structural unit and the nitrogen-containing heterocyclic structural unit, respectively, and m:n = (1-9):1; Z is a linking group capable of undergoing a coupling reaction with the nucleoside phosphoramidite; and * represents a bond end.

2. The polymer-loaded glass carrier according to claim 1, characterized in that The particle size of the glass carrier is 120 mesh to 200 mesh.

3. The polymer-loaded glass carrier according to claim 1, characterized in that: The average pore size of the glass carrier is 4. The polymer-loaded glass carrier according to any one of claims 1 to 3, 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 5,000 to 20,000.

5. The polymer-loaded glass carrier according to any one of claims 1 to 3, characterized in that: A is N, and B and Y are independently N or C.

6. The polymer-loaded glass carrier according to any one of claims 1 to 3, characterized in that: Z is derived from at least one of the following linkers: Wherein, R is H, halogen or C1-C5 alkoxy, and R' is a group derived from adenine, guanine, cytosine or thymine.

7. The method for preparing a polymer-loaded glass carrier according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: S1: coating the styrene / nitrogen-containing heterocyclic copolymer having the structure shown in formula (2) on the surface of a glass carrier to obtain a glass carrier loaded with the styrene / nitrogen-containing heterocyclic copolymer; S2: When X3 is -N3, the glass support loaded with styrene / nitrogen-containing heterocyclic copolymer is subjected to a reduction reaction to obtain a glass support loaded with aminostyrene / nitrogen-containing heterocyclic copolymer; when X3 is -OH, the glass support loaded with styrene / nitrogen-containing heterocyclic copolymer is a glass support loaded with hydroxystyrene / nitrogen-containing heterocyclic copolymer, and step S3 is directly performed; S3: reacting the glass support loaded with the aminostyrene / nitrogen-containing heterocyclic copolymer and / or the glass support loaded with the hydroxystyrene / nitrogen-containing heterocyclic copolymer with a linker to bond a linking group capable of undergoing a coupling reaction with nucleoside phosphoramidites to the amino position of the aminostyrene / nitrogen-containing heterocyclic copolymer and / or the hydroxy position of the hydroxystyrene / nitrogen-containing heterocyclic copolymer, thereby obtaining the polymer-loaded glass support; In formula (4), A, B and Y are independently N, C, S or O; R1 is a single bond or a C1-C5 alkylene group; X3 is -N3 or -OH; m and n represent the degree of polymerization of the styrene structural unit and the nitrogen-containing heterocyclic structural unit, respectively, and m:n = (1-9):1; * represents the bond end.

8. The method for preparing a polymer-loaded glass carrier according to claim 7, characterized in that: In step S1, the method for coating the styrene / nitrogen-containing heterocyclic copolymer on the surface of the glass carrier comprises sequentially subjecting the styrene / nitrogen-containing heterocyclic copolymer and the glass carrier to a reflux reaction and a room temperature reaction in the presence of triethylamine and an organic solvent, followed by filtration, and optionally washing and removing impurities from the obtained solid product to obtain a glass carrier loaded with the styrene / nitrogen-containing heterocyclic copolymer.

9. The method for preparing a polymer-loaded glass carrier according to claim 7, wherein: In step S2, the reduction reaction conditions include a temperature of 0°C to 40°C and a time of 10 hours to 48 hours.

10. Use of the polymer-loaded glass carrier according to any one of claims 1 to 6 as a carrier for oligonucleotide synthesis.

11. A method for synthesizing an oligonucleotide, characterized in that: The method comprises sequentially subjecting a nucleoside phosphoramidite synthesis unit to coupling reaction, capping reaction and oxidation reaction using a polymer-loaded glass carrier as a solid phase carrier, and then cutting the oligonucleotide chain from the solid phase carrier to obtain the oligonucleotide.

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