CN114874376B

CN114874376B - Porous resin beads, and preparation method and application thereof

Info

Publication number: CN114874376B
Application number: CN202210667591.2A
Authority: CN
Inventors: 卢晋华; 张阳; 李刚
Original assignee: Hebei Dinaxingke Biotechnology Co ltd; Tsingke Biotechnology Co Ltd
Current assignee: Hebei Dinaxingke Biotechnology Co ltd; Tsingke Biotechnology Co Ltd
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2023-07-11
Anticipated expiration: 2042-06-14
Also published as: CN114874376A

Abstract

The invention provides a porous resin bead, a preparation method and application thereof, wherein the porous resin bead comprises a substituted or unsubstituted styrene unit, a dimethacrylate unit, an acetoxystyrene unit and a swelling balance unit; because the porous resin beads comprise a swelling balance unit, the porous resin beads can reach higher swelling performance than common polystyrene porous resin in a solvent with larger polarity, and the porous resin beads have higher reaction efficiency and improve the purity and yield of long-fragment oligonucleotides when the synthesis of the long-fragment oligonucleotides is carried out; meanwhile, the swelling volume of the porous resin beads in the solvent with large polarity is increased, the swelling volume in the solvent with small polarity is reduced, the difference of swelling properties in the solvents with different polarities is reduced, and further the fluctuation of pressure in the reactor is reduced, so that the yield of the long-fragment oligonucleotide is stable.

Description

Porous resin beads, and preparation method and application thereof

Technical Field

The invention relates to the technical field of copolymer preparation, in particular to a porous resin bead, a preparation method and application thereof.

Background

The synthesis ability of an oligonucleotide can be evaluated by yield and the full length ratio (purity) of the synthesized gene fragment of interest, and the length of the synthesized oligonucleotide is generally determined by the pore size of the synthetic vector. The existing synthetic carrier is controllable aperture nano-pore glass, which is a rigid, non-swelling inorganic material, and the main component is silicon dioxide. The pore diameter of the nano-pore glass can be controlled to be about 0-400nm, but the specific surface area and the surface hydroxyl content of the nano-pore glass are reduced along with the increase of the pore diameter, the loading capacity and the base synthesis length of the nano-pore glass are directly related to the pore size, and if the synthesis length is larger than the corresponding parameters, the purity of gene synthesis is greatly reduced, so that the nano-pore glass is unfavorable for large-scale synthesis of long-fragment oligonucleotides when the nano-pore glass is used as a carrier.

With the development of nucleic acid drugs, porous resins are used as solid carriers in the oligonucleotide synthesis process, the loading of the porous resins can be controlled to 400umol/g, but the porous resins have smaller swelling property in acetonitrile and larger swelling property in toluene, and when the porous resins are used as solid carriers, the porous resins can cause unstable yield among batches, and the yield and purity of the porous resins can be greatly reduced when DNA more than 20-mers is synthesized, so that the porous resins are not beneficial to large-scale synthesis of long-chain oligonucleotides.

Disclosure of Invention

Based on this, it is necessary to provide a porous resin bead which can be used for large-scale, high-purity long-fragment oligonucleotide synthesis, and a method for producing the same and applications thereof.

One embodiment provides a porous resin bead comprising a substituted or unsubstituted styrene unit, a dimethacrylate unit, an acetoxystyrene unit and a swelling balance unit;

the swelling balance unit includes one or more of an acrylate unit and a substituted or unsubstituted acrylonitrile unit.

In some embodiments, the substituted or unsubstituted acrylonitrile units comprise one or more of acrylonitrile units and methacrylonitrile units; and/or the acrylate units comprise one or more of methyl acrylate units and butyl acrylate units;

Optionally, the substituted or unsubstituted styrene units include one or more of styrene units, halogenated styrene units, and alkyl substituted styrene units; and/or the dimethacrylate units comprise one or more of diethylene glycol dimethacrylate units and triethylene glycol dimethacrylate units.

In some embodiments, the porous resin beads comprise a plurality of hydroxyl groups, a plurality of the hydroxyl groups being located on the surface of the porous resin beads and/or within a plurality of cells of the surface, respectively;

optionally, the content of a plurality of hydroxyl groups is 550-580 mu mol/g;

alternatively, the porous resin beads have a particle size of 100 to 400 mesh.

An embodiment provides a method for preparing porous resin beads, comprising polymerizing a substituted or unsubstituted styrene monomer, a dimethacrylate monomer, an acetoxystyrene monomer and a swelling balance monomer to form a copolymer.

In some embodiments, the method specifically comprises the following steps:

mixing a stabilizer, methylene blue and water to prepare a dispersion medium;

mixing the substituted or unsubstituted styrene monomer, the dimethacrylate monomer, the acetoxystyrene monomer, the swelling balance monomer, a pore-forming agent and an initiator to prepare an oil phase;

Mixing the oil phase with the dispersion medium, introducing nitrogen, stirring, and heating to obtain the copolymer;

removing the porogen contained in the copolymer to obtain a porous copolymer;

and (3) hydrolyzing the porous copolymer in an alkaline alcohol aqueous solution to obtain the hydroxyl porous resin beads.

In some embodiments, the swelling balance monomer comprises 2-5% by mass of the total monomer; and/or the mass percentage of the dimethacrylate monomer to the total monomer is 3-7%; and/or the acetoxystyrene monomer accounts for 5-12% of the total monomer by mass.

In some embodiments, the swelling balance monomer comprises one or more of a substituted or unsubstituted acrylonitrile monomer and an acrylate monomer;

optionally, the swelling balance monomer comprises a substituted or unsubstituted acrylonitrile monomer and an acrylate monomer; the mass ratio of the substituted or unsubstituted acrylonitrile monomer to the acrylic ester monomer is (1-8) 1;

optionally, the substituted or unsubstituted acrylonitrile monomer includes one or more of an acrylonitrile monomer and a methacrylonitrile monomer; and/or the acrylate monomer comprises one or more of methyl acrylate monomer and butyl acrylate monomer;

Optionally, the substituted or unsubstituted styrene monomer includes one or more of a styrene monomer, a halogenated styrene monomer, and an alkyl substituted styrene monomer; and/or the dimethacrylate monomer comprises one or more of a diethylene glycol dimethacrylate monomer and a triethylene glycol dimethacrylate monomer.

In some embodiments, the stabilizer comprises polyvinyl alcohol;

optionally, the polyvinyl alcohol comprises at least one of the following conditions:

(1) The alcoholysis degree is 75-89%;

(2) The molecular weight is 1700-2000 Da;

optionally, the stabilizer accounts for 0.02-1% of the water by mass; and/or the mass percentage of the water of the methylene Lan Zhan is 0.01-0.03%.

In some embodiments, the initiator comprises benzoyl peroxide, the initiator comprising 1-6% by mass of the total monomer; and/or the pore-foaming agent comprises one or more of isooctane, isooctanol, undecanol, n-decanol and 1-chlorodecane, wherein the pore-foaming agent accounts for 1.0-2.6% of the total monomer by mass.

In some embodiments, the aqueous alkaline alcohol solution includes sodium hydroxide, ethanol, and water;

Optionally, the mass ratio of the sodium hydroxide to the water in the alkaline alcohol aqueous solution is (1-5): 100; and/or the volume ratio of water to ethanol in the alkaline aqueous alcohol solution is 1:2;

optionally, the heating is performed at a temperature of 70 to 90 ℃.

An embodiment provides an application of the porous resin beads or the porous resin beads prepared by the preparation method of the porous resin beads in the preparation of oligonucleotides.

The porous resin beads provided by the invention have the advantages that as the porous resin beads comprise the swelling balance unit, the porous resin beads can reach higher swelling performance than the common polystyrene porous resin in a solvent with larger polarity, and the porous resin beads have higher reaction efficiency and improve the purity and yield of long-fragment oligonucleotides when the long-fragment oligonucleotides are synthesized; meanwhile, the swelling volume of the porous resin beads in the solvent with large polarity is increased, the swelling volume in the solvent with small polarity is reduced, the difference of swelling properties in the solvents with different polarities is reduced, and further the fluctuation of pressure in the reactor is reduced, so that the yield of the long-fragment oligonucleotide is stable.

Drawings

FIG. 1 is a topography of porous resin beads provided in example 1;

FIG. 2 is a topography of the porous resin beads provided in comparative example 1;

FIG. 3 is a topography of the porous resin beads provided in comparative example 2.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

Herein, "one or more" refers to any one or any two or a combination of any two or more of the listed items.

In this context, reference to a numerical interval is to be construed as continuous and includes the minimum and maximum values of the range, and each value between such minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

In this context, referring to units of data range, if a unit is only carried after the right endpoint, the units representing the left and right endpoints are identical. For example, 70-90℃means that the units of the left end point "70" and the right end point "90" are both℃respectively.

The temperature parameter herein is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other.

The synthesis ability of an oligonucleotide can be evaluated by yield and the full length ratio (purity) of the synthesized gene fragment of interest, and the length of the synthesized oligonucleotide is generally determined by the pore size of the synthetic vector. The common synthetic carrier in the market at present is controllable aperture nano-pore glass, which is a rigid, non-swelling inorganic material, and the main component is silicon dioxide. The aperture of the nano-pore glass can be controlledAbout 0-400nm, but its specific surface area and surface hydroxyl content decrease with increasing pore size. The commonly used nanoporous glass for oligonucleotide synthesis comprises 500A, 1000A and 2000A,500A of Hebeidixing family Biotech Co.Ltd with a specific surface area of 80m ² About/g, a loading of up to about 100. Mu. Mol/g, a base length of generally about 20, a specific surface area of 1000A of 35m ² The loading per gram is about 40 mu mol/g at most, the base length is generally less than 80, the specific surface area of 2000A is 20m ² The highest loading per gram is about 25 mu mol/g, and the length of the base is generally less than 120; the loading and base synthesis length of the nanopore glass are directly related to the pore size, and if the synthesis length is larger than the corresponding parameters, the purity of gene synthesis is greatly reduced, so that the nanopore glass is unfavorable for large-scale synthesis of long-fragment oligonucleotides when the nanopore glass is used as a carrier.

With the development of nucleic acid drugs, porous resins are used as solid carriers in oligonucleotide synthesis; the porous resin has a loading capacity of about 250. Mu. Mol/g and 400. Mu. Mol/g for RNA and DNA when used as a solid support, but when used as a solid support, the porous resin causes a decrease in yield and purity when used for synthesizing DNA of 20-mer or more, and is disadvantageous for synthesizing long-chain oligonucleotides.

The swelling pore size of the porous resin determines the length of oligonucleotide synthesis and the surface hydroxyl loading size determines the yield of oligonucleotide synthesis. The porous resin can only swell but not dissolve in the phosphoramidite solid phase synthesis solvent, and when the porous resin is used as a carrier, the low molecular compound containing a functional group is combined with the porous resin in a covalent bond form, then a single-step or multi-step coupling reaction is carried out, and the byproducts of the reaction can be removed by filtration.

The porous resin has sparse distribution of functional groups, can avoid side reaction of macromolecules, and is favorable for synthesizing oligonucleotides with larger molecular weight. Although the yield of the synthesized oligonucleotide can be improved when the functional groups of the porous resin are densely distributed, there is a limit to the molecular chain length.

Most of the porous resins used in the prior art are PS crosslinked resins, which have relatively low polarity and low swelling properties in solvents with high polarity, and thus cannot be fully stretched. In order to obtain a large number of oligonucleotides of target length, it is necessary to swell to a certain pore size in a solvent of great polarity to achieve a high reaction efficiency. However, the porous resin has good swelling performance in a solvent with small polarity, and the reaction in various organic solvents can cause the yield of the reaction column and the oligonucleotide to be changed after multiple steps in the solid phase synthesis process of phosphoramidite.

In order to solve the above problems, an embodiment provides a porous resin bead, which may include a substituted or unsubstituted styrene unit, a dimethacrylate unit, an acetoxystyrene unit, and a swelling balance unit.

It is to be noted that the unit mentioned herein means a segment in the porous resin bead. The substituted or unsubstituted styrene unit means a segment composed of a substituted or unsubstituted styrene monomer participating in polymerization; the dimethacrylate units refer to chain segments formed by the polymerization of dimethacrylate monomers; the acetoxystyrene unit refers to a chain segment formed by the polymerization reaction of acetoxystyrene monomers; the swelling balance unit refers to a chain segment formed by the participation of a swelling balance monomer in polymerization reaction, and the swelling balance monomer refers to a monomer capable of reducing the swelling volume difference of the porous resin beads in different solvents.

The porous resin beads comprise a swelling balance unit, so that the porous resin beads can reach higher swelling performance than common polystyrene porous resin in a solvent with larger polarity, and have higher reaction efficiency when long-fragment oligonucleotide synthesis is carried out, and the purity and yield of the long-fragment oligonucleotide are improved; meanwhile, the swelling volume of the porous resin beads in the solvent with large polarity is increased, the swelling volume in the solvent with small polarity is reduced, the difference of swelling properties in the solvents with different polarities is reduced, and further the fluctuation of pressure in the reactor is reduced, so that the yield of the long-fragment oligonucleotide is stable.

When the porous resin beads are used for oligonucleotide solid phase synthesis carriers, the loading capacity can reach 350-400 mu mol/g, the synthesis efficiency of synthesizing 20 bases is 89% or more, the synthesis efficiency of synthesizing 40 bases is 80% or more, the synthesis efficiency of synthesizing 60 bases is 73% or more, and the synthesis efficiency of synthesizing 80 bases is 65% or more. And has stable synthetic purity and batch-to-batch yield differences when synthesizing oligonucleotides in large quantities.

In some embodiments, the swelling balance units may include one or more of acrylate units and substituted or unsubstituted acrylonitrile units.

When the porous resin beads contain substituted or unsubstituted acrylonitrile units or acrylic acid ester units, the difference in swelling properties of the porous resin beads in solvents of different polarities can be reduced; however, when the porous resin beads contain both an acrylic acid ester unit and a substituted or unsubstituted acrylonitrile unit, the difference in swelling properties of the porous resin beads in solvents of different polarities is minimized.

In some embodiments, the substituted or unsubstituted acrylonitrile units include one or more of acrylonitrile units and methacrylonitrile units.

In some of these embodiments, the acrylate units may include one or more of methyl acrylate units and butyl acrylate units.

In some embodiments, the substituted or unsubstituted styrene units can include one or more of styrene units, halogenated styrene units, and alkyl substituted styrene units. For example, the halogenated styrene unit may include chlorostyrene unit or dichlorostyrene unit, etc., and is not particularly limited; the alkyl-substituted styrene may include a methylstyrene unit and the like, and is not particularly limited.

In some embodiments, the dimethacrylate units may include one or more of diethylene glycol dimethacrylate units and triethylene glycol dimethacrylate units.

In some embodiments, the porous resin beads may include a plurality of hydroxyl groups, and a plurality of the hydroxyl groups may be located on the surface of the porous resin beads and/or within a plurality of cells of the surface, respectively. Hydroxyl groups are functional groups, and the amount of hydroxyl groups affects the length and purity of the synthesized oligonucleotides.

In some of these embodiments, the plurality of hydroxyl groups may be present in an amount of 550 to 580. Mu. Mol/g; for example, 550. Mu. Mol/g, 555. Mu. Mol/g, 560. Mu. Mol/g, 5655. Mu. Mol/g, 570. Mu. Mol/g, 575. Mu. Mol/g, 580. Mu. Mol/g, etc., are not particularly limited.

In some embodiments, the porous resin beads may have a particle size of 100 to 400 mesh.

In some embodiments, the swelling balance monomer may include one or more of a substituted or unsubstituted acrylonitrile monomer and an acrylate monomer. When the substituted or unsubstituted acrylonitrile monomer or acrylic ester monomer is added in the preparation of the porous resin beads, the swelling difference of the porous resin beads in solvents with different polarities can be reduced; however, when the porous resin beads contain both the acrylate monomer and the substituted or unsubstituted acrylonitrile monomer, the difference in swelling properties of the porous resin beads in solvents of different polarities is minimized.

In some embodiments, the swelling balance monomer may be 2-5% by mass of the total monomer; for example, it may be 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%. When the mass percentage of the swelling balance monomer to the total monomer is higher than 5%, irregular spherical porous resin beads are obtained; when the mass percentage of the swelling balance monomer to the total monomer is less than 2%, the difference in swelling properties of the porous resin beads in solvents of different polarities cannot be effectively reduced.

It is understood that the total monomers refer to substituted or unsubstituted styrene monomers, dimethacrylate monomers, acetoxystyrene monomers and swelling balance monomers employed in preparing the porous resin beads.

In some embodiments, the swelling balance monomer includes a substituted or unsubstituted acrylonitrile monomer and an acrylate monomer; the mass ratio of the substituted or unsubstituted acrylonitrile monomer to the acrylic acid ester monomer may be (1 to 8): 1. For example, it may be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1, etc., preferably 5:2.

In some embodiments, the substituted or unsubstituted acrylonitrile monomer may include one or more of an acrylonitrile monomer and a methacrylonitrile monomer.

In some embodiments, the acrylate monomer may include one or more of a methyl acrylate monomer and a butyl acrylate monomer.

In some embodiments, the substituted or unsubstituted styrene monomer may include one or more of styrene monomer, halogenated styrene monomer, and alkyl substituted styrene monomer; for example, the halogenated styrene monomer may include chlorostyrene, dichlorostyrene, etc., and is not particularly limited; the alkyl-substituted styrene may include methylstyrene and the like, and is not particularly limited.

In some embodiments, the dimethacrylate monomer may include one or more of a diethylene glycol dimethacrylate monomer and a triethylene glycol dimethacrylate monomer. In the preparation of the porous resin beads, the dimethacrylate monomer has the function of a crosslinking agent.

In some embodiments, the dimethacrylate monomer comprises 3-7% by mass of the total monomer; for example, it may be 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5% or 7%, etc. When the mass percentage of the dimethacrylate monomer to the total monomer is lower than 3%, the swelling performance of the porous resin beads in the solvent with small polarity is remarkably improved, and the yield of the porous resin beads is reduced; when the mass percentage of the dimethacrylate monomer to the total monomer is higher than 7%, the swelling performance of the porous resin beads in the solvent with small polarity is remarkably reduced, and the swelling performance of the porous resin beads in the solvent with large polarity is also reduced, thus reducing the synthesis efficiency of the long-chain oligonucleotide.

In some embodiments, the acetoxystyrene monomer comprises 5-12% by mass of the total monomer; for example, the content may be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5% or 12%, etc., and is not particularly limited. In the preparation of porous resin beads, the acetoxystyrene monomer provides a hydroxyl source, and when the mass percentage of the acetoxystyrene monomer in the total monomer is lower than 5%, the hydroxyl site for synthesis is too sparse, and the yield of the synthesized oligonucleotide is too low; when the acetoxystyrene monomer is more than 12% by mass of the total monomer, the hydroxyl sites for synthesis are too dense, and excessive hydroxyl groups do not significantly increase the intermediate content, but rather excessive hydroxyl groups may cause a decrease in the purity of the synthesized oligonucleotide.

In some embodiments, the method specifically includes the following steps:

mixing a stabilizer, methylene blue and water to prepare a dispersion medium;

removing the porogen contained in the copolymer to obtain a porous copolymer;

the porous copolymer is hydrolyzed in an alkaline alcohol aqueous solution to obtain porous resin beads.

In the present invention, the polymerization between monomers can be promoted by mixing the oil phase with the dispersion medium and then introducing nitrogen.

In some embodiments, the stabilizer may include polyvinyl alcohol; the addition of the stabilizer allows the polymerization reaction to run through the critical period during the preparation of the porous resin, eliminating the tendency of the tacky droplets to aggregate in a dispersion-aggregation dynamic equilibrium state, not only allowing more uniform dispersion of the various monomers, but also protecting the tacky droplets from agglomerations due to mutual adhesion.

In some of these embodiments, the polyvinyl alcohol may have an alcoholysis degree of 75 to 89%; for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, etc., preferably 88 to 89%. The alcoholysis degree of the polyvinyl alcohol has a direct influence on the dispersion stability of the resin beads obtained by suspension polymerization; when the alcoholysis degree of the polyvinyl alcohol is lower than 75%, the dispersion capability and the protection capability of each monomer are weaker, and the prepared polymer is coarse and has wide particle distribution; when the alcoholysis degree of the polyvinyl alcohol is higher than 89%, the solution viscosity is high in the preparation process, the heat transfer is difficult, and the polymer aggregation is easy to cause.

In some of these embodiments, the polyvinyl alcohol may have a molecular weight of 1700 to 2000Da; preferably 1788Da. The molecular weight of the polyvinyl alcohol also has a direct influence on the dispersion stability of the resin beads obtained by suspension polymerization.

In some embodiments, the stabilizer may comprise 0.02-1% by mass of the water; for example, it may be 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95% or 1%, etc., and preferably 0.5 to 1%.

In some embodiments, the methylene Lan Zhan may comprise 0.01 to 0.03% by mass of the water; for example, the content may be 0.01%, 0.02% or 0.03%, etc., and is not particularly limited. When the porous resin beads are prepared, the protective capability of the polyvinyl alcohol is weakened in the later stage of the polymerization reaction, and the addition of methylene blue can ensure that the resin beads with good dispersibility can still be obtained when the protective capability of the polyvinyl alcohol is weakened.

In some embodiments, the initiator may include benzoyl peroxide. The initiator is used for initiating the polymerization reaction of each monomer.

In some of these embodiments, the initiator may be 1-6% by mass of the total monomer; for example, the content may be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5% or 6%, etc., and is not particularly limited.

In some embodiments, the porogen may include one or more of isooctane, isooctanol, undecanol, n-decanol, and 1-chlorodecane. The porogen is used to form pores in the copolymer during the preparation process.

In some of these embodiments, the porogen may comprise 1.0 to 2.6 mass percent of the total monomer; for example, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5% or 2.6% may be used, and the present invention is not particularly limited.

In some embodiments, the aqueous alkaline alcohol solution may include sodium hydroxide, ethanol, and water.

In some of these embodiments, the volume ratio of water to ethanol in the basic aqueous alcohol solution may be 1:2; the mass ratio of the sodium hydroxide to the water in the alkaline alcohol aqueous solution may be (1-5): 100, for example, may be 1:100, 1.5:100, 2:100, 2.5:100, 3:100, 3.5:100, 4:100, 4.5:100 or 5:100, etc., and is not particularly limited. When the mass of sodium hydroxide and water in the alkaline alcohol aqueous solution is lower than 1:100, the acetoxy group cannot be completely hydrolyzed, and the amount of exposed hydroxyl groups is small; when the mass of sodium hydroxide and water in the alkaline aqueous alcohol solution is higher than 5:100, the alkalinity is too strong, and the structure of the copolymer will be destroyed.

In some embodiments, the mass ratio of the sodium hydroxide to the substituted or unsubstituted styrene monomer is (2-3) 20; for example, it may be 2:20, 2.1:20, 2.2:20, 2.3:20, 2.4:20, 2.5:20, 2.6:20, 2.7:20, 2.8:20, 2.9:20 or 3:20, etc., without being limited in particular.

In some embodiments, the heating temperature may be 70-90 ℃ when heating; for example, the temperature may be 70 ℃, 72 ℃, 74 ℃, 76 ℃, 78 ℃, 80 ℃, 82 ℃, 84 ℃, 86 ℃, 88 ℃, or 90 ℃, and the like, and is not particularly limited.

When heating is carried out, sectional heating can be adopted, and the method specifically comprises the following steps: heating at 80 deg.c for 3-5 hr, heating at 85 deg.c for 3-5 hr and then heating at 90 deg.c for 3-5 hr.

In some embodiments, the stirring speed may be 350rpm and the stirring time may be 8-15 hours; the copolymer with the particle size of 100-400 meshes can be obtained by stirring.

In some embodiments, the porogen contained in the copolymer may be removed by heating and stirring in acetone.

In some embodiments, when heating and stirring in acetone, the temperature of the heating and stirring may be 50 to 70 ℃, for example, may be 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, or the like, and is not particularly limited; the heating and stirring time can be 10 to 15 hours, for example, 11 hours, 12 hours, 13 hours, 14 hours or 15 hours, etc., and is not particularly limited; the volume ratio of the acetone to the copolymer can be (1.5-2): 1, for example, can be 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1, etc., and the method is not particularly limited; the porogen contained in the copolymer may be dissolved by heating and stirring in acetone to form a porous copolymer.

The porous resin beads or the porous resin beads prepared by the preparation method of the porous resin beads provided by an embodiment are applied to the preparation of oligonucleotides. When the porous resin beads are used for oligonucleotide solid phase synthesis carriers, the carrying capacity can reach 350-400 mu mol/g, the synthesis efficiency of synthesizing 20 bases is 89% or more, the synthesis efficiency of synthesizing 40 bases is 80% or more, the synthesis efficiency of synthesizing 60 bases is 73% or more, and the synthesis efficiency of synthesizing 80 bases is 65% or more. And has stable synthetic purity and batch-to-batch yield differences when synthesizing oligonucleotides in large quantities.

The porous resin beads of the present invention, and the preparation method and application thereof are described in detail with reference to specific examples.

In the following examples and comparative examples, acetonitrile was used as a more polar solvent and toluene was used as a less polar solvent in performance measurement of porous resin beads, since acetonitrile and toluene were commonly used as reagents in oligonucleotide synthesis.

The BPO mentioned in the examples below refers to benzoyl peroxide.

The following examples and comparative examples were conducted in order to determine the hydroxyl group content of the porous resin beads: accurately weighing 25g of acetic anhydride, quantifying to 100mL by using anhydrous pyridine, accurately weighing 1g to 15mL of resin beads, sealing a reaction bottle, taking 0.5mL to 1g of resin beads of the quantified pyridine acetic anhydride solution, adding 9.5mL of pyridine, reacting for 2 hours at 95-100 ℃, adding 1mL of water, continuously heating for 10min, titrating by using 0.2mol/l sodium hydroxide, wherein the blank titration volume of the number of unadded resin is A, the titration volume of the added resin beads is B, and the hydroxyl content is 2000 [ mu ] mol/g (A-B)

The method used in measuring the swelling volume of the porous resin beads in each of the following examples and comparative examples was: accurately weighing 1g of resin, placing the resin into 2 measuring cylinders with accurate quantitative and 10mL, recording the dry volume, respectively adding acetonitrile and toluene, uniformly stirring, and observing the volume of the resin beads after swelling after 24 hours.

In the following examples and comparative examples, it is necessary to couple the porous resin beads of each example and comparative example with the intermediate in order to determine the intermediate loading of the porous resin beads, and the structure after coupling is shown in formula I:

wherein, in the formula I, the black solid sphere represents a porous resin bead, and the part connected with the black solid sphere is an intermediate.

Example 1

Dissolving 10g of polyvinyl alcohol with molecular weight of 1788Da and alcoholysis degree of 87-88% in 1L of water, adding 0.2g of methylene blue, heating the water phase to 70 ℃ for dissolution for 12h, and then cooling to 50 ℃ to prepare a dispersion medium;

introducing nitrogen for 15min, and uniformly mixing 160g of styrene, 10g of diethylene glycol dimethacrylate, 20g of acetoxystyrene, 5g of acrylonitrile, 2g of methyl acrylate, 70g of isooctane, 160g of isooctanol and 4.5g of BPO to prepare an oil phase;

adding the oil phase into the water phase, adjusting the rotating speed to 350rpm to ensure that the resin beads are 100-400 meshes, and stirring for 15min; then heating to 80 ℃ for 2h,85 ℃ for 3h and 90 ℃ for 3h; then filtering with 70-degree hot water; after draining, 1.5L of acetone is added, stirring is carried out at 65 ℃ overnight, washing is carried out with alcohol, drying is carried out at 60 ℃ for 20 hours, and 100-400 mesh resin beads are sieved.

20g of the resin obtained above was dissolved in 200mL of alcohol, 2g of sodium hydroxide was dissolved in 100mL of water and added to the above alcohol solution, followed by stirring at 75℃for 6 hours, neutralization with hydrochloric acid, washing of the resin with deionized water and alcohol 3 times, and vacuum drying at 60℃to obtain hydroxyl group-containing porous resin beads.

The hydroxyl group content was 580. Mu. Mol/g as measured by the method described above; the dry volume was 2.8mL, the swelling volume in acetonitrile was 4.9mL, and the swelling volume in toluene was 5.0mL; the intermediate loading is 352 mu mol/g; the morphology of the product is observed by an SEM scanning electron microscope as shown in FIG. 1.

Example 2

Example 2 differs from example 1 only in that: methyl acrylate was not added, and the other was the same.

The hydroxyl content is 582 mu mol/g by the method; the dry volume was 2.8mL, the swelling volume in acetonitrile was 3.4mL, and the swelling volume in toluene was 5.6mL; the intermediate loading was 354. Mu. Mol/g.

Example 3

Example 3 differs from example 1 only in that: acrylonitrile was not added, and the other was the same.

The hydroxyl group content was 581. Mu. Mol/g as measured by the method described above; the dry volume was 2.7mL, the swelling volume in acetonitrile was 3.6mL, and the swelling volume in toluene was 5.8mL; the intermediate loading was 351. Mu. Mol/g.

Example 4

Example 4 differs from example 1 in that: triethylene glycol dimethacrylate is used for replacing diethylene glycol dimethacrylate, methacrylonitrile is used for replacing acrylonitrile, chlorostyrene is used for replacing styrene, and all the other components are the same.

The hydroxyl content is 578 mu mol/g measured by the method; the dry volume was 2.6mL, the swelling volume in acetonitrile was 4.7mL, and the swelling volume in toluene was 4.9mL; wherein the intermediate loading was 349. Mu. Mol/g.

Example 5

Example 5 differs from example 1 in that: butyl acrylate is used to replace methyl acrylate, methyl styrene is used to replace styrene, and all the others are the same.

The hydroxyl content of the catalyst is 584 mu mol/g by the method; the dry volume was 3.0mL, the swelling volume in acetonitrile was 4.7mL, and the swelling volume in toluene was 5.0mL; the intermediate loading was 354. Mu. Mol/g.

Comparative example 1

Comparative example 1 and example 1 differ only in that: methylene blue was not added, all other things being equal.

The hydroxyl group content was measured to be 467. Mu. Mol/g by the method described above; the dry volume was 3.0mL, the swelling volume in acetonitrile was 4.9mL, and the swelling volume in toluene was 5.2mL; the intermediate loading is 281 mu mol/g; the morphology is observed by SEM scanning electron microscopy as shown in figure 2.

Comparative example 2

Comparative example 2 and example 1 differ only in that: acrylonitrile and methyl acrylate were not added, and the other was the same.

The hydroxyl content of the polymer is 579 mu mol/g by the method; the dry volume was 2.7mL, the swelling volume in acetonitrile was 3.2mL, and the swelling volume in toluene was 6.2mL; the intermediate loading is 350 mu mol/g; the morphology is observed by SEM scanning electron microscopy as shown in figure 3.

Comparative example 3

Comparative example 3 differs from example 1 only in that: the amount of diethylene glycol dimethacrylate added was 5g, and the other matters were the same. The reduction in the amount of diethylene glycol dimethacrylate causes a decrease in the specific surface area, a further decrease in the hydroxyl content, and a decrease in the yield of the resin beads.

The hydroxyl content was 465. Mu. Mol/g as measured by the method described above; the dry volume was 2.8mL, the swelling volume in acetonitrile was 3.3mL, and the swelling volume in toluene was 8.4mL; the intermediate loading was 270. Mu. Mol/g.

Comparative example 4

Comparative example 4 differs from example 1 only in that: the amount of diethylene glycol dimethacrylate added was 20g, and the other matters were the same.

The hydroxyl content of the polymer is 577 mu mol/g by the method; the dry volume was 2.8mL, the swelling volume in acetonitrile was 3.0mL, and the swelling volume in toluene was 4.5mL; wherein the intermediate loading was 349. Mu. Mol/g.

Comparative example 5

Comparative example 5 differs from example 1 only in that: the amount of acetoxystyrene added was 5g, and the other was the same.

The hydroxyl group content was 340. Mu. Mol/g, the dry volume was 2.8mL, the swelling volume in acetonitrile was 4.8mL, and the swelling volume in toluene was 4.9mL as measured by the method described above; the intermediate loading was 167. Mu. Mol/g.

Comparative example 6

Comparative example 6 differs from example 1 only in that: the amount of acetoxystyrene added was 30g, and the other was the same.

The hydroxyl group content was 750. Mu. Mol/g, the dry volume was 2.8mL, the swelling volume in acetonitrile was 4.9mL, and the swelling volume in toluene was 5.0mL, as measured by the method described above; the intermediate loading was 354. Mu. Mol/g.

Performance testing

The porous resin beads prepared in each example and each comparative example were subjected to synthesis efficiency test using a 24-pass synthesizer of the family Optimaceae, the synthesis conditions are shown in Table 1,

TABLE 1

The synthesis steps are as follows:

step 1, removing a protecting group (dimethoxy trityl group) from a nucleotide monomer which is connected with the protecting group and is pre-connected to a carrier to obtain a free 5' -hydroxyl;

step 2, using phosphoramidite and an activator to activate the 3 'end of a new base monomer on a carrier to obtain a nucleoside phosphite activated intermediate, and carrying out condensation reaction on the nucleoside phosphite activated intermediate and the free 5' -hydroxyl in the step 1 to obtain a nucleoside phosphite intermediate;

step 3, capping reaction is carried out to eliminate unreacted free 5' -hydroxyl;

step 4, oxidizing the phosphorous acid of the nucleoside phosphite intermediate of step 2 to a phosphotriester;

Step 5, cleaning the residual reagent in the steps 1 to 4 by using a cleaning solution;

step 6, repeating the steps 1 to 5;

and 7, separating the deoxynucleotide primer pre-product from the carrier to obtain the deoxynucleotide primer.

20, 40, 60, 80 gene fragments were synthesized separately, and then the OD yield of the oligonucleotides was determined by measuring the UV absorbance at 260nm of each gene fragment, and the full length ratio of the resulting oligonucleotides was calculated using the waters 2695 test.

The results of the hydroxyl group content, swelling volume, intermediate loading test of the porous resin beads prepared in each example and each comparative example are summarized in table 2; the OD yields and full length ratio results of oligonucleotides prepared using the porous resin beads prepared in each example and each comparative example as a carrier are summarized in Table 3.

TABLE 2

TABLE 3 Table 3

/>

From the results of example 1, example 2, example 3 and comparative example 2, it is evident that the introduction of the acrylonitrile monomer or the acrylic ester monomer can increase the swelling volume of the porous resin beads in acetonitrile and reduce the swelling volume of the porous resin beads in toluene, but the effect is optimal when the acrylonitrile monomer and the acrylic ester monomer are simultaneously added; the method shows that when the porous resin beads are prepared, the substituted or unsubstituted acrylonitrile monomer or acrylic ester monomer is added, and the swelling difference of the porous resin beads in solvents with different polarities can be reduced; however, when the porous resin beads contain both the acrylate monomer and the substituted or unsubstituted acrylonitrile monomer, the difference in swellability of the porous resin beads in solvents of different polarities is minimized

As can be seen from comparison of FIGS. 1, 2 and 3, the addition of methylene blue during the preparation of the porous resin beads can give spherical polystyrene resin beads having good monodispersity; the method shows that when the porous resin beads are prepared, the protective capability of the polyvinyl alcohol is weakened in the late stage of the polymerization reaction, and the addition of the methylene blue can ensure that the resin beads with good dispersibility can be still obtained when the protective capability of the polyvinyl alcohol is weakened. From the data of comparative example 1 in Table 3, it is found that the polymerized resin beads caused a decrease in hydroxyl group content, and the synthesis effect was seriously deteriorated.

From the results of example 1 and comparative examples 3 and 4, it is understood that when the mass percentage of diethylene glycol dimethacrylate to the total monomer is less than 3%, the swelling volume of the prepared porous resin beads in toluene is significantly increased, and when the mass percentage of diethylene glycol dimethacrylate to the total monomer is more than 7%, the swelling volume of the prepared porous resin beads in toluene is reduced, but the swelling volume in acetonitrile is also significantly reduced, and the synthesis efficiency of long-chain oligonucleotide is lowered; it is shown that when the mass percentage of dimethacrylate monomer to the total monomer is less than 3%, the swelling property of the porous resin beads in a solvent having a small polarity is remarkably improved, but the swelling property in acetonitrile is not greatly changed, and the yield of the resin beads becomes low; when the mass percentage of the dimethacrylate monomer to the total monomer is higher than 7%, the swelling performance of the porous resin beads in the solvent with small polarity is remarkably reduced, and the swelling performance of the porous resin beads in the solvent with large polarity is also reduced, thus reducing the synthesis efficiency of the long-chain oligonucleotide.

From the results of example 1 and comparative examples 5 and 6, it is understood that when the mass percentage of the acetoxystyrene monomer to the total monomer is less than 5%, the hydroxyl group content of the produced porous resin beads is low, the yield of the synthetic oligonucleotides is unchanged, but the loading is decreased, and the amount of synthetic resin required to synthesize the same amount of oligonucleotides is increased; when the mass percentage of the acetoxystyrene monomer to the total monomer is higher than 12%, the hydroxyl content of the prepared porous resin beads is too high, but the loading of the intermediate is not increased along with the increase of the hydroxyl content, the loading of the intermediate is about 400 mu mol/g at most, and the residual hydroxyl has serious influence on the synthesis efficiency, so that the synthesis purity is reduced when the porous resin beads are used for synthesis; indicating that when the mass percentage of the acetoxystyrene monomer in the total monomer is less than 5%, the hydroxyl sites for synthesis are too sparse, the yield of the synthesized oligonucleotide is unchanged, but the yield is too low; when the acetoxystyrene monomer is more than 12% by mass of the total monomer, the hydroxyl sites for synthesis are too dense, the intermediate loading can only bind to the hydroxyl sites in a limited manner, and the remaining excess hydroxyl groups participate in the reaction during synthesis, resulting in a decrease in the purity of the synthesized oligonucleotide.

As can be seen from Table 3, the porous resin beads prepared from the substituted or unsubstituted styrene monomer, dimethacrylate monomer, acetoxystyrene monomer, substituted or unsubstituted acrylonitrile monomer, and acrylate monomer, when used in oligonucleotide solid phase synthesis support, have a synthesis efficiency of 89% or more for synthesizing 20 bases, 80% or more for synthesizing 40 bases, 73% or more for synthesizing 60 bases, 65% or more for synthesizing 80 bases, and can satisfy a stable synthesis purity and a lot-to-lot yield difference when synthesizing a large amount of nucleic acids.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A porous resin bead characterized by comprising a substituted or unsubstituted styrene unit, a dimethacrylate unit, an acetoxystyrene unit and a swelling balance unit;

the swelling balance unit comprises one or more of an acrylate unit and a substituted or unsubstituted acrylonitrile unit;

the preparation raw materials of the porous resin beads comprise substituted or unsubstituted styrene monomer, dimethacrylate monomer, acetoxystyrene monomer and swelling balance monomer; the weight percentage of the dimethacrylate monomer in the total monomer is 3-7%; the acetoxyl styrene monomer accounts for 5-12% of the total monomer by mass percent;

the preparation of the porous resin beads comprises the step of mixing a stabilizer, methylene blue and water to prepare a dispersion medium;

the substituted or unsubstituted styrene units include one or more of styrene units, halogenated styrene units, and alkyl substituted styrene units.

2. The porous resin bead according to claim 1, wherein the substituted or unsubstituted acrylonitrile units comprise one or more of acrylonitrile units and methacrylonitrile units; and/or the acrylate units comprise one or more of methyl acrylate units and butyl acrylate units;

Optionally, the dimethacrylate units include one or more of diethylene glycol dimethacrylate units and triethylene glycol dimethacrylate units.

3. The porous resin bead according to any one of claims 1 to 2, wherein the porous resin bead comprises a plurality of hydroxyl groups, the plurality of hydroxyl groups being located on the surface of the porous resin bead and/or within a plurality of cells of the surface, respectively;

optionally, the content of a plurality of hydroxyl groups is 550-580 mu mol/g;

alternatively, the porous resin beads have a particle size of 100 to 400 mesh.

4. A preparation method of porous resin beads is characterized by comprising the steps of carrying out polymerization reaction on substituted or unsubstituted styrene monomer, dimethacrylate monomer, acetoxystyrene monomer and swelling balance monomer to generate a copolymer;

the swelling balance monomer comprises one or more of an acrylate monomer and a substituted or unsubstituted acrylonitrile monomer;

the weight percentage of the dimethacrylate monomer in the total monomer is 3-7%; the acetoxyl styrene monomer accounts for 5-12% of the total monomer by mass percent;

the preparation of the copolymer comprises the steps of mixing a stabilizer, methylene blue and water to prepare a dispersion medium;

The substituted or unsubstituted styrene monomer includes one or more of styrene monomer, halogenated styrene monomer and alkyl substituted styrene monomer.

5. The method for producing a porous resin bead according to claim 4, comprising the steps of:

mixing a stabilizer, methylene blue and water to prepare a dispersion medium;

removing the porogen contained in the copolymer to obtain a porous copolymer;

6. The method for producing a porous resin bead according to claim 4 or 5, wherein the swelling balance monomer is 2 to 5% by mass of the total monomer.

7. The method for producing a porous resin bead according to claim 4 or 5, wherein the swelling balance monomer comprises a substituted or unsubstituted acrylonitrile monomer and an acrylate monomer; the mass ratio of the substituted or unsubstituted acrylonitrile monomer to the acrylic ester monomer is (1-8) 1;

optionally, the dimethacrylate monomer comprises one or more of a diethylene glycol dimethacrylate monomer and a triethylene glycol dimethacrylate monomer.

8. The method for producing a porous resin bead according to claim 5, wherein the stabilizer comprises polyvinyl alcohol;

(1) The alcoholysis degree is 75-89%;

(2) The molecular weight is 1700-2000 Da;

9. The method for producing porous resin beads according to claim 5, wherein the initiator comprises benzoyl peroxide, and the initiator accounts for 1 to 6% by mass of the total monomer; and/or the pore-foaming agent comprises one or more of isooctane, isooctanol, undecanol, n-decanol and 1-chlorodecane, wherein the pore-foaming agent accounts for 1.0-2.6% of the total monomer by mass.

10. The method for producing porous resin beads according to claim 5, wherein the aqueous alkaline alcohol solution comprises sodium hydroxide, ethanol and water;

optionally, the heating is performed at a temperature of 70 to 90 ℃.

11. Use of the porous resin beads according to any one of claims 1 to 3 or the porous resin beads prepared by the method for preparing porous resin beads according to any one of claims 4 to 10 for preparing oligonucleotides.