CN114874376A

CN114874376A - Porous resin bead and preparation method and application thereof

Info

Publication number: CN114874376A
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: 2022-08-09
Anticipated expiration: 2042-06-14
Also published as: CN114874376B

Abstract

The invention provides a porous resin bead and a preparation method and application thereof, wherein the porous resin comprises a substituted or unsubstituted styrene unit, a dimethacrylate unit, an acetoxystyrene unit and a swelling balance unit; because the porous resin beads comprise the swelling balance unit, the porous resin beads can achieve 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, so that the purity and the yield of the long-fragment oligonucleotide are improved; meanwhile, the swelling volume of the porous resin beads in a solvent with high polarity is increased, the swelling volume in a solvent with low polarity is reduced, the difference of swelling properties in solvents with different polarities is reduced, and further the fluctuation of pressure in a reactor is reduced, so that the yield of the long-fragment oligonucleotide is stable.

Description

Porous resin bead 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 and a preparation method and application thereof.

Background

The ability of an oligonucleotide to be synthesized can be evaluated by the yield and the ratio of the total length (purity) of the synthesized gene fragment of interest, and the length of the synthesized oligonucleotide is usually determined by the pore size of the synthetic vector. The existing synthetic carrier is nano-pore glass with controllable pore diameter, is a rigid and non-swelling inorganic material, and mainly comprises silicon dioxide. The aperture 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 aperture, the loading capacity and the base synthesis length are directly related to the aperture size, and if the synthesis length is larger than corresponding parameters, the purity of gene synthesis is greatly reduced, so that the nano-pore glass is not beneficial to the large-scale synthesis of long-fragment oligonucleotides when being used as a carrier.

With the development of nucleic acid drugs, porous resins are used as solid carriers in oligonucleotide synthesis processes, and the loading capacity can be controlled to 400umol/g, but the swelling capacity of the porous resins in acetonitrile is small, the swelling capacity of the porous resins in toluene is large, when the porous resins are used as solid carriers, batch-to-batch yield is unstable, when DNA with more than 20-mer is synthesized, the yield and purity are greatly reduced, and the large-scale synthesis of long-chain oligonucleotides is not facilitated.

Disclosure of Invention

Based on this, there is a need for a porous resin bead that can be used for large-scale, high-purity, long-fragment oligonucleotide synthesis, and a method for preparing and using the same.

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

the swelling equilibrium units include one or more of acrylate units and substituted or unsubstituted acrylonitrile units.

In some embodiments, the substituted or unsubstituted acrylonitrile units include 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 bead comprises a plurality of hydroxyl groups, the plurality of hydroxyl groups being located at the surface of the porous resin bead and/or within a plurality of channels of the surface, respectively;

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

optionally, the particle size of the porous resin beads is 100-400 meshes.

An embodiment provides a method for preparing porous resin beads, which includes polymerizing a substituted or unsubstituted styrene monomer, a dimethacrylate monomer, an acetoxystyrene monomer, and a swelling equilibrium 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 prepare the copolymer;

removing a pore-foaming agent contained in the copolymer to prepare a porous copolymer;

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

In some embodiments, the swelling equilibrium monomer is 2 to 5% by mass of the total monomers; and/or the mass percentage of the dimethacrylate monomer in 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 equilibrium monomers include one or more of substituted or unsubstituted acrylonitrile monomers and acrylate monomers;

optionally, the swelling equilibrium monomers include substituted or unsubstituted acrylonitrile monomers and acrylate monomers; the mass ratio of the substituted or unsubstituted acrylonitrile monomer to the acrylate monomer is (1-8) to 1;

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

optionally, the substituted or unsubstituted styrene monomer comprises 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 methylene blue in the water is 0.01-0.03%.

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

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

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

optionally, the heating temperature is 70-90 ℃.

An embodiment provides an application of the porous resin bead prepared by the preparation method of the porous resin bead or the porous resin bead in preparing oligonucleotide.

According to the porous resin bead and the preparation method and application thereof, as the porous resin bead comprises the swelling balance unit, the porous resin bead can achieve higher swelling performance than that of common polystyrene porous resin in a solvent with higher polarity, and has higher reaction efficiency when long-fragment oligonucleotide is synthesized, so that the purity and the yield of the long-fragment oligonucleotide are improved; meanwhile, the swelling volume of the porous resin beads in a solvent with high polarity is increased, the swelling volume in a solvent with low polarity is reduced, the difference of swelling properties in solvents with different polarities is reduced, and further the fluctuation of pressure in a reactor is reduced, so that the yield of the long-fragment oligonucleotide is stable.

Drawings

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

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

fig. 3 is a morphology diagram of the porous resin beads provided in comparative example 2.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying 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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.

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

Herein, reference to numerical intervals is deemed continuous within the numerical intervals, unless otherwise stated, and includes the minimum and maximum values of the range, as well as each and every value between such minimum and maximum values. 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-describing 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 encompass any and all subranges subsumed therein.

In this context, referring to units of the data range, if only with units after the right end point, the units representing the left end point and the right end point are the same. For example, 70 to 90 ℃ means that the units of the left end point "70" and the right end point "90" are both in degrees centigrade.

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

As used herein, the term "and/or", "and/or" includes any one of two or more of the associated listed items, as well as any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all combinations of the associated listed items.

The ability of an oligonucleotide to be synthesized can be evaluated by the yield and the ratio of the total length (purity) of the synthesized gene fragment of interest, and the length of the synthesized oligonucleotide is usually determined by the pore size of the synthetic vector. The synthesis carrier generally existing in the market at present is nano-pore glass with controllable pore diameter, the nano-pore glass is a rigid and 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. Commonly used nanopore glasses for oligonucleotide synthesis include 500A, 1000A and 2000A of Hebei Dinarxing Biotech, Inc., 500A having a specific surface area of 80m ² About/g, the maximum loading amount is about 100 mu mol/g, the length of the basic group is about 20, the specific surface area of 1000A is 35m ² The loading capacity 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 maximum loading amount is about 25 mu mol/g, and the length of the basic group is generally less than 120; the carrying capacity and the base synthesis length of the nanopore glass are directly related to the pore size, and if the synthesis length is larger than corresponding parameters, the purity of gene synthesis is greatly reduced, so that the nanopore glass is not beneficial to large-scale synthesis of long-fragment oligonucleotides when being used as a carrier.

With the development of nucleic acid drugs, porous resins have been used as solid supports during oligonucleotide synthesis; when the porous resin is used as a solid support, the loading capacity for RNA and DNA is about 250. mu. mol/g and 400. mu. mol/g, but when the porous resin is used as a solid support, the yield and purity are reduced when DNA of 20-mer or more is synthesized, and the synthesis of long-chain oligonucleotides is also not facilitated.

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

When the functional groups of the porous resin are sparsely distributed, side reactions of macromolecules can be avoided, and the synthesis of oligonucleotides with larger molecular weights is facilitated. When the functional groups of the porous resin are densely distributed, the yield of the synthesized oligonucleotide can be increased, but there is a limit to the molecular chain length.

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

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

It should be noted that, in this specification, the unit refers to a segment in the porous resin bead. The substituted or unsubstituted styrene unit refers to a chain segment formed by the polymerization reaction of a substituted or unsubstituted styrene monomer; the dimethacrylate unit is a chain segment formed by polymerization reaction of dimethacrylate monomer; the acetoxystyrene unit refers to a chain segment formed by acetoxystyrene monomer participating in polymerization reaction; the swelling equilibrium unit refers to a segment composed of a swelling equilibrium monomer participating in polymerization, and the swelling equilibrium monomer refers to a monomer capable of reducing the difference in swelling volumes of the porous resin beads in different solvents.

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

When the porous resin bead is used for an oligonucleotide solid phase synthesis carrier, 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 synthesis purity and lot-to-lot yield variation when oligonucleotides are synthesized in large quantities.

In some embodiments, the swelling equilibrium 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 acrylate 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 acrylate 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 thereof, the substituted or unsubstituted acrylonitrile units comprise 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 may include one or more of styrene units, halogenated styrene units, and alkyl-substituted styrene units. For example, the halogenated styrene unit may include a chlorostyrene unit, a dichlorostyrene unit, or the like, and is not particularly limited; the alkyl-substituted styrene may include a methyl styrene unit, etc., 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 bead may include a plurality of hydroxyl groups, and the plurality of hydroxyl groups may be located at a surface of the porous resin bead and/or within a plurality of pores 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 embodiments, the hydroxyl groups can be present in an amount of 550 to 580 μmol/g; for example, the concentration may be 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, or the like, and the concentration is not particularly limited.

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

One embodiment provides a method for preparing porous resin beads, which includes polymerizing a substituted or unsubstituted styrene monomer, a dimethacrylate monomer, an acetoxystyrene monomer, and a swelling equilibrium monomer to form a copolymer.

In some embodiments, the swelling equilibrium monomers may include one or more of substituted or unsubstituted acrylonitrile monomers and acrylate monomers. When the substituted or unsubstituted acrylonitrile monomer or acrylate monomer is added during the preparation of the porous resin beads, the difference of the swelling properties of the porous resin beads in solvents with different polarities can be reduced; however, when the porous resin beads contain both an acrylate monomer and a 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 swollen equilibrium monomers can be 2-5% by mass of the total monomers; for example, it may be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or the like. When the mass percentage of the swelling equilibrium monomer in the total monomer is higher than 5%, regular spherical porous resin beads cannot be obtained; when the mass percentage of the swelling equilibrium monomer to the total monomers is less than 2%, the difference in the 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 used in preparing the porous resin beads.

In some embodiments, the swelling equilibrium monomers include substituted or unsubstituted acrylonitrile monomers and acrylate monomers; the mass ratio of the substituted or unsubstituted acrylonitrile monomer to the acrylate monomer can be (1-8): 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 acrylonitrile monomers and methacrylonitrile monomers.

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

In some embodiments, the substituted or unsubstituted styrene monomer may include one or more of a styrene monomer, a halogenated styrene monomer, and an alkyl-substituted styrene monomer; for example, the halogenated styrene monomer may include chlorostyrene, dichlorostyrene, or the like, and is not particularly limited; the alkyl-substituted styrene may include methyl styrene, etc., 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 is 3 to 7% by mass of the total monomers; for example, it may be 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, or the like. When the mass percentage of the dimethacrylate monomer in the total monomers is less than 3%, the swelling performance of the porous resin beads in a solvent with low polarity is remarkably improved, and the yield of the porous resin beads is reduced; when the mass percentage of the dimethacrylate monomer in the total monomers is higher than 7%, the swelling performance of the porous resin beads in a solvent with low polarity is remarkably reduced, and the swelling performance of the porous resin beads in a solvent with high polarity is also reduced, so that the synthesis efficiency of the long-chain oligonucleotide is reduced.

In some embodiments, the acetoxystyrene monomer is 5-12% by weight of the total monomers; for example, the concentration 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%, and the like, and is not particularly limited. When the mass percentage of the acetoxystyrene monomer in the total monomers is less than 5%, the hydroxyl sites for synthesis are too sparse, and the yield of the synthesized oligonucleotide is too low; when the mass percentage of the acetoxystyrene monomer in the total monomers is higher than 12%, the hydroxyl sites used for synthesis are too dense, the content of the intermediate cannot be increased significantly due to excessive hydroxyl groups, and the purity of the synthesized oligonucleotide is reduced due to excessive hydroxyl groups.

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

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

and hydrolyzing the porous copolymer in an alkaline alcohol aqueous solution to prepare the porous resin beads.

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

In some embodiments, the stabilizer may include polyvinyl alcohol; when the porous resin is prepared, the stabilizer is added, so that the polymerization reaction can be carried out for a dangerous period, the aggregation tendency of sticky droplets in a dispersion-aggregation dynamic equilibrium state is eliminated, various monomers can be dispersed more uniformly, the sticky droplets can be protected, and the agglomeration caused by mutual adhesion can be prevented.

In some of these embodiments, the degree of alcoholysis of the polyvinyl alcohol can be from 75% to 89%; for example, it may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and preferably 88 to 89%. The alcoholysis degree of the polyvinyl alcohol has 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 dispersing ability and the protective ability of each monomer are weak, the prepared polymer is large, and the particle distribution is wide; 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 easily caused.

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

In some embodiments, the stabilizer may be 0.02-1% by weight 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%, and preferably 0.5 to 1%.

In some embodiments, the methylene blue may be 0.01 to 0.03% by mass of the water; for example, the content may be 0.01%, 0.02%, 0.03%, etc., and is not particularly limited. When the porous resin beads are prepared, the protection capability of polyvinyl alcohol is weakened in the later period of polymerization reaction, and the methylene blue is added, so that the resin beads with good dispersibility can be obtained when the protection 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 monomers; for example, the concentration may be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6%, and the like, and is not particularly limited.

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

In some embodiments, the mass percent of the pore-foaming agent in the total monomers can be 1.0-2.6%; for example, the concentration may be 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%, and the like, and is not particularly limited.

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

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

In some of these embodiments, the mass ratio of the sodium hydroxide to the substituted or unsubstituted styrene monomer is (2-3): 20; for example, the ratio 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, and the like, and is not particularly limited.

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

When heating is performed, segmented heating can be adopted, and the method specifically comprises the following steps: heating at 80 ℃ for 3-5 h, heating at 85 ℃ for 3-5 h, and then heating at 90 ℃ for 3-5 h.

In some embodiments, the stirring speed may be 350rpm, and the stirring time may be 8-15 h; the copolymer having a particle size of 100 to 400 mesh can be obtained by stirring.

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

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

An embodiment provides an application of the porous resin bead or the porous resin bead prepared by the preparation method of the porous resin bead in preparing an oligonucleotide. When the porous resin beads are used for an oligonucleotide solid phase synthesis carrier, 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 synthesis purity and lot-to-lot yield variation when oligonucleotides are synthesized in large quantities.

The porous resin beads of the present invention, the preparation method and the use thereof will be described in detail with reference to the following examples.

It should be noted that, since acetonitrile and toluene are used as reagents commonly used in the oligonucleotide synthesis process, acetonitrile is used as a solvent having a relatively high polarity and toluene is used as a solvent having a relatively low polarity when the performance of the porous resin beads is measured in the following examples and comparative examples.

The BPO referred to in the following examples refers to benzoyl peroxide.

The method used in the measurement of the hydroxyl group content of the porous resin beads in each of the following examples and comparative examples was: accurately weighing 25g of acetic anhydride, then quantifying to 100mL by using anhydrous pyridine, accurately weighing 1g to 15mL of resin beads in a closed reaction bottle, taking 0.5mL to 1g of the quantified pyridine acetic anhydride solution of the pyridine acetic anhydride solution, then adding 9.5mL of pyridine, reacting for 2h at 95-100 ℃, then adding 1mL of water, continuing heating for 10min, titrating by using 0.2mol/l concentration sodium hydroxide, adding resin beads with a blank titration volume of A, adding resin beads with a titration volume of B, and adding resin beads with a hydroxyl content of 2000 (A-B) mu mol/g

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

In the following examples and comparative examples, the intermediate loading of the porous resin beads is measured, and the porous resin beads in the examples and comparative examples are coupled with the intermediate, wherein the structure of the coupled porous resin beads is shown as formula I:

wherein, the black solid sphere in formula I 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 the molecular weight of 1788Da and the alcoholysis degree of 87-88% in 1L of water, adding 0.2g of methylene blue, heating the water phase to 70 ℃, dissolving for 12 hours, and then cooling to 50 ℃ to prepare a dispersion medium;

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

adding the oil phase into the water phase, adjusting the rotation speed to 350rpm, enabling the resin bead particles to be in a size of 100-400 meshes, and stirring for 15 min; then heating to 80 ℃ and preserving heat for 2h, preserving heat for 3h at 85 ℃ and preserving heat for 3h at 90 ℃; then filtering with hot water of 70 ℃; after being dried by pumping, 1.5L of acetone is added to be stirred at 65 ℃ overnight, washed by alcohol, dried at 60 ℃ for 20h, and screened with 400-mesh resin beads.

Dissolving 20g of the obtained resin in 200mL of alcohol, dissolving 2g of sodium hydroxide in 100mL of water, adding the solution into the alcohol solution, stirring for 6h at 75 ℃, then neutralizing with hydrochloric acid, washing the resin with deionized water and alcohol for 3 times, and drying in vacuum at 60 ℃ to obtain the hydroxyl-containing porous resin beads.

The hydroxyl content is 580 mu mol/g by adopting the method; the dry volume was 2.8mL, the swell volume in acetonitrile was 4.9mL, and the swell volume in toluene was 5.0 mL; the intermediate loading is 352 mu mol/g; the morphology of the film observed by SEM scanning electron microscope is shown in FIG. 1.

Example 2

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

The hydroxyl content of the product is measured to be 582 mu mol/g by the method; the dry volume was 2.8mL, the swell volume in acetonitrile was 3.4mL, and the swell volume in toluene was 5.6 mL; 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 rest were the same.

The hydroxyl content was measured to be 581 μmol/g using the method described previously; the dry volume was 2.7mL, the swell volume in acetonitrile was 3.6mL, and the swell volume in toluene was 5.8 mL; the intermediate loading was 351. mu. mol/g.

Example 4

Example 4 differs from example 1 in that: triethylene glycol dimethacrylate is adopted to replace diethylene glycol dimethacrylate, methacrylonitrile is adopted to replace acrylonitrile, chlorostyrene is adopted to replace styrene, and the rest are the same.

The hydroxyl content of the hydroxyl group was measured to be 578. mu. mol/g by the method described above; the dry volume was 2.6mL, the swell volume in acetonitrile was 4.7mL, and the swell volume in toluene was 4.9 mL; 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, and methyl styrene is used to replace styrene, all the other things being equal.

The hydroxyl content of the hydroxyl solution is 584 mu mol/g measured by the method; the dry volume was 3.0mL, the swell volume in acetonitrile was 4.7mL, and the swell volume in toluene was 5.0 mL; 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, and the rest was the same.

The hydroxyl content of the product is measured to be 467 mu mol/g by the method; the dry volume was 3.0mL, the swell volume in acetonitrile was 4.9mL, and the swell volume in toluene was 5.2 mL; the intermediate loading capacity is 281 mu mol/g; the morphology of the sample observed by SEM scanning electron microscope is shown in FIG. 2.

Comparative example 2

Comparative example 2 differs from example 1 only in that: acrylonitrile and methyl acrylate were not added, and the rest were the same.

The hydroxyl content of the product is measured to be 579 mu mol/g by the method; the dry volume was 2.7mL, the swell volume in acetonitrile was 3.2mL, and the swell volume in toluene was 6.2 mL; the intermediate loading is 350 mu mol/g; the morphology of the film observed by SEM scanning electron microscope is shown in FIG. 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 components were the same. The reduction in the amount of diethylene glycol dimethacrylate resulted in a decrease in the specific surface area, a further decrease in the hydroxyl group content and a decrease in the yield of resin beads.

The hydroxyl content of the hydroxyl group is measured to be 465 mu mol/g by adopting the method; the dry volume was 2.8mL, the swell volume in acetonitrile was 3.3mL, and the swell volume in toluene was 8.4 mL; 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 components were the same.

The hydroxyl content of the product is measured to be 577 mu mol/g by the method; the dry volume was 2.8mL, the swell volume in acetonitrile was 3.0mL, and the swell volume in toluene was 4.5 mL; 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 rest was the same.

The hydroxyl content was measured to be 340. mu. mol/g, the dry volume was 2.8mL, the swell volume in acetonitrile was 4.8mL, and the swell volume in toluene was 4.9mL using the method described previously; 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 same was true for all the cases.

The hydroxyl content was 750. mu. mol/g, the dry volume was 2.8mL, the swell volume in acetonitrile was 4.9mL, the swell volume in toluene was 5.0mL, as measured by the method described previously; 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 a synthesis efficiency test using a Pomaceae 24-pass synthesizer under the synthesis conditions shown in Table 1,

TABLE 1

The synthesis steps are as follows:

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

step 2, activating the 3 'end of a new basic group monomer on the carrier by using phosphoramidite and an activating agent 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, carrying out cap reaction to eliminate unreacted free 5' -hydroxyl;

step 4, the phosphorouside of the nucleoside phosphite intermediate in the step 2 is oxidized into phosphotriester;

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

step 6, repeating the step 1 to the step 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, respectively, and then OD yield of the oligonucleotide was determined by measuring ultraviolet absorbance at 260nm of each gene fragment, and the full length ratio of the resulting oligonucleotide was calculated using waters 2695 test.

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

TABLE 2

TABLE 3

From the results of example 1, example 2, example 3 and comparative example 2, it can be seen that the introduction of acrylonitrile monomer or acrylate monomer can increase the swelling volume of the porous resin beads in acetonitrile and decrease the swelling volume of the porous resin beads in toluene, but the addition of acrylonitrile monomer and acrylate monomer at the same time is most effective; the results show that when the porous resin beads are prepared, the swelling difference of the porous resin beads in solvents with different polarities can be reduced by adding substituted or unsubstituted acrylonitrile monomers or acrylate monomers; however, when the porous resin beads contain both an acrylate monomer and a substituted or unsubstituted acrylonitrile monomer, the difference in swelling properties of the porous resin beads in solvents of different polarities is minimized

As can be seen from a comparison of fig. 1, 2 and 3, when methylene blue was added to prepare porous resin beads, spherical polystyrene resin beads having good monodispersity could be obtained; the method shows that when the porous resin beads are prepared, the protection capability of polyvinyl alcohol at the later stage of polymerization reaction is weakened, and the methylene blue is added, so that the resin beads with good dispersibility can be obtained when the protection capability of the polyvinyl alcohol is weakened. As shown in Table 3, the data of comparative example 1 show that the polymerized resin beads cause a decrease in the hydroxyl group content and the synthesis effect is 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 monomers is less than 3%, the swelling volume of the obtained porous resin beads in toluene is significantly increased, and when the mass percentage of diethylene glycol dimethacrylate to the total monomers is more than 7%, the swelling volume of the obtained porous resin beads in toluene is decreased, but the swelling volume in acetonitrile is also significantly decreased, and the synthesis efficiency of long-chain oligonucleotides is decreased; it is shown that when the mass percentage of the 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 in the total monomers is higher than 7%, the swelling performance of the porous resin beads in a solvent with low polarity is remarkably reduced, and the swelling performance of the porous resin beads in a solvent with high polarity is also reduced, so that the synthesis efficiency of the long-chain oligonucleotide is reduced.

From the results of example 1 and comparative examples 5 and 6, it can be seen that when the mass percentage of acetoxystyrene monomer to the total monomers is less than 5%, the hydroxyl group content of the resulting porous resin beads is low, the yield of the synthesized oligonucleotide is not changed, but the loading is decreased, and the amount of the synthetic resin required to synthesize the same amount of oligonucleotide is increased; when the mass percentage of the acetoxystyrene monomer in the total monomers is higher than 12%, the hydroxyl content of the prepared porous resin beads is too high, but the loading capacity of the intermediate is not improved along with the increase of the hydroxyl content, the highest loading capacity of the intermediate is about 400 mu mol/g, the residual hydroxyl has serious influence on the synthesis efficiency, and the synthesis purity is reduced when the acetoxystyrene monomer is used for synthesis; it is shown that when the porous resin beads are prepared, the acetoxystyrene monomer provides a hydroxyl source, and when the mass percentage of the acetoxystyrene monomer in the total monomers is less than 5%, the hydroxyl sites for synthesis are too sparse, the yield of the synthesized oligonucleotide is not changed, but the yield is too low; when the mass percentage of the acetoxystyrene monomer in the total monomers is higher than 12%, the hydroxyl sites used for synthesis are too dense, the intermediate loading capacity can only be limited to combine with the hydroxyl sites, and the residual excessive hydroxyl participates in the reaction in the synthesis process, so that the purity of the synthetic oligonucleotide is reduced.

As can be seen from Table 3, when the porous resin beads prepared from substituted or unsubstituted styrene monomer, dimethacrylate monomer, acetoxystyrene monomer, substituted or unsubstituted acrylonitrile monomer, and acrylate monomer are used for oligonucleotide solid phase synthesis of a carrier, the synthesis efficiency of 20 bases is 89% or more, the synthesis efficiency of 40 bases is 80% or more, the synthesis efficiency of 60 bases is 73% or more, and the synthesis efficiency of 80 bases is 65% or more, and the stable synthesis purity and the batch-to-batch yield difference can be satisfied when nucleic acid is synthesized in large quantities.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

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

2. The porous resin bead defined in 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;

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, and the plurality of hydroxyl groups are respectively located on the surface of the porous resin bead and/or in a plurality of pores of the surface;

optionally, the particle size of the porous resin beads is 100-400 meshes.

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

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

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

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

6. The method for preparing porous resin beads according to claim 4 or 5, wherein the swelling balance monomer is 2 to 5% by mass of the total monomers; and/or the mass percentage of the dimethacrylate monomer in the total monomer is 3-7%; and/or the acetoxystyrene monomer accounts for 5-12% of the total monomer by mass.

7. The method for preparing porous resin beads according to claim 4 or 5, wherein the swelling equilibrium monomer comprises one or more of a substituted or unsubstituted acrylonitrile monomer and an acrylate monomer;

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

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

optionally, the heating temperature is 70-90 ℃.

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 according to any one of claims 4 to 10 for preparing oligonucleotides.