Porous resin beads, a method for producing the same, and a method for producing nucleic acids using the porous resin beads

Abstract

To provide a porous resin bead for suppressing a rise in back pressure in a reaction vessel and stably producing a nucleic acid without liquid feeding failure in a process of synthesizing a nucleic acid. A porous resin bead characterized by consisting essentially of a styrene-hydroxystyrene-divinylbenzene-phenyl methylene malononitrile copolymer.

Description

Technical Field

[0001] This invention relates to porous resin beads, methods for manufacturing the same, and methods for manufacturing nucleic acids using the porous resin beads. Background Technology

[0002] The chemical synthesis of nucleic acids widely utilizes solid-phase synthesis methods, such as the phosphorous amide process. When using porous resin beads as carriers for solid-phase synthesis, these beads are preferably swollen to a certain extent in an organic solvent to efficiently synthesize nucleic acids on the carrier. However, when synthesizing nucleic acids sequentially on a carrier in various organic solvents, if the degree of swelling of the porous resin beads differs in each solvent, for example, when synthesizing nucleic acids on a carrier filled in a reaction vessel of a certain capacity, the volume change of the porous resin beads during liquid delivery may cause an increase in back pressure within the reaction vessel, leading to poor liquid delivery. Furthermore, these problems may reduce the yield of the target nucleic acid.

[0003] An attempt was made to improve the ability to synthesize nucleic acids by using (meth)acrylonitrile to suppress the variation in the swelling rate of porous resin beads in various organic solvents (see Patent Document 1). However, in the case of the porous resin beads in that document, although the variation in the swelling rate of the porous resin beads in various organic solvents was suppressed, the effect of suppressing the rise in back pressure within the reaction vessel was not sufficient. Existing technical documents Patent documents

[0004] Patent Document 1: Japanese Patent Application Publication No. 2008-74979 Summary of the Invention The problem that the invention aims to solve

[0005] The objective of this invention is to provide porous resin beads for the stable manufacture of nucleic acids in a reaction vessel without poor liquid delivery, thus suppressing back pressure rise during the synthesis of nucleic acids. Methods for solving problems

[0006] In order to solve the above-mentioned problems, the inventors conducted repeated in-depth research and found that a specific copolymer can become a porous resin bead. By using the porous resin bead, the back pressure rise in the reaction vessel can be suppressed and nucleic acid can be stably manufactured without poor liquid delivery during the nucleic acid synthesis process. Further research led to the completion of this invention.

[0007] That is, the present invention relates to the following. (1) A porous resin bead, characterized in that it is essentially composed of a styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer. (2) The porous resin beads according to (1), wherein the amount of benzenemethylene malononitrile structural units is 0.1 to 5.0 mmol / g relative to the total amount of structural units in the copolymer. (3) A method for manufacturing porous resin beads, characterized in that an organic solvent and water are used to perform suspension copolymerization of styrene monomers, acyloxystyrene monomers, divinylbenzene monomers and benzenemethylene malononitrile monomers to obtain a styrene-acyloxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer, and the obtained styrene-acyloxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer is hydrolyzed to transform it into a styrene-hydroxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer. (4) A method for manufacturing nucleic acid, characterized in that an oligonucleotide is obtained by sequentially binding a nucleoside or nucleotide to a porous resin bead described in (1) or (2) via a cleavable linker. Invention Effects

[0008] By using porous resin beads containing the specific copolymer of the present invention, it is possible to suppress back pressure rise within the reaction vessel and stably manufacture nucleic acids without poor liquid delivery during the nucleic acid synthesis process. Furthermore, the yield of the target nucleic acid can be increased. Detailed Implementation

[0009] The present invention will now be described in detail with reference to preferred embodiments, but the invention is not limited to these embodiments.

[0010] The porous resin beads of the present invention are characterized in that they are essentially composed of a styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer.

[0011] Typical examples of the styrene, hydroxystyrene, divinylbenzene, and benzenemethylene malononitrile structural units of the porous resin beads of the present invention are described below.

[0012] [Chemical Formula 1] Styrene structural unit

[0013] [Chemical Formula 2] Hydroxystyrene structural unit

[0014] [Chemical Formula 3] or Divinylbenzene structural unit

[0015] [Chemical Formula 4] benzenemethylene malononitrile structural unit

[0016] One or more hydrogen atoms (including hydrogen atoms in the benzene ring) in a styrene structural unit can be replaced by substituents such as alkyl groups, halogen atoms, carboxyl groups, sulfonic acid groups (-SO3H), cyano groups, alkoxy groups, and nitro groups with 1 to 5 carbon atoms. Among these, hydroxyl groups are excluded as substituents.

[0017] One or more hydrogen atoms in the hydroxystyrene structural unit (including hydrogen atoms in the benzene ring but excluding hydrogen atoms in the hydroxyl group) can be substituted by substituents such as alkyl, halogen, carboxyl, sulfonic acid, cyano, alkoxy, and nitro groups with 1 to 5 carbon atoms.

[0018] One or more hydrogen atoms (including hydrogen atoms in the benzene ring) in the divinylbenzene structural unit can be substituted by substituents such as alkyl, halogen, carboxyl, sulfonic acid, cyano, alkoxy, and nitro groups with 1 to 5 carbon atoms.

[0019] One or more hydrogen atoms (including hydrogen atoms in the benzene ring) in the benzenemethylene malononitrile structural unit can be substituted by substituents such as alkyl, halogen, carboxyl, sulfonic acid, cyano, alkoxy, and nitro groups with 1 to 5 carbon atoms.

[0020] The amount of styrene structural units is preferably 1.0 to 10.0 mmol / g, more preferably 4.0 to 8.0 mmol / g, and even more preferably 5.5 to 6.4 mmol / g, relative to the total amount of structural units in the porous resin beads of the present invention.

[0021] The amount of hydroxystyrene structural units is preferably 0.01 to 1.5 mmol / g, more preferably 0.1 to 1.0 mmol / g, and even more preferably 0.4 to 0.6 mmol / g, relative to the total amount of structural units in the porous resin beads of the present invention. If the amount of hydroxystyrene structural units relative to the total amount of structural units in the porous resin beads is too low, the final amount of hydroxyl groups will be low, thus tending to result in a smaller amount of the synthetic reactants obtained when used as a support for solid-phase synthesis. If the amount is too high, the distance between adjacent hydroxyl groups is insufficient, thus tending to cause adjacent chemical reactions to easily hinder each other, and reducing the purity of the synthetic reactants obtained when used as a support for solid-phase synthesis.

[0022] The amount of divinylbenzene structural units is preferably 0.1 to 2.0 mmol / g, more preferably 0.2 to 1.0 mmol / g, and even more preferably 0.3 to 0.5 mmol / g, relative to the total amount of structural units in the porous resin beads of the present invention. If the amount of divinylbenzene structural units is too low relative to the total amount of structural units in the porous resin beads, the specific surface area of ​​the obtained porous resin beads becomes smaller, thus tending to result in a smaller amount of synthetic reactants obtained when used as a support for solid-phase synthesis. If the amount is too high, the swelling degree of the obtained porous resin beads in organic solvents becomes lower, thus tending to result in a smaller amount of synthetic reactants obtained when used as a support for solid-phase synthesis.

[0023] The amount of benzenemethylene malononitrile structural units relative to the total amount of structural units in the porous resin beads of the present invention is preferably 0.1 to 5.0 mmol / g, more preferably 0.5 to 3.0 mmol / g, and even more preferably 1.0 to 1.6 mmol / g. If the amount of benzenemethylene malononitrile structural units relative to the total amount of structural units in the porous resin beads is within the above range, it has the effect of suppressing the variation of the swelling rate of the porous resin beads in various organic solvents and suppressing the rise of back pressure in the reaction vessel.

[0024] For the structural units in the porous resin beads of the present invention, without affecting the solid-phase synthesis reaction using the obtained porous resin beads, structural units that do not belong to any of the following structural units: styrene, hydroxystyrene, divinylbenzene, or benzenemethylene malononitrile. For example, other structural units may be added to the styrene-hydroxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer, or any structural unit in the styrene-hydroxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer may be replaced with other structural units.

[0025] The median pore size of the porous resin beads used in the solid-phase synthesis support of the present invention, as determined by mercury porosimetry, is not particularly limited, but is preferably 1 to 200 nm, more preferably 5 to 100 nm, and even more preferably 20 to 70 nm. When the median pore size is too small, the swelling caused by the organic solvent is low when used as a solid-phase synthesis support, thus reducing the site for the synthesis reaction. As a result, it tends to be difficult to induce the desired reaction, or the number of base sequences in the oligonucleotide synthesis may be less than desired. Conversely, when the median pore size is too large, the hydroxyl groups on the surface of the resin beads, which serve as the reaction site, have fewer opportunities to contact with reaction-related substances, tending to reduce the yield.

[0026] The median pore size of the porous resin beads of the present invention was determined by mercury intrusion porosimetry. Specifically, 0.1 g of the test sample was placed into an Autopore V 9620 (manufactured by Micromeritics) fine pore distribution measuring device, and the measurement was performed by mercury intrusion porosimetry under conditions of a mercury contact angle of 130°C and a mercury surface tension of 485 dyn / cm.

[0027] The swelling degree of the porous resin beads of the present invention is calculated by dividing (swelling volume) by (drying volume). Regarding the drying volume, 1.00 g of the solid-phase synthesis carrier is placed in a 10 ml graduated cylinder, and its apparent volume is measured. Regarding the swelling volume, a large excess of various organic solvents is added to the graduated cylinder containing the above-mentioned solid-phase synthesis carrier, and after standing at room temperature for 24 hours, its apparent volume is measured. The swelling ratio is the ratio of the swelling degree in each organic solvent; the swelling ratio of toluene to acetonitrile is calculated by dividing (swelling degree of toluene) by (swelling degree of acetonitrile).

[0028] The liquid delivery performance of the porous resin beads of this invention was determined using AKTA (registered trademark (oligopilot) plus 10 (manufactured by Cytiva). The synthesis equipment pressure when the column was filled with toluene and then delivered with acetonitrile (100~800 cm / h) and the synthesis equipment pressure when the column was filled with acetonitrile and then delivered with toluene (100~800 cm / h) were calculated.

[0029] The method for manufacturing the porous resin bead-shaped solid-phase synthesis carrier of the present invention is not particularly limited, and hydroxystyrene can be used instead of acyloxystyrene monomers to directly manufacture the porous resin bead-shaped solid-phase synthesis carrier of the present invention. However, hydroxystyrene is a very easily polymerized and unstable monomer, so it is not easy to handle and store. Therefore, it is preferable to obtain it by hydrolyzing the copolymer obtained by suspending copolymerization of each monomer.

[0030] In this invention, styrene monomers refer to styrene or its substitutes (wherein, acyloxy groups are excluded as substituents), preferably unsubstituted styrene. Examples of styrene substitutes include compounds in which one or more hydrogen atoms of styrene are substituted with alkyl groups having 1 to 5 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl), halogen atoms, carboxyl groups, sulfonic acid groups, cyano groups, alkoxy groups having 1 to 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy), nitro groups, etc.

[0031] As styrene monomers, specific examples include alkylstyrene such as styrene, ethylstyrene, methylstyrene, dimethylstyrene, trimethylstyrene, and butylstyrene; halostyrene such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, and bromostyrene; haloalkylstyrene such as chloromethylstyrene and fluoromethylstyrene; vinyl benzoate, sodium styrene sulfonate, cyanostyrene, methoxystyrene, ethoxystyrene, butoxystyrene, and nitrostyrene. It should be noted that when using divinylbenzene as a divinylbenzene monomer (described below), commercially available divinylbenzene contains ethylstyrene as an impurity; in fact, this ethylstyrene sometimes also functions as a styrene monomer.

[0032] In this invention, acyloxystyrene monomers refer to acyloxystyrene or its substitutes, preferably p-acetoxystyrene. Examples of acyloxystyrene substitutes include compounds in which one or more hydrogen atoms other than the acyloxy group are substituted with an alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group (e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy), or a nitro group. Furthermore, the acyloxy group refers to a substituent represented by the general formula X-CO-O- (where X is an alkyl or phenyl group), preferably an acyloxy group of an alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl) having 1 to 5 carbon atoms, and more preferably an acetoxy group. The acyloxy group is preferably positioned para-positioned relative to the vinyl group, but may also be positioned ortho- or meta-positioned. Specifically, examples of acyloxystyrene monomers include p-acetoxystyrene and p-benzoyloxystyrene.

[0033] In this invention, the divinylbenzene monomer is divinylbenzene or its substitutes, preferably divinylbenzene. Examples of divinylbenzene substitutes include compounds in which one or more hydrogen atoms of divinylbenzene are substituted with alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl), halogen atoms, carboxyl groups, sulfonic acid groups, cyano groups, alkoxy groups (e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy), nitro groups, etc., having substituted one or more hydrogen atoms of divinylbenzene with 1 to 5 carbon atoms. The two vinyl groups can be positioned at any of the para, meta, or ortho positions. Specifically, examples of divinylbenzene monomers include p-divinylbenzene, m-divinylbenzene, o-divinylbenzene, or mixtures thereof.

[0034] In this invention, the benzenemethylmalononitrile monomer is benzenemethylmalononitrile or its substitutes, preferably benzenemethylmalononitrile. Examples of substitutes for benzenemethylmalononitrile include compounds in which one or more hydrogen atoms of benzenemethylmalononitrile are replaced by alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl), halogen atoms, carboxyl groups, sulfonic acid groups, cyano groups, alkoxy groups (e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy), nitro groups, etc., having substituted one or more hydrogen atoms of benzenemethylmalononitrile with 1 to 5 carbon atoms.

[0035] Compared to the total amount of monomers including styrene monomers, acyloxystyrene monomers, divinylbenzene monomers and benzenemethylene malononitrile monomers in suspension copolymerization, the amount of styrene monomers added is preferably 40.0 to 85.0% by weight, more preferably 48.0 to 73.0% by weight, and even more preferably 56.8 to 66.8% by weight.

[0036] Compared to the total amount of monomers including styrene monomers, acyloxystyrene monomers, divinylbenzene monomers and benzenemethylene malononitrile monomers in suspension copolymerization, the amount of acyloxystyrene monomers is preferably 3.0 to 20.0% by weight, more preferably 6.0 to 10.0% by weight, and even more preferably 8.2% by weight.

[0037] Relative to the total amount of monomers including styrene monomers, acyloxystyrene monomers, divinylbenzene monomers and benzenemethylene malononitrile during suspension copolymerization, the amount of divinylbenzene monomers is preferably 1.0 to 18.0 wt%, more preferably 3.0 to 10.0 wt%, and even more preferably 5.0 to 6.0 wt%.

[0038] The amount of benzenemium malononitrile monomer added is preferably 10.0 to 30.0% by weight, more preferably 15.0 to 25.0% by weight, relative to the total amount of monomers including styrene monomers, acyloxystyrene monomers, divinylbenzene monomers and benzenemium malononitrile monomers in suspension copolymerization.

[0039] In this invention, the suspension copolymerization system may contain monomer components that do not belong to any of the following categories: styrene monomers, acyloxystyrene monomers, divinylbenzene monomers, or benzenemethylene malononitrile monomers, without affecting the solid-phase synthesis reaction using the obtained porous resin beads.

[0040] Suspension copolymerization is carried out by emulsifying the mixture of the above monomers and organic solvents in water by stirring. The organic solvent in this invention refers to a solvent other than water in the suspension copolymerization system, and preferably a structural isomer of a ketone or heptanone with 5 to 12 carbon atoms (e.g., 4-heptanone (dipropyl ketone)) and an alcohol.

[0041] As an alcohol, for example, an aliphatic alcohol can be used, preferably an aliphatic alcohol with 5 to 12 carbon atoms, more preferably 2-ethylhexanol, tert-amyl alcohol, heptanol, nonanol, 2-octanol, decol, lauryl alcohol, cyclohexanol, etc., and most preferably 1-heptanol.

[0042] Furthermore, during suspension polymerization, the porosity of the resulting porous resin beads increases; therefore, it is preferable to coexist 4-heptanone and an alcohol. The weight ratio of 4-heptanone to alcohol is appropriately varied depending on the specific combination of 4-heptanone and alcohol, thereby increasing the specific surface area of ​​the resulting porous resin beads. The preferred mixing ratio of 4-heptanone to alcohol, by weight, is preferably 1:9 to 6:4, more preferably 4:6 to 5:5. In this invention, 4-heptanone and alcohol are used as pore-forming agents, preferably in a weight ratio of 1:9 to 6:4, more preferably in a weight ratio of 4:6 to 5:5.

[0043] The weight of the organic solvent during suspension copolymerization is preferably 0.5 to 2.0 times, more preferably 0.8 to 1.8 times, relative to the total weight of the monomers. If this value deviates from the above range, the specific surface area of ​​the resulting porous resin beads may be smaller, and the amount of synthetic reactants produced by the chemical reaction may be less.

[0044] There are no particular limitations on its use as a dispersing stabilizer. For example, hydrophilic protective colloids such as polyvinyl alcohol, polyacrylic acid, gelatin, starch, and carboxymethyl cellulose, as well as insoluble powders such as calcium carbonate, magnesium carbonate, calcium phosphate, barium sulfate, calcium sulfate, and bentonite can be used.

[0045] As a polymerization initiator, there are no particular limitations. For example, previously known peroxides such as benzoyl peroxide, dilauroyl peroxide, distearyl peroxide, 1,1-di(tert-butylperoxide)-2-methylcyclohexane, 1,1-di(tert-hexylperoxide)-3,3,5-trimethylcyclohexane, 1,1-di(tert-hexylperoxide)cyclohexane, 1,1-di(tert-butylperoxide)cyclohexane, di-tert-hexylperoxide, tert-butylcumyl peroxide, di-tert-butylperoxide, 1,1,3,3-tetramethylbutylperoxide-2-ethylhexanoate, tert-hexylperoxide-2-ethylhexanoate, tert-butylperoxide-2-ethylhexanoate, tert-butylperoxide isopropyl monocarbonate, etc., as well as azo compounds such as 2,2'-azobisisobutyronitrile, 2,2'-azobis-2-methylbutyronitrile, and 2,2'-azobis-2,4-dimethylpentanonitrile, etc.

[0046] The reaction conditions for suspension copolymerization can be set appropriately, such as stirring at 60~90℃ for 30 minutes to 48 hours.

[0047] A styrene-acyloxystyrene-divinylbenzene-benzylmalonium nitrile copolymer can be obtained through the suspension copolymerization described above. After the obtained copolymer is appropriately cleaned by removing fine particles and filtration, it is subjected to the hydrolysis treatment described below.

[0048] Hydrolysis for converting acyloxystyrene-divinylbenzene-benzylmalonium nitrile copolymers to hydroxyl groups can be carried out using known methods and conditions. Acid catalysts such as hydrochloric acid, hydrobromic acid, or combinations of these acids with organic solvents such as methanol or dioxane, or base catalysts such as sodium hydroxide, potassium hydroxide, ammonia, hydrazine hydrate, or combinations of sodium hydroxide and ethanol can be used. The amount of acid catalyst is at least one equivalent of acyloxystyrene, preferably at least two equivalents, and the amount of base catalyst is at least one equivalent of acyloxystyrene, preferably at least two equivalents. It should be noted that it is not necessary to convert all acyloxy groups to hydroxyl groups during hydrolysis; the residual acyloxy groups are preferably about 10% or less relative to the hydroxystyrene structural units in the copolymer. Such copolymers with residual acyloxy groups are also included in this invention.

[0049] The above method yields a solid-phase carrier for the synthesis of porous resin beads according to the present invention. Further appropriate treatments such as drying and grading can then be performed.

[0050] The solid-phase synthesis support of the present invention can be used as a support for various chemical synthesis reactions. By using the solid-phase synthesis support of the present invention, even when using multiple organic solvents in a series of steps of a chemical synthesis reaction, it is possible to suppress the variation in the degree of swelling in each organic solvent and suppress the rise in back pressure, thus eliminating problems such as poor liquid delivery.

[0051] The solid-phase synthesis support of the present invention can be used particularly effectively for the synthesis of oligonucleotides or their derivatives. Preferably, the solid-phase synthesis support of the present invention is for the synthesis of oligonucleotides or their derivatives using toluene and acetonitrile.

[0052] The method for synthesizing oligonucleotides using the solid-phase synthesis carrier of the present invention can employ conventionally known methods. A linker is attached to the hydroxyl group of the solid-phase synthesis carrier of the present invention, and then an amidation is attached stepwise from the end of the linker in a manner forming a predetermined base sequence. This synthesis reaction can be carried out using an automated synthesis apparatus. For example, various organic solvents such as acetonitrile and amidation solutions are sequentially fed into a flow cytometer within an apparatus filled with the solid-phase synthesis carrier with the linker attached, and the reaction is repeated. Finally, the target oligonucleotide can be obtained by cleaving the linker portion through hydrolysis or the like. Conventionally known linkers are used, such as solid-phase synthesis carriers with nucleotide linkers having the following structure.

[0053] [Chemical Formula 5]

[0054] Regarding the yield in the method for synthesizing oligonucleotides using the solid-phase synthesis carrier of the present invention, the 20-mer yield per synthesis scale (OD / μmol) is calculated by (OD yield) × (total length %) / synthesis scale. The 20-mer yield per bead volume (OD / ml) is calculated by (20-mer yield per synthesis scale) × (binding amount of DMT-dT-3'-succinate) / (maximum swelling volume (toluene swelling volume)). Example

[0055] The following examples illustrate the invention in more detail, but the invention is not limited to the examples described below.

[0056] <Bead Making> Example 1 〇Suspension copolymer A 500ml separable flask equipped with a cooler, stirrer, and nitrogen inlet tube was placed in a constant temperature water bath. 1.9g of polyvinyl alcohol (made by Kuraray) and 188.5g of distilled water were added and stirred and dissolved at 175rpm. Separately, 34.9 g of styrene (manufactured by Fujifilm and Kohsen Pure Chemical Industries), 7.8 g of benzenemethylene malononitrile (manufactured by Tokyo Chemical Industries), 4.3 g of p-acetoxystyrene (manufactured by TOSOH·FINECHEM), 5.2 g of 55% divinylbenzene (isomer mixture, manufactured by Fujifilm and Kohsen Pure Chemical Industries, 2.9 g based on divinylbenzene), 46.8 g of 1-heptanol (manufactured by Fujifilm and Kohsen Pure Chemical Industries), 39.0 g of 4-heptanone (manufactured by Tokyo Chemical Industries), 1.2 g of benzoyl peroxide (manufactured by Nippon Yushi), and 0.3 g of 0.5 mol / L sodium nitrite solution (manufactured by Fujifilm and Kohsen Pure Chemical Industries) were mixed and dissolved, and this solution was added to the aforementioned separable flask. Under a nitrogen flow and at room temperature, the mixture was stirred at 440 rpm using a cross-shaped propeller blade, then heated to 80°C, and subjected to suspension copolymerization for 10 hours. 〇 Cleaning The film is filtered and cleaned using distilled water and acetone (Fujifilm and Kojun Pharmaceutical) and methanol (Fujifilm and Kojun Pharmaceutical). Hydrolysis In a 500 ml separable flask, the powder of the above-mentioned styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer and 210 g of 2-propanol (manufactured by Fujifilm and Koei Tecmo Chemicals) were added and stirred at 280 rpm to disperse the copolymer. A mixed solution of 2.5 g of sodium hydroxide (manufactured by Fujifilm and Koei Tecmo Chemicals) and 50 g of distilled water was added, and the mixture was heated to 80 °C for 1 hour for hydrolysis. After neutralization with hydrochloric acid, the mixture was filtered and washed with distilled water, acetone, and methanol, and then dried under reduced pressure to obtain a powdered porous resin bead-like solid-phase synthesis carrier composed of the styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer.

[0057] Example 2 〇Suspension copolymer A 500ml separable flask equipped with a cooler, stirrer, and nitrogen inlet tube was placed in a constant temperature water bath. 1.9g of polyvinyl alcohol (made by Kuraray) and 188.5g of distilled water were added and stirred and dissolved at 175rpm. Separately, 32.3 g of styrene (manufactured by Fujifilm and Kohsen Pure Chemical Industries), 10.5 g of benzenemethylene malononitrile (manufactured by Tokyo Chemical Industry), 4.3 g of p-acetoxystyrene (manufactured by TOSOH·FINECHEM) (8.2 wt% relative to all monomers), 5.2 g of 55% divinylbenzene (isomer mixture, manufactured by Fujifilm and Kohsen Pure Chemical Industries) (2.9 g based on divinylbenzene), 45.9 g of 1-heptanol (manufactured by Fujifilm and Kohsen Pure Chemical Industries), 39.9 g of 4-heptanone (manufactured by Tokyo Chemical Industry), 1.2 g of benzoyl peroxide (manufactured by Nippon Yushi), and 0.3 g of 0.5 mol / L sodium nitrite solution (manufactured by Fujifilm and Kohsen Pure Chemical Industries) were mixed and dissolved, and this solution was added to the aforementioned separable flask. Under a nitrogen flow and at room temperature, the mixture was stirred at 440 rpm using a cross-shaped propeller blade, then heated to 80°C, and subjected to suspension copolymerization for 10 hours. 〇 Cleaning The filtration and cleaning process was performed in the same manner as in Example 1. Hydrolysis After hydrolysis, neutralization, filtration and washing in the same manner as in Example 1, the mixture was dried under reduced pressure to obtain a powdered solid-phase synthesis carrier composed of porous resin beads made of a styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer.

[0058] Example 3 〇Suspension copolymer A 500ml separable flask equipped with a cooler, stirrer, and nitrogen inlet tube was placed in a constant temperature water bath. 1.9g of polyvinyl alcohol (made by Kuraray) and 188.5g of distilled water were added and stirred and dissolved at 175rpm. Separately, 29.7 g of styrene (manufactured by Fujifilm and Kohsen Pure Chemical Industries), 13.1 g of benzenemethylene malononitrile (manufactured by Tokyo Chemical Industry), 4.3 g of p-acetoxystyrene (manufactured by TOSOH·FINECHEM), 5.2 g of 55% divinylbenzene (isomer mixture, manufactured by Fujifilm and Kohsen Pure Chemical Industries, 2.9 g based on divinylbenzene), 44.6 g of 1-heptanol (manufactured by Fujifilm and Kohsen Pure Chemical Industries), 41.2 g of 4-heptanone (manufactured by Tokyo Chemical Industry), 1.2 g of benzoyl peroxide (manufactured by Nippon Yushi), and 0.3 g of 0.5 mol / L sodium nitrite solution (manufactured by Fujifilm and Kohsen Pure Chemical Industries) were mixed and dissolved, and this solution was added to the aforementioned separable flask. Under a nitrogen flow and at room temperature, the mixture was stirred at 440 rpm using a cross-shaped propeller blade, then heated to 80°C, and subjected to suspension copolymerization for 10 hours. 〇 Cleaning The filtration and cleaning process was performed in the same manner as in Example 1. Hydrolysis After hydrolysis, neutralization, filtration and washing in the same manner as in Example 1, the mixture was dried under reduced pressure to obtain a powdered solid-phase synthesis carrier composed of porous resin beads made of a styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer.

[0059] Comparative Example 1 〇Suspension copolymer A 500ml separable flask equipped with a cooler, stirrer, and nitrogen inlet tube was placed in a constant temperature water bath. 1.9g of polyvinyl alcohol (made by Kuraray) and 188.9g of distilled water were added and stirred at 175rpm to dissolve. Separately, 34.9 g of styrene (manufactured by Fujifilm and Kohsen Pure Chemicals), 8.4 g of methacrylonitrile (manufactured by Fujifilm and Kohsen Pure Chemicals), 4.0 g of p-acetoxystyrene (manufactured by TOSOH·FINECHEM, 7.7 wt% relative to all monomers), 5.0% of 55% divinylbenzene (isomer mixture, manufactured by Fujifilm and Kohsen Pure Chemicals, 2.8 g based on divinylbenzene), 60.1 g of 2-ethylhexanol (manufactured by Fujifilm and Kohsen Pure Chemicals), 25.7 g of isooctane (manufactured by Fujifilm and Kohsen Pure Chemicals), 1.2 g of benzoyl peroxide (manufactured by Nippon Yushi), and 0.3 g of 0.5 mol / L sodium nitrite solution (manufactured by Fujifilm and Kohsen Pure Chemicals) were mixed and dissolved, and this solution was added to the aforementioned separable flask. Under a nitrogen flow and at room temperature, the mixture was stirred at 440 rpm using a cross-shaped propeller blade, then heated to 80°C, and subjected to suspension copolymerization for 10 hours. 〇 Cleaning The film is filtered and cleaned using distilled water and acetone (Fujifilm and Kojun Pharmaceutical) and methanol (Fujifilm and Kojun Pharmaceutical). Hydrolysis In a 500 ml separable flask, the powder of the above-mentioned styrene-hydroxystyrene-divinylbenzene-methacrylonitrile copolymer and 210 g of 2-propanol (manufactured by Fujifilm and Kohsen Pure Chemicals) were added and stirred at 280 rpm to disperse the copolymer. A mixed solution of 2.5 g of sodium hydroxide (manufactured by Fujifilm and Kohsen Pure Chemicals) and 50 g of distilled water was added, and the mixture was heated to 80 °C for 1 hour for hydrolysis. After neutralization with hydrochloric acid, the mixture was filtered and washed with distilled water, acetone, and methanol, and then dried under reduced pressure to obtain a porous resin bead-like solid-phase synthesis carrier composed of powdered styrene-hydroxystyrene-divinylbenzene-methacrylonitrile copolymer.

[0060] The amounts (g) of monomers and pore-forming agents used in the suspension copolymerization of Examples 1-3 and Comparative Example 1, the weight ratios of each pore-forming agent (pore-forming agent 1 (P1): pore-forming agent 2 (P2)) and the weight ratio of the total amount of pore-forming agent to the total amount of monomers (the ratio of the amount of pore-forming agent (P1+P2) to the total amount of monomers) are shown in Table 1 below. Additionally, the amounts (mmol / g) of each structural unit in the porous resin beads obtained in Examples 1-3 and Comparative Example 1 are shown in Table 2 below. It should be noted that the amount (mmol / g) of each structural unit is calculated by (amount of each monomer (g)) / (molecular weight of each monomer (g / mol)) × 1000 / (total amount of monomers (g)). Table 1 The numbers in parentheses represent the amount of each monomer added (by weight %) relative to the total amount of monomers.

[0061] Table 2

[0062] <Experimental Methods> The following analysis was performed on the porous resin bead-like solid-phase synthesis carriers obtained in Examples 1-3 and Comparative Example 1. [Mid-position fine aperture] A 0.1g sample was placed into an Autopore V 9620 micropore distribution measuring device (manufactured by Micromeritics) and the mercury porosimetry was performed under the conditions of a mercury contact angle of 130°C and a mercury surface tension of 485 dyn / cm. [Swellability (dry volume, swollen volume, degree of swelling, swelling ratio)] The degree of swelling is calculated by dividing the (swollen volume) by the (dry volume). Here, the dry volume is determined by adding 1.00 g of the solid-phase synthesis carrier to a 10 ml graduated cylinder and measuring its apparent volume. The swelling volume is determined by adding a large excess of various organic solvents to a graduated cylinder containing the aforementioned solid-phase synthesis carrier, allowing it to stand at room temperature for 24 hours, and then measuring its apparent volume. The swelling ratio is the ratio of the degree of swelling in each organic solvent. The swelling ratio of toluene to acetonitrile is calculated by dividing the (swelling degree of toluene) by the (swelling degree of acetonitrile). [Liquid delivery capability; back pressure increase] 0.36–0.48 g of porous resin beads were packed into a 3.8 ml synthesis column and placed in an AKTA (registered trademark (oligopilot) plus 10 (manufactured by Cytiva)) container. The synthesis apparatus pressures were determined when the column was filled with toluene and then fed with acetonitrile (100–800 cm⁻¹ / h), and when the column was filled with acetonitrile and then fed with toluene (100–800 cm⁻¹ / h). The apparatus was stopped when the synthesis apparatus pressure exceeded 20 bar.

[0063] [Yield] 〇 Connector load response For the porous resin beads obtained in Examples 1, 2, 3 and Comparative Example 1, 1.0 g of porous resin beads, 287 mg of DMT-dT-3'-succinate (manufactured by ChemGenes), 161 mg of HBTU (manufactured by Sigma-Aldrich), 0.13 ml of N,N-diisopropylethylamine (manufactured by Tokyo Chemical Industry), and 10 ml of acetonitrile (manufactured by Fujifilm and Kohden Chemical Co., Ltd.) were mixed and reacted at room temperature for 12 hours with stirring. After filtration and washing with acetonitrile and methanol, the mixture was dried. 〇 End-capping reaction In these porous resin beads, 2.5 ml of CapA (20% acetic anhydride (manufactured by Fujifilm and Koichi Chemical) / 80% acetonitrile (manufactured by Fujifilm and Koichi Chemical)), 2.5 ml of CapB (20% N-methylimidazole (manufactured by Tokyo Chemical Industry) / 30% pyridine (manufactured by Fujifilm and Koichi Chemical) / 50% acetonitrile (manufactured by Fujifilm and Koichi Chemical)), 25 mg of 4-dimethylaminopyridine (manufactured by Fujifilm and Koichi Chemical), and 5 ml of acetonitrile were mixed and reacted at room temperature for 12 hours with stirring. After filtration and washing with acetonitrile and methanol, the mixture was dried under reduced pressure to obtain a solid-phase synthesis support bound to DMT-dT-3'-succinate. The amount of DMT-dT-3'-succinate bound was determined by absorbance measurement (412 nm) of the DMT groups after deprotection using a p-toluenesulfonic acid / acetonitrile solution. Synthesis and analysis 3-4 mg of the DMT-dT-3'-succinate-bound porous resin beads obtained above were loaded into a synthesis column (capacity 0.56 ml) and placed in a DNA / RNA synthesizer-NTS M-series (Techno, Japan). 20-mer mixed-sequence DNA oligonucleotides were synthesized under conditions of 5 eq / synthesis scale with nucleoside phosphoramide concentration and DMT-off. After synthesis, the dried porous resin beads were immersed in 0.35 ml of 28% ammonia solution and reacted at 55°C for 18 hours to excise the DNA oligonucleotides and deprotect the amino groups. The OD yield (equivalent to the amount of nucleic acid synthesized) was determined by UV absorbance measurement (260 nm) of the filtrate after filtering the porous resin beads. Additionally, UPLC analysis (Waters) of the filtrate was performed to determine the percentage of total length (the proportion of DNA oligonucleotides with the target sequence length). The 20-mer yield (OD / μmol) per synthesis scale was calculated by (OD yield) × (total length %) / synthesis scale. The 20mer yield per bead volume (OD / ml) is calculated by (20mer yield per synthetic scale) × (binding amount of DMT-dT-3'-succinate) / (maximum swelling volume (toluene swelling volume)).

[0064] The median pore size, swelling properties, liquid delivery properties, back pressure rise, and oligonucleotide yield of the porous resin bead-like solid-phase synthesis carriers obtained in Examples 1-3 and Comparative Example 1 are shown in Table 3 below. The porous resin bead-shaped solid-phase synthesis carrier of the present invention exhibits low swelling inhibition, small swelling difference, and increased liquid delivery in various organic solvents (acetonitrile, toluene), thus reducing back pressure rise and increasing oligonucleotide yield.

[0065] Table 3 ACN: Acetonitrile TOL: Toluene.

Claims

1. A porous resin bead, characterized in that, It is essentially composed of a styrene-hydroxystyrene-divinylbenzene-benzylmalonium nitrile copolymer.

2. The porous resin bead according to claim 1, wherein, The amount of benzenemethylene malononitrile structural units relative to the total amount of structural units in the copolymer is 0.1~5.0 mmol / g.

3. A method for manufacturing porous resin beads, characterized in that, Using organic solvents and water, styrene monomers, acyloxystyrene monomers, divinylbenzene monomers, and benzenemethylene malononitrile monomers are subjected to suspension copolymerization to obtain a styrene-acyloxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer. The obtained styrene-acyloxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer is then hydrolyzed to transform it into a styrene-hydroxystyrene-divinylbenzene-benzenemethylene malononitrile copolymer.

4. A method for manufacturing nucleic acids, characterized in that, Oligonucleotides are obtained by sequentially binding nucleosides or nucleotides to porous resin beads as described in claim 1 or 2 via cleavable linkers.

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