Porous resin bead, method for producing same, and method for producing nucleic acid using said porous resin bead

Abstract

The present invention addresses the problem of providing porous resin beads for stably producing a nucleic acid without causing a liquid feeding failure by suppressing an increase in back pressure in a reaction vessel in a step for synthesizing the nucleic acid. Provided are porous resin beads characterized by essentially comprising a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile-based copolymer.

Description

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

[0001] The present invention relates to porous resin beads, a method for producing the same, and a method for producing nucleic acids using the porous resin beads.

[0002] Solid-phase synthesis using the phosphoramidite method is widely used for the chemical synthesis of nucleic acids. When porous resin beads are used as a support for solid-phase synthesis, it is desirable for the porous resin beads to swell to a certain extent in an organic solvent so that nucleic acids can be efficiently synthesized on the support. However, when nucleic acids are synthesized sequentially on a support in various different organic solvents, if the degree of swelling of the porous resin beads in the various organic solvents differs, for example, when nucleic acids are chemically synthesized on a support filled in a reaction vessel of a fixed volume, the volume of the porous resin beads serving as the support may change during solution delivery, causing an increase in back pressure within the reaction vessel, which may result in poor solution delivery. Furthermore, these problems may result in a decrease in the yield of the desired nucleic acid.

[0003] Attempts have been made to improve nucleic acid synthesis performance by using (meth)acrylonitrile to suppress fluctuations in the swelling ratio of porous resin beads in various organic solvents (see Patent Document 1). However, although the porous resin beads in this document suppress fluctuations in the swelling ratio of porous resin beads in various organic solvents, they are not effective enough in suppressing an increase in back pressure in a reaction vessel.

[0004] JP 2008-74979 A

[0005] An object of the present invention is to provide porous resin beads that can suppress an increase in back pressure in a reaction vessel during a nucleic acid synthesis process, and that can stably produce nucleic acids without poor liquid transfer.

[0006] As a result of extensive research to solve the above-mentioned problems, the present inventors discovered that a specific copolymer can be made into porous resin beads, and that the use of such porous resin beads can suppress an increase in back pressure in a reaction vessel during the nucleic acid synthesis process, enabling stable production of nucleic acids without poor liquid delivery. Further research led to the completion of the present invention.

[0007] That is, the present invention relates to the following: (1) Porous resin beads consisting essentially of a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer; (2) Porous resin beads according to (1), in which the amount of benzalmalononitrile structural units relative to the total amount of structural units in the copolymer is 0.1 to 5.0 mmol / g; (3) A method for producing porous resin beads, comprising suspension copolymerizing a styrene-based monomer, an acyloxystyrene-based monomer, a divinylbenzene-based monomer, and a benzalmalononitrile-based monomer using an organic solvent and water to obtain a styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile copolymer, and then hydrolyzing the obtained styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile copolymer to obtain a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer; and (4) A method for producing nucleic acids, in which nucleosides or nucleotides are sequentially bound to the porous resin beads according to (1) or (2) via a cleavable linker to obtain an oligonucleotide.

[0008] By using porous resin beads containing the specific copolymer of the present invention, an increase in back pressure in a reaction vessel can be suppressed in the nucleic acid synthesis step, and nucleic acids can be stably produced without liquid delivery failure.Furthermore, the yield of the target nucleic acid can be improved.

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

[0010] The porous resin beads of the present invention are characterized by consisting essentially of a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer.

[0011] Typical examples of the structural units of styrene, hydroxystyrene, divinylbenzene and benzalmalononitrile in the porous resin beads of the present invention are as follows:

[0012] Styrene structural unit

[0013] Hydroxystyrene structural unit

[0014] Divinylbenzene structural unit

[0015] Benzalmalononitrile structural unit

[0016] One or more hydrogen atoms in the styrene structural unit (including hydrogen atoms in the benzene ring) may be substituted with an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group (—SO ３ It may be substituted with a substituent such as a hydroxyl group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group, provided that a hydroxyl group is excluded as a substituent.

[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) may be substituted with a substituent such as an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group.

[0018] One or more hydrogen atoms in the divinylbenzene structural unit (including hydrogen atoms in the benzene ring) may be substituted with a substituent such as an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group.

[0019] One or more hydrogen atoms (including hydrogen atoms of the benzene ring) in the benzalmalononitrile structural unit may be substituted with a substituent such as an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group.

[0020] The amount of styrene structural units relative to the total amount of structural units in the porous resin beads of the present invention 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.

[0021] The amount of hydroxystyrene structural units relative to the total amount of structural units in the porous resin beads of the present invention 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. If the amount of hydroxystyrene structural units relative to the total amount of structural units in the porous resin beads is too small, the amount of hydroxyl groups finally obtained will be low, and the amount of synthesis reaction product obtained when used as a support for solid-phase synthesis will tend to be small. If the amount is too large, the distance between adjacent hydroxyl groups will be insufficient, and chemical reactions occurring adjacent to each other will likely be inhibited, and the purity of the synthesis reaction product obtained when used as a support for solid-phase synthesis will tend to be low.

[0022] The amount of divinylbenzene structural units relative to the total amount of structural units in the porous resin beads of the present invention 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. If the amount of divinylbenzene structural units relative to the total amount of structural units in the porous resin beads is too small, the specific surface area of ​​the resulting porous resin beads will be small, and the amount of synthetic reaction product obtained when used as a support for solid-phase synthesis will tend to be small. If the amount is too large, the swelling degree of the resulting porous resin beads in organic solvents will be low, and the amount of synthetic reaction product obtained when used as a support for solid-phase synthesis will tend to be small.

[0023] The amount of benzalmalononitrile 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. When the amount of benzalmalononitrile structural units relative to the total amount of structural units in the porous resin beads is within the above range, the effect of suppressing the variation in swelling ratio of the porous resin beads in various organic solvents and suppressing an increase in back pressure in a reaction vessel can be achieved.

[0024] The structural units in the porous resin beads of the present invention may contain structural units that do not fall under any of the styrene structural unit, hydroxystyrene structural unit, divinylbenzene structural unit, and benzalmalononitrile structural unit, to the extent that they do not affect the solid-phase synthesis reaction using the resulting porous resin beads. For example, a different structural unit may be additionally contained in a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer, or any structural unit of a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer may be replaced with another structural unit.

[0025] The median pore diameter of the porous resin beads used in the solid-phase synthesis support of the present invention, as measured 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. If the median pore diameter is too small, when used as a solid-phase synthesis support, the degree of swelling with organic solvents will be low, resulting in a small synthetic reaction site, which tends to make it difficult for the desired reaction to occur or to result in a smaller-than-desired number of base sequences in oligonucleotide synthesis. On the other hand, if the median pore diameter is too large, there will be less opportunity for contact between the hydroxyl groups on the surface of the resin beads, which are the reaction site, and substances involved in the reaction, which tends to result in a lower yield.

[0026] The median pore diameter of the porous resin beads of the present invention is measured by mercury intrusion porosimetry. Specifically, 0.1 g of a measurement sample is placed in a pore size distribution analyzer, Autopore V 9620 (manufactured by Micromeritics), and the measurement is performed by mercury intrusion porosimetry under the 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 the swollen volume by the dry volume. Regarding the dry volume, 1.00 g of the support for solid phase synthesis is placed in a 10 ml measuring cylinder, and the apparent volume is measured. Regarding the swelling volume, a large excess of various organic solvents is added to the measuring cylinder containing the support for solid phase synthesis, and the cylinder is left to stand at room temperature for 24 hours, after which the apparent volume is measured. The swelling ratio is the ratio of the swelling degrees in various organic solvents, and the swelling ratio of toluene to acetonitrile is calculated by dividing the swelling degree in toluene by the swelling degree in acetonitrile.

[0028] The liquid transportability of the porous resin beads of the present invention is measured using an AKTA (registered trademark) oligopilot plus 10 (manufactured by Cytiva). The synthesizer pressure when the column is filled with toluene and then acetonitrile is transported (100 to 800 cm / h) and the synthesizer pressure when the column is filled with acetonitrile and then toluene is transported (100 to 800 cm / h) are measured.

[0029] The method for producing the porous resin bead support for solid-phase synthesis of the present invention is not particularly limited, and the porous resin bead support for solid-phase synthesis of the present invention can also be produced directly by using hydroxystyrene instead of an acyloxystyrene-based monomer. However, since hydroxystyrene is an unstable monomer that polymerizes very easily, it is not easy to handle or store. Therefore, a method of obtaining the support by suspension copolymerizing each monomer and hydrolyzing the copolymer obtained is preferred.

[0030] In the present invention, the styrene-based monomer refers to styrene or a substituted styrene (excluding acyloxy groups as a substituent), preferably unsubstituted styrene. Examples of the substituted styrene include compounds in which one or more hydrogen atoms of styrene are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy group, ethoxy group, propoxy group, butoxy group, pentyloxy group), a nitro group, or the like.

[0031] Specific examples of the styrene-based monomer include styrene, alkylstyrenes such as ethylstyrene, methylstyrene, dimethylstyrene, trimethylstyrene, and butylstyrene, halogenated styrenes such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, and bromostyrene, halogenated alkylstyrenes such as chloromethylstyrene and fluoromethylstyrene, vinyl benzoate, sodium styrenesulfonate, cyanostyrene, methoxystyrene, ethoxystyrene, butoxystyrene, and nitrostyrene. When divinylbenzene is used as the divinylbenzene-based monomer described below, commercially available divinylbenzene contains ethylstyrene as an impurity, and this ethylstyrene may actually function as the styrene-based monomer.

[0032] In the present invention, the term "acyloxystyrene-based monomer" refers to acyloxystyrene or a substituted acyloxystyrene, preferably p-acetoxystyrene. Examples of substituted acyloxystyrene include compounds in which one or more hydrogen atoms other than the acyloxy group are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy, or pentyloxy), or a nitro group. The acyloxy group is a substituent represented by the general formula X-CO-O- (wherein X is an alkyl group or a phenyl group), preferably an acyloxy group in which X is an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), and more preferably an acetoxy group. The acyloxy group is preferably positioned para to the vinyl group, but may also be positioned ortho or meta. Specific examples of the acyloxystyrene monomer include p-acetoxystyrene and p-benzoxystyrene.

[0033] In the present invention, the divinylbenzene-based monomer refers to divinylbenzene or a substituted divinylbenzene, preferably divinylbenzene. Examples of substituted divinylbenzene include compounds in which one or more hydrogen atoms of divinylbenzene are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy, or pentyloxy), or a nitro group. The two vinyl groups may be located at the para, meta, or ortho positions. Specific examples of the divinylbenzene-based monomer include p-divinylbenzene, m-divinylbenzene, o-divinylbenzene, or a mixture thereof.

[0034] In the present invention, the benzalmalononitrile-based monomer refers to benzalmalononitrile or a substituted compound thereof, preferably benzalmalononitrile. Examples of the substituted benzalmalononitrile include compounds in which one or more hydrogen atoms of benzalmalononitrile are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy, or pentyloxy), or a nitro group.

[0035] In the suspension copolymerization, the amount of the styrene-based monomer charged relative to the total amount of monomers including the styrene-based monomer, the acyloxystyrene-based monomer, the divinylbenzene-based monomer, and the benzalmalononitrile-based monomer 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] In the suspension copolymerization, the amount of the acyloxystyrene-based monomer charged relative to the total amount of monomers including the styrene-based monomer, the acyloxystyrene-based monomer, the divinylbenzene-based monomer, and the benzalmalononitrile-based monomer 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] In the suspension copolymerization, the amount of the divinylbenzene-based monomer charged relative to the total amount of monomers including the styrene-based monomer, the acyloxystyrene-based monomer, the divinylbenzene-based monomer, and benzalmalononitrile is preferably 1.0 to 18.0% by weight, more preferably 3.0 to 10.0% by weight, and even more preferably 5.0 to 6.0% by weight.

[0038] In the suspension copolymerization, the amount of the benzalmalononitrile-based monomer charged relative to the total amount of monomers including the styrene-based monomer, the acyloxystyrene-based monomer, the divinylbenzene-based monomer, and the benzalmalononitrile-based monomer is preferably 10.0 to 30.0% by weight, more preferably 15.0 to 25.0% by weight.

[0039] In the present invention, the suspension copolymerization system may contain a monomer component that does not fall into any of the styrene-based monomers, acyloxystyrene-based monomers, divinylbenzene-based monomers, and benzalmalononitrile-based monomers, as long as it does not affect the solid-phase synthesis reaction using the resulting porous resin beads.

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

[0041] As the alcohol, for example, an aliphatic alcohol can be used, preferably an aliphatic alcohol having 5 to 12 carbon atoms, more preferably 2-ethylhexyl alcohol, t-amyl alcohol, heptanol, nonyl alcohol, 2-octanol, decanol, lauryl alcohol, cyclohexanol, etc., and most preferably 1-heptanol.

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

[0043] The weight of the organic solvent used in suspension copolymerization is preferably 0.5 to 2.0 times, more preferably 0.8 to 1.8 times, the total weight of the monomers. If this value is outside the above range, the specific surface area of ​​the resulting porous resin beads may become small, and the amount of synthetic reactant produced by the chemical reaction may become small.

[0044] The dispersion stabilizer is not particularly limited, and examples thereof include conventionally known hydrophilic protective colloids such as polyvinyl alcohol, polyacrylic acid, gelatin, starch, and carboxymethyl cellulose, and sparingly soluble powders such as calcium carbonate, magnesium carbonate, calcium phosphate, barium sulfate, calcium sulfate, and bentonite.

[0045] The polymerization initiator is not particularly limited, and examples thereof include conventionally known dibenzoyl peroxide, dilauroyl peroxide, distearoyl peroxide, 1,1-di(t-butylperoxy)-2-methylcyclohexane, 1,1-di(t-hexylperoxy)-3,3,5-trimethylcyclohexane, 1,1-di(t-hexylperoxy)cyclohexane, 1,1-di(t-butylperoxy)cyclohexane, di-t-hexyl peroxide, t-butylcumyl peroxide, and the like. Peroxides such as di-t-butyl peroxide, 1,1,3,3-tetramethylbutylperoxy-2-ethylhexanoate, t-hexylperoxy-2-ethylhexanoate, t-butylperoxy-2-ethylhexanoate, and t-butylperoxyisopropyl monocarbonate, and azo compounds such as 2,2'-azobisisobutyronitrile, 2,2'-azobis-2-methylbutyronitrile, and 2,2'-azobis-2,4-dimethylvaleronitrile can be used.

[0046] The reaction conditions for suspension copolymerization can be appropriately set, and examples thereof include stirring at 60 to 90° C. for 30 minutes to 48 hours.

[0047] The above-described suspension copolymerization can produce a styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile copolymer. After removing fine particles and washing the obtained copolymer by filtration or the like, the copolymer is subjected to the hydrolysis treatment described below.

[0048] Hydrolysis to convert acyloxy groups in a styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile copolymer to hydroxyl groups can be carried out by known means and conditions. Examples of such catalysts include hydrochloric acid, hydrobromic acid, or combinations of these acids with organic solvents such as methanol or dioxane; and alkali catalysts such as sodium hydroxide, potassium hydroxide, ammonia, hydrazine hydrate, or combinations of sodium hydroxide and ethanol. The amount of the acid catalyst is at least one equivalent, preferably at least two equivalents, of the acyloxystyrene. The amount of the alkali catalyst is at least one equivalent, preferably at least two equivalents, of the acyloxystyrene. It is not necessary to convert all acyloxy groups to hydroxyl groups in the hydrolysis treatment; the remaining acyloxy groups are preferably about 10% or less of the hydroxystyrene structural units in the copolymer. Such copolymers containing remaining acyloxy groups are also encompassed by the present invention.

[0049] By the above method, the porous resin beads of the present invention can be obtained as a support for solid-phase synthesis. In this case, further treatments such as drying and classification may be carried out as appropriate.

[0050] The support for solid-phase synthesis of the present invention can be used as a support for various chemical synthesis reactions. By using the support for solid-phase synthesis of the present invention, even when multiple organic solvents are used in succession in a series of steps of a chemical synthesis reaction, fluctuations in swelling degree in each organic solvent are suppressed and back pressure increases are also suppressed, thereby eliminating problems such as poor liquid delivery.

[0051] The solid phase synthesis support of the present invention can be particularly effectively used 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 or acetonitrile.

[0052] Conventional methods can be used to synthesize oligonucleotides using the solid-phase synthesis support of the present invention. A linker is attached to the hydroxyl group of the solid-phase synthesis support of the present invention, and then amidites are attached stepwise from the end of the linker to form a predetermined base sequence. This synthesis reaction can be carried out using an automated synthesizer. For example, various organic solvents such as acetonitrile and amidite solutions are sequentially fed into a flow reactor in an apparatus filled with a solid-phase synthesis support to which a linker has been attached, and the reaction is repeated. Finally, the linker portion is cleaved by hydrolysis or the like to obtain the desired oligonucleotide. Conventional linkers can be used, including, for example, solid-phase synthesis supports to which a nucleotide linker having the following structure has been attached:

[0053]

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

[0055] The present invention will be described in more detail below by showing examples, but the present invention is not limited to the examples described below.

[0056] <Bead Production> Example 1 Suspension Copolymerization A 500 ml separable flask equipped with a cooler, a stirrer, and a nitrogen inlet tube was placed in a thermostatic water bath, and 1.9 g of polyvinyl alcohol (manufactured by Kuraray) and 188.5 g of distilled water were added and dissolved by stirring at 175 rpm. Separately, 34.9 g of styrene (Fujifilm Wako Pure Chemical Industries, Ltd.), 7.8 g of benzalmalononitrile (Tokyo Chemical Industry Co., Ltd.), 4.3 g of p-acetoxystyrene (Tosoh Finechem Co., Ltd.), 5.2 g of 55% divinylbenzene (isomer mixture, Fujifilm Wako Pure Chemical Industries, Ltd.) (2.9 g as divinylbenzene), 46.8 g of 1-heptanol (Fujifilm Wako Pure Chemical Industries, Ltd.), 39.0 g of 4-heptanone (Tokyo Chemical Industry Co., Ltd.), 1.2 g of benzoyl peroxide (NOF Corporation), and 0.3 g of 0.5 mol / L sodium nitrite solution (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved, and the resulting solution was added to the separable flask. Under a nitrogen stream, the mixture was stirred at 440 rpm using a cross-shaped propeller blade at room temperature, then heated to 80 °C and subjected to suspension copolymerization for 10 hours. Washing: Filtration and washing were performed using distilled water, acetone (Fujifilm Wako Pure Chemical Industries, Ltd.), and methanol (Fujifilm Wako Pure Chemical Industries, Ltd.). Hydrolysis: The above styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile copolymer powder and 210 g of 2-propanol (Fujifilm Wako Pure Chemical Industries, Ltd.) were placed in a 500 ml separable flask and stirred at 280 rpm to disperse. A mixed solution of 2.5 g of sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) and 50 g of distilled water was added, and the temperature was raised to 80°C and hydrolysis reaction was carried out for 1 hour. After neutralization with hydrochloric acid, the mixture was filtered and washed using distilled water, acetone, and methanol, and then dried under reduced pressure to obtain a porous resin bead-shaped support for solid-phase synthesis consisting of a powdered styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer.

[0057] Example 2 Suspension Copolymerization A 500 ml separable flask equipped with a cooler, a stirrer, and a nitrogen inlet tube was placed in a thermostatic water bath, and 1.9 g of polyvinyl alcohol (Kuraray) and 188.5 g of distilled water were added and dissolved by stirring at 175 rpm. Separately, 32.3 g of styrene (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 10.5 g of benzalmalononitrile (manufactured by Tokyo Chemical Industry Co., Ltd.), 4.3 g of p-acetoxystyrene (manufactured by Tosoh Finechem Co., Ltd.) (8.2 wt % based on the total monomers), 5.2 g of 55% divinylbenzene (isomer mixture, Fujifilm Wako Pure Chemical Industries, Ltd.) (2.9 g as divinylbenzene), 45.9 g of 1-heptanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 39.9 g of 4-heptanone (manufactured by Tokyo Chemical Industry Co., Ltd.), 1.2 g of benzoyl peroxide (manufactured by Nippon Oil & Fats Co., Ltd.), and 0.3 g of 0.5 mol / L sodium nitrite solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved, and the resulting solution was added to the separable flask. Under a nitrogen stream, the mixture was stirred at 440 rpm using a cross-shaped propeller blade at room temperature, and then heated to 80°C, where suspension copolymerization was carried out for 10 hours. ○ Washing: Filtration and washing were carried out in the same manner as in Example 1. ○ Hydrolysis: After hydrolysis, neutralization, filtration and washing were carried out in the same manner as in Example 1, followed by drying under reduced pressure, to obtain a porous resin bead-shaped support for solid-phase synthesis consisting of a powdered styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer.

[0058] Example 3 Suspension Copolymerization A 500 ml separable flask equipped with a cooler, a stirrer, and a nitrogen inlet tube was placed in a thermostatic water bath, and 1.9 g of polyvinyl alcohol (manufactured by Kuraray) and 188.5 g of distilled water were added and dissolved by stirring at 175 rpm. Separately, 29.7 g of styrene (Fujifilm Wako Pure Chemical Industries, Ltd.), 13.1 g of benzalmalononitrile (Tokyo Chemical Industry Co., Ltd.), 4.3 g of p-acetoxystyrene (Tosoh Finechem Co., Ltd.), 5.2 g of 55% divinylbenzene (isomer mixture, Fujifilm Wako Pure Chemical Industries, Ltd.) (2.9 g as divinylbenzene), 44.6 g of 1-heptanol (Fujifilm Wako Pure Chemical Industries, Ltd.), 41.2 g of 4-heptanone (Tokyo Chemical Industry Co., Ltd.), 1.2 g of benzoyl peroxide (NOF Corporation), and 0.3 g of 0.5 mol / L sodium nitrite solution (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved, and the resulting solution was added to the separable flask. Under a nitrogen stream, the mixture was stirred at 440 rpm using a cross-shaped propeller blade at room temperature, then heated to 80 °C and subjected to suspension copolymerization for 10 hours. Washing: Filtration and washing were carried out in the same manner as in Example 1. Hydrolysis: After hydrolysis, neutralization, filtration and washing were carried out in the same manner as in Example 1, and drying under reduced pressure was carried out to obtain a porous resin bead-shaped support for solid-phase synthesis made of a powdered styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer.

[0059] Comparative Example 1 Suspension Copolymerization A 500 ml separable flask equipped with a cooler, a stirrer, and a nitrogen inlet tube was placed in a thermostatic water bath, and 1.9 g of polyvinyl alcohol (manufactured by Kuraray) and 188.9 g of distilled water were added and dissolved by stirring at 175 rpm. Separately, 34.9 g of styrene (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 8.4 g of methacrylonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 4.0 g of p-acetoxystyrene (manufactured by Tosoh Finechem Co., Ltd.) (7.7 wt % based on the total monomers), 5.0 g of 55% divinylbenzene (isomer mixture, Fujifilm Wako Pure Chemical Industries, Ltd.) (2.8 g as divinylbenzene), 60.1 g of 2-ethylhexanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 25.7 g of isooctane (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1.2 g of benzoyl peroxide (manufactured by Nippon Oil & Fats Co., Ltd.), and 0.3 g of 0.5 mol / L sodium nitrite solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved, and the resulting solution was added to the separable flask. The mixture was stirred at 440 rpm using a cross-shaped propeller blade under a nitrogen stream at room temperature, then heated to 80°C and subjected to suspension copolymerization for 10 hours. Washing: Filtration and washing were performed using distilled water, acetone (Fujifilm Wako Pure Chemical Industries, Ltd.), and methanol (Fujifilm Wako Pure Chemical Industries, Ltd.). Hydrolysis: The styrene-acyloxystyrene-divinylbenzene-methacrylonitrile copolymer powder and 210 g of 2-propanol (Fujifilm Wako Pure Chemical Industries, Ltd.) were placed in a 500 ml separable flask and stirred at 280 rpm to disperse the mixture. A mixed solution of 2.5 g of sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) and 50 g of distilled water was added, and the mixture was heated to 80°C and subjected to hydrolysis for 1 hour. The mixture was neutralized with hydrochloric acid, filtered, washed with distilled water, acetone, and methanol, and then dried under reduced pressure to obtain a porous resin bead-shaped support for solid-phase synthesis consisting of a powdered styrene-hydroxystyrene-divinylbenzene-methacrylonitrile copolymer.

[0060] The amounts (g) of the monomers and porogens used in the suspension copolymerization of Examples 1 to 3 and Comparative Example 1, as well as the weight ratio of each porogen (porogen 1 (P1) : porogen 2 (P2)), and the weight ratio of the total amount of porogens to the total amount of monomers (ratio of the amount of porogens (P1 + P2) to the total amount of monomers) are shown in Table 1 below. The amount (mmol / g) of each structural unit in the porous resin beads obtained in Examples 1 to 3 and Comparative Example 1 is shown in Table 2 below. The amount (mmol / g) of each structural unit was calculated by (amount (g) of each monomer) / (molecular weight (g / mol) of each monomer) × 1000 / (total amount of monomers (g)).

[0061]

[0062] Test Methods The porous resin bead-like supports for solid-phase synthesis obtained in Examples 1 to 3 and Comparative Example 1 were analyzed as follows. [Median pore size] 0.1 g of a measurement sample was placed in an Autopore V 9620 pore size distribution analyzer (Micromeritics), and measurements were made by mercury intrusion porosimetry under conditions of a mercury contact angle of 130°C and a mercury surface tension of 485 dyn / cm. [Swellability (dry volume, swollen volume, swelling degree, swelling ratio)] The swelling degree was calculated by dividing the swollen volume by the dry volume. The dry volume was determined by placing 1.00 g of the support for solid-phase synthesis in a 10 ml graduated cylinder and measuring its apparent volume. The swollen volume was determined by adding a large excess of various organic solvents to the cylinder containing the support for solid-phase synthesis, allowing it to stand at room temperature for 24 hours, and then measuring its apparent volume. The swelling ratio is the ratio of swelling degrees in various organic solvents, and the swelling ratio of toluene to acetonitrile was calculated by dividing the swelling degree in toluene by the swelling degree in acetonitrile. [Flow characteristics and back pressure increase] 0.36-0.48 g of porous resin beads were packed into a synthesis column (volume 3.8 ml) and placed in an AKTA® oligopilot Plus 10 (manufactured by Cytiva). After filling the column with toluene, the synthesizer pressure was measured when acetonitrile was pumped (100-800 cm / h), and after filling the column with acetonitrile, the synthesizer pressure was measured when toluene was pumped (100-800 cm / h). Note that the synthesizer shuts down when the synthesizer pressure exceeds 20 bar.

[0063] [Yield] Linker loading reaction The porous resin beads obtained in Examples 1, 2, 3, and Comparative Example 1 were prepared by mixing 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 Co., Ltd.), and 10 ml of acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), reacting the mixture at room temperature for 12 hours with stirring, filtering and washing the mixture with acetonitrile and methanol, and then drying the mixture. Capping reaction: These porous resin beads were mixed with 2.5 ml of CapA (20% acetic anhydride (Fujifilm Wako Pure Chemical Industries, Ltd.) / 80% acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.)), 2.5 ml of CapB (20% N-methylimidazole (Tokyo Chemical Industry Co., Ltd.) / 30% pyridine (Fujifilm Wako Pure Chemical Industries, Ltd.) / 50% acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.)), 25 mg of 4-dimethylaminopyridine (Fujifilm Wako Pure Chemical Industries, Ltd.), and 5 ml of acetonitrile. The mixture was stirred at room temperature for 12 hours, filtered, washed with acetonitrile and methanol, and then dried under reduced pressure to obtain a solid-phase synthesis support bearing DMT-dT-3'-succinate. The amount of DMT-dT-3'-succinate bound was determined by absorbance measurement (412 nm) of the DMT group deprotected using p-toluenesulfonic acid / acetonitrile solution. Synthesis and Analysis: 3-4 mg of the DMT-dT-3'-succinate-conjugated porous resin beads obtained above were packed into a synthesis column (0.56 ml volume) and inserted into a DNA / RNA Synthesizer - NTS M-Series (Nihon Techno). A 20-mer mixed-sequence DNA oligonucleotide was synthesized using a nucleoside phosphoramidite concentration of 5 eq / synthesis scale and DMT-off conditions. After synthesis, the dried porous resin beads were immersed in 0.35 ml of 28% aqueous ammonia and incubated at 55°C for 18 hours to cleave the DNA oligonucleotide and deprotect the base amino groups. The OD yield of the nucleic acid (equivalent to the amount of nucleic acid synthesis) was determined from UV absorbance (260 nm) of the filtrate separated from the porous resin beads by filtration.The filtrate was analyzed by UPLC (Waters) to determine the full-length percentage (the percentage of DNA oligonucleotides with the desired sequence length). The 20-mer yield (OD / μmol) per synthesis scale was calculated by multiplying the OD yield by the full-length percentage per synthesis scale. The 20-mer yield (OD / ml) per bead volume was calculated by multiplying the 20-mer yield by the amount of DMT-dT-3'-succinate bound to the bead by the maximum swelling volume (toluene swelling volume).

[0064] The median pore size, swelling property, liquid transport property / back pressure increase, and oligonucleotide yield of the porous resin bead-shaped solid phase synthesis supports obtained in Examples 1 to 3 and Comparative Example 1 are shown in Table 3 below. The porous resin bead-shaped solid phase synthesis support of the present invention has low swelling property in various organic solvents (acetonitrile, toluene), with little difference in swelling degree, and improved liquid transport property, which resulted in a reduced back pressure increase and an increased oligonucleotide yield.

[0065]

Claims

1. Porous resin beads consisting essentially of a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile copolymer.

2. The porous resin beads according to claim 1, wherein the amount of the benzalmalononitrile structural unit relative to the total amount of structural units in said copolymer is 0.1 to 5.0 mmol / g.

3. A method for producing porous resin beads, comprising the steps of: suspension copolymerizing a styrene-based monomer, an acyloxystyrene-based monomer, a divinylbenzene-based monomer, and a benzalmalononitrile-based monomer using an organic solvent and water to obtain a styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile-based copolymer; and hydrolyzing the obtained styrene-acyloxystyrene-divinylbenzene-benzalmalononitrile-based copolymer to convert it into a styrene-hydroxystyrene-divinylbenzene-benzalmalononitrile-based copolymer.

4. A method for producing a nucleic acid, comprising sequentially binding nucleosides or nucleotides to the porous resin beads according to claim 1 or 2 via cleavable linkers to obtain an oligonucleotide.

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