Method for producing oligonucleotide
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Filing Date
- 2026-01-21
- Publication Date
- 2026-06-17
Smart Images
Figure 2025028591000001 
Figure 2025028591000002
Abstract
Description
Methods for producing oligonucleotides
[0001] The present invention relates to a method for producing oligonucleotides.
[0002] Various methods for producing oligonucleotides are known. For example, an oligonucleotide prepared on a solid support via a cleavable linker is hydrolyzed with aqueous ammonia, methylamine solution, or the like to cleave the oligonucleotide from the solid support. Known cleavable linkers include those having a cyclic vicinal diol structure, such as a universal linker.
[0003] Regarding the cleavage of an oligonucleotide from a complex containing a cleavable linker having a cyclic vicinal diol structure and a support, Patent Document 1 describes a method for cleaving an oligonucleotide by contacting the complex with an aqueous solution containing an alkylamine, a monovalent inorganic salt, and an alcohol. However, as shown in Comparative Examples 4 and 5 of this specification, by-products and decomposition products to which the cleavable linker is bound are generated, and the purity and yield of the oligonucleotide are not high.
[0004] Patent Document 2 describes the cleavage of oligonucleotides using a concentrated ammonium hydroxide solution. Patent Document 3 describes the cleavage of oligonucleotides using gaseous hydrated ammonia. However, in the method of Patent Document 2, when the terminal nucleoside of the oligonucleotide bound to the solid support is other than LNA, by-products and decomposition products to which a cleavable linker is bound are generated, and therefore the purity and yield of the oligonucleotide are not high. Furthermore, the method of Patent Document 3 requires a special high-temperature, high-pressure reaction vessel that can withstand the use of gaseous ammonia.
[0005] Japanese Patent Application Laid-Open No. 2017-008038 Special Publication No. 2020-515599 Special Publication No. 2004-513629
[0006] The problem to be solved by the present invention is to provide a method for obtaining oligonucleotides with high purity and yield, by suppressing the production of by-products and / or other decomposition products to which the cleavable linker is attached in the step of cleaving the oligonucleotide from a complex comprising the oligonucleotide, the cleavable linker, and a support.
[0007] As a result of intensive research aimed at solving this problem, the present inventors have unexpectedly found that by using an organic solvent selected from ethers, cyclic amides, and cyclic ureas and / or a metal salt of an organic acid in the cleavage step, it is possible to suppress the production of by-products and / or other decomposition products and obtain oligonucleotides with high purity and yield, and have completed the present invention.
[0008] [1] A method for producing an oligonucleotide, comprising the steps of cleaving the oligonucleotide from a complex comprising the oligonucleotide, a cleavable linker, and a support in a solution containing an alkylamine and an organic solvent, and optionally deprotecting a protecting group on the oligonucleotide, wherein the cleavable linker has a cyclic vicinal diol structure, and the organic solvent is selected from ethers, cyclic amides, and cyclic ureas. [2] The method according to [1], wherein the solution further contains a metal salt of an organic acid. [3] The method according to [2], wherein the organic acid is selected from substituted or unsubstituted alkylsulfonic acids, substituted or unsubstituted arylsulfonic acids, substituted or unsubstituted alkanoic acids, and substituted or unsubstituted alkanedioic acids. [4] The method according to [2] or [3], wherein the metal salt is an alkali metal salt.
[0009] [5] A method for producing an oligonucleotide, comprising the steps of cleaving the oligonucleotide from a complex comprising the oligonucleotide, a cleavable linker, and a support in a solution containing an alkylamine, and optionally deprotecting a protecting group on the oligonucleotide, wherein the cleavable linker has a cyclic vicinal diol structure, and the solution further contains a metal salt of an organic acid. [6] The method according to [5], wherein the organic acid is selected from the group consisting of a substituted or unsubstituted alkylsulfonic acid, a substituted or unsubstituted arylsulfonic acid, a substituted or unsubstituted alkanoic acid, and a substituted or unsubstituted alkanedioic acid. [7] The method according to [5] or [6], wherein the metal salt is an alkali metal salt.
[0010] According to the production method of the present invention, it is possible to obtain an oligonucleotide with high purity and yield from a complex comprising an oligonucleotide, a cleavable linker, and a support, while suppressing the production of by-products and / or other decomposition products to which the cleavable linker is attached.
[0011] The method for producing an oligonucleotide of the present invention comprises the steps of cleaving the oligonucleotide from a complex comprising an oligonucleotide, a cleavable linker, and a support in a solution comprising an alkylamine and an organic solvent, and optionally deprotecting the protecting group of the oligonucleotide, wherein the cleavable linker has a cyclic vicinal diol structure, and the organic solvent is selected from ethers, cyclic amides, and cyclic ureas. In the method for producing an oligonucleotide of the present invention, the step of cleaving the oligonucleotide from the complex and the step of deprotecting the protecting group of the oligonucleotide may be performed in the same step, or the latter step may be performed after the former step, or the former step may be performed after the latter step. Furthermore, the complex comprising an oligonucleotide, a cleavable linker, and a support of the present invention particularly refers to an embodiment in which the cleavable linker is covalently bonded to the support and the oligonucleotide, respectively.
[0012] The "support" is not particularly limited as long as it is a structure capable of immobilizing the 3'-end or 5'-end of the oligonucleotide to be synthesized via a linker molecule. The support may be a solid-phase support or a liquid-phase support, preferably a solid-phase support. Examples of solid-phase supports that can be used include porous supports such as glass-based supports, polystyrene-based supports, silica gel-based supports, and acrylamide-based supports, and are preferably glass-based supports, polystyrene-based supports, or silica gel-based supports, and particularly preferably glass-based supports or polystyrene-based supports. An acrylamide-based support is a porous support made of a resin primarily composed of structural units of acrylamide or its derivatives. A polystyrene-based support is a porous support made of a resin primarily composed of structural units of styrene or its derivatives.
[0013] The support has a functional group for bonding with the cleavable linker, such as an amino group or a hydroxyl group. Specific solid-phase supports include those described in JP 2011-088843 A and JP 2013-177371 A. In a preferred embodiment, low-swelling polystyrene particles commercially available as NittoPhase (registered trademark) (manufactured by Kinovate) or Primer Support (registered trademark) (manufactured by Cytiva) can be used. The average particle size of the porous support used in oligonucleotide synthesis is preferably 1 to 1000 μm, more preferably 5 to 500 μm, and even more preferably 10 to 200 μm.
[0014] The "cleavable linker having a cyclic vicinal diol structure" is also called a universal linker, and examples thereof include a cleavable linker having bicyclo[2.2.1]heptane in which the methylene may be substituted with an oxygen atom, a sulfur atom, or an imino group, and a cleavable linker having bicyclo[3.2.1]octane in which the methylene may be substituted with an oxygen atom, a sulfur atom, or an imino group. Specific examples include Unilinker, the cleavable linkers described in Patent Documents 1 to 3, and the cleavable linkers shown below. A cleavable linker selected from any one of formulas (A), (B), and (C) below is preferred, a cleavable linker of formula (B) below is more preferred, and a cleavable linker of formula (B) below (X = NH or O, R = phenyl) is even more preferred.
[0015]
[0016] [In the formula, X is an oxygen atom or NH. Y is methylene, an oxygen atom, a sulfur atom, NH, a divalent group of an aromatic hydrocarbon which may be substituted (e.g., an optionally substituted phenylene), or the like. The dashed line represents an absent ring or a ring fused to a bicyclo ring. R is an optionally substituted alkyl, an optionally substituted aryl, or the like, preferably methyl, isopropyl, or phenyl. Z is an alkyl, alkoxy, aryl, acyl, or the like, preferably methoxy.]
[0017] One hydroxyl group of the cyclic vicinal diol structure of the cleavable linker is bound to the support via an ester bond with, for example, succinic acid, and the other hydroxyl group is bound to the oligonucleotide via a phosphate ester bond.
[0018] Examples of "oligonucleotides" include RNA oligonucleotides, DNA oligonucleotides, and those containing modified sugar moieties or modified bases, and preferably include RNA oligonucleotides or RNA oligonucleotides containing modified sugar moieties and / or modified bases. The number of bases in the oligonucleotide may be, for example, 2 to 200, preferably 3 to 150, more preferably 5 to 100, and even more preferably 7 to 50. Examples of the modified sugar moiety include those having ribose, hexitol, morpholine, or N-(2-aminoethyl)glycine as the backbone. Modifications to ribose may be, for example, modifications at the 2'-position or 5'-position or cross-linked modifications. Specific examples of modifications at the 2'-position include 2'-F, 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-arabinosyl-F (2'-FANA), and the like. Specific examples of the modification at the 5' position include 5'-methyl (5'-Me), 5'-cyclopropylene (5'-CP), etc. Specific examples of the bridged modification include those in which a bridge structure is introduced between the 2' and 4' positions, such as 2',4'-BNA (LNA), 2',4'-BNACOC, 2',4'-BNANC, 2'-aminoLNA, ENA, AmNA, scpBNA, cEt, GuNA, etc.
[0019] The modified bases include, for example, those having a heterocycle as the skeleton, and examples of the heterocycle include purine derivatives and pyrimidine derivatives. Purine derivatives include, for example, adenine, guanine, isoguanine, xanthine, hypoxanthine, and the like, which may have a substituent. Pyrimidine derivatives include, for example, cytosine, thymine, uracil, and the like, which may have a substituent. Examples of the substituent include a hydroxyl group, an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a fluoro group, a chloro group, a bromo group, an iodo group, a carboxyl group, and the like. Preferred examples of substituted purine derivative bases include an 8-bromoadenyl group, an 8-bromoguanyl group, and an 8-oxoguanyl group. Preferred examples of the substituted pyrimidine derivative base include a 5-methylcytosyl group (mC), a 5-bromocytosyl group, a 5-bromouracil group, a 5-iodouracil group, a 5-iodocytosyl group, a 5-fluorouracil group, and a 5-(N-aminohexyl-3-acrylimide) group. The oligonucleotide of the present invention has a structure in which nucleosides are linked by, for example, a phosphodiester bond, a thiophosphate bond, a dithiophosphate bond, a phosphoramidate bond, or a boranophosphate bond, and preferably a phosphodiester bond or a thiophosphate bond.
[0020] Oligonucleotides are produced by sequentially binding nucleotides to the support-bound cleavable linker starting from the other hydroxyl group. If necessary, the functional group of the base moiety (e.g., amino group), the functional group of the sugar moiety (e.g., 2'-hydroxyl group), and the phosphate moiety of the nucleotide are protected with protecting groups. Various protecting groups used in the production of oligonucleotides are known in the art, and suitable protecting groups can be appropriately selected from these various protecting groups. Examples of protecting groups for the functional group of the base moiety include acyl groups and dimethylformamidyl groups. Preferably, these protecting groups are selected from the group consisting of isobutyryl, acetyl, benzoyl, phenoxyacetyl, isopropylphenoxyacetyl, tert-butylphenoxyacetyl, and dimethylformamidyl groups. Benzoyl, acetyl, or isobutyryl groups are more preferred. Examples of the protecting group for the functional group of the sugar moiety include silyl protecting groups and optionally substituted alkyl groups, and are preferably tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and triisopropylsiloxymethyl groups. Examples of the protecting group for the phosphate ester moiety include a cyanoethyl group.
[0021] The reaction mechanism of oligonucleotide cleavage is thought to be as follows: (1) an alkylamine cleaves the ester bond between the support and the cleavable linker, removing the support; and (2) a negative charge generated at one hydroxyl group of the vicinal diol of the cleavable linker undergoes a nucleophilic substitution reaction with the phosphate ester at the other hydroxyl group, producing a cyclic phosphate diester containing the cleavable linker, resulting in cleavage of the oligonucleotide from which the terminal phosphate group has been removed. If step (2) does not proceed and the reaction terminates at step (1), by-products in which the cleavable linker is bonded to the oligonucleotide are produced, resulting in a decrease in the purity and yield of the oligonucleotide. If the reaction conditions are made more severe to promote cleavage of the phosphate ester bond between the hydroxyl group of the cleavable linker and the oligonucleotide in order to suppress the formation of the by-products, decomposition of the oligonucleotide also proceeds, resulting in the formation of decomposition products, and a decrease in the purity and yield of the oligonucleotide. In the production method of the present invention, the formation of by-products and decomposition products in which the cleavable linker is bonded in the cleavage step is suppressed, thereby enabling the production of oligonucleotides with high purity and yield.
[0022] Examples of "alkylamines" include monoamines and diamines. Examples of monoamines include primary alkylamines, secondary alkylamines, and tertiary alkylamines. Primary amines are, for example, linear or branched monoalkylamines, preferably having 1 to 10 carbon atoms in the alkyl moiety, more preferably methylamine, ethylamine, n-propylamine, n-butylamine, tert-butylamine, n-pentylamine, and n-hexylamine, and even more preferably methylamine, ethylamine, and tert-butylamine. Secondary amines are, for example, linear or branched dialkylamines, preferably having 1 to 10 carbon atoms in the alkyl moiety, more preferably dimethylamine, diethylamine, methylethylamine, di-n-propylamine, di-n-butylamine, and di-tert-butylamine. Tertiary amines are, for example, linear or branched trialkylamines, preferably having 1 to 10 carbon atoms in the alkyl moiety, more preferably trimethylamine, triethylamine, and the like. Preferred monoamines are primary amines.
[0023] The diamine is preferably a straight-chain diamine, more preferably a straight-chain diaminoalkane, even more preferably 1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopropane, or 1,4-diaminobutane, and even more preferably ethylenediamine. The alkylamine may be used, for example, as an aqueous solution. The alkylamine is usually used in a large excess amount relative to the complex containing the oligonucleotide, the cleavable linker, and the support; for example, 5 to 100 equivalents, more preferably 10 to 50 equivalents per base length per mole of oligonucleotide.
[0024] It is preferable to select appropriate reaction conditions for the reaction temperature and reaction time according to the oligonucleotide and cleavable linker. The reaction temperature is selected, for example, from the range of 10 to 60°C, more preferably from the range of 25 to 50°C, and even more preferably from the range of 30 to 40°C. If the reaction temperature is too high, decomposition of the oligonucleotide proceeds, producing decomposition products and reducing the purity and yield of the oligonucleotide. If the reaction temperature is too low, a long reaction time is required. It is preferable to check the progress of the reaction and stop the reaction at the optimal time.
[0025] The "ethers, cyclic amides, and cyclic ureas" used as organic solvents are preferably those that can dissolve the reactants and do not decompose under the reaction conditions, and more preferably ethers. Examples of "ethers" include chain ethers and cyclic ethers. Examples of chain ethers include diethyl ether, diisopropyl ether, dibutyl ether, dibenzyl ether, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), methyl tert-amyl ether (MTAE), ethyl tert-amyl ether (ETAE), cyclopentyl methyl ether (CPME), dimethoxyethane, and dipropylene glycol dimethyl ether. Preferred are diisopropyl ether, MTBE, ETBE, CPME, and dimethoxyethane, and more preferably dimethoxyethane. Examples of cyclic ethers include tetrahydrofuran (THF), tetrahydropyran, 2-methyltetrahydrofuran, 4-methyltetrahydropyran, 1,4-dioxane, dihydrolevoglucosenone, etc., with THF and 1,4-dioxane being preferred, and THF being more preferred. Examples of "cyclic amides" include those having a five- or six-membered ring, with N-methylpyrrolidone being preferred. Examples of "cyclic ureas" include those having a five- or six-membered ring, with 1,3-dimethyl-2-imidazolidinone and N,N'-dimethylpropyleneurea being preferred, and 1,3-dimethyl-2-imidazolidinone being more preferred.
[0026] As will be seen from a comparison between Example 1 and Comparative Examples 3 to 5 described below, the present invention, which uses an organic solvent selected from ethers, cyclic amides, and cyclic ureas, differs from the invention of Patent Document 1, which uses ethanol or isopropanol, in that it is possible to efficiently cleave oligonucleotides while suppressing the generation of by-products, decomposition products, and the like, and to obtain oligonucleotides with high purity and yield.
[0027] The "organic acid" in "metal salt of organic acid" is preferably one that can be dissolved in the reaction solution of this reaction in the form of a metal salt of the organic acid and can be separated and removed from the oligonucleotide. Examples of organic acids include substituted or unsubstituted alkylsulfonic acids, substituted or unsubstituted arylsulfonic acids, substituted or unsubstituted alkanoic acids, substituted or unsubstituted alkanedioic acids, substituted or unsubstituted unsaturated carboxylic acids, and substituted or unsubstituted arylcarboxylic acids, and the like, preferably selected from alkylsulfonic acids, arylsulfonic acids, and alkanoic acids, more preferably arylsulfonic acids or alkanoic acids. Examples of substituents used in the substitution include mercapto, amino, hydroxy, alkyl, alkenyl, alkynyl, alkanoyl, aryl, nitro, cyano, fluoro, chloro, bromo, iodo, and the like, and preferably mercapto, amino, hydroxy, alkyl, and the like. Examples of the alkylsulfonic acids include, for example, linear or branched C １～１０ Examples of the alkylsulfonic acids include alkylsulfonic acids and their substituted derivatives, and preferred examples include methanesulfonic acid, ethanesulfonic acid, isethionic acid, taurine, N-methyltaurine, 3-mercapto-1-propanesulfonic acid, 10-camphorsulfonic acid, etc. Examples of the arylsulfonic acids include benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc., and preferred examples include p-toluenesulfonic acid, etc. Examples of the alkanoic acids include linear or branched C ２～１０ Examples of the alkanedioic acid include alkanoic acids and their substituted derivatives, and preferred examples include acetic acid, propionic acid, butanoic acid, trifluoroacetic acid, glycine, lactic acid, and pyruvic acid. ３～１０Examples of the unsaturated carboxylic acid include alkyl diacids and their substituted derivatives, and preferred examples include oxalic acid, malonic acid, succinic acid, glutaric acid, glutamic acid, etc. Examples of the unsaturated carboxylic acid include linear or branched C ３ ~ １０ Examples of the arylcarboxylic acid include unsaturated carboxylic acids and their substitution products, and preferred examples thereof include acrylic acid, methacrylic acid, crotonic acid, butenoic acid, pentenoic acid, propiolic acid, maleic acid, etc. Examples of the arylcarboxylic acid include benzoic acid and phthalic acid, and preferred examples include benzoic acid.
[0028] Examples of the "metal salt" in "metal salt of organic acid" include alkali metal salts (lithium salt, sodium salt, potassium salt, rubidium salt, etc.), alkaline earth metal salts (magnesium salt, etc.), etc., preferably alkali metal salts (lithium salt, sodium salt, potassium salt, etc.), more preferably lithium salt, sodium salt, potassium salt. The amount of metal salt of organic acid added in the reaction mixture can be, for example, an amount that results in a concentration of 10 to 1000 mM, preferably an amount that results in a concentration of 20 to 500 mM, more preferably an amount that results in a concentration of 50 to 300 mM. By using these metal salts of organic acid, it is possible to suppress the production of by-products and decomposition products to which the cleavable linker is bonded from a complex containing an oligonucleotide, a cleavable linker, and a support, and to cleave the oligonucleotide with high purity and yield.
[0029] Deprotection conditions for the protecting groups of oligonucleotides are widely known in the art for each protecting group. In the production method of the present invention, suitable deprotection conditions can be appropriately selected from these deprotection conditions.
[0030] Another method for producing an oligonucleotide of the present invention includes the steps of cleaving the oligonucleotide from a complex containing an oligonucleotide, a cleavable linker, and a support in a solution containing an alkylamine and a metal salt of an organic acid, and optionally deprotecting a protecting group of the oligonucleotide. The cleavable linker and the metal salt of the organic acid can be preferably the same as those used in the above-mentioned production methods. This production method can be carried out according to the description of the above-mentioned production method.
[0031] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the sugar moiety of the RNA oligonucleotides in the examples is a ribose ring, the base moiety is selected from adenine, cytosine, guanine, and uracil, and the bond between nucleotides is a phosphodiester bond. 2'-F (2'-fluoro RNA nucleotide) in the examples has a structure in which the hydroxyl group at the 2' position of the ribose ring is substituted with a fluorine atom, and the base moiety is selected from adenine, cytosine, guanine, and uracil, unless otherwise specified. 2'-OMe (2'-methoxy RNA nucleotide) has a structure in which the sugar moiety is a ribose ring with a methoxy group, and the base moiety is selected from adenine, cytosine, guanine, and uracil, unless otherwise specified. In addition, unless otherwise specified, 2'-MOE (2'-O-methoxyethyl RNA nucleotide) has a sugar moiety in which the hydroxyl group at the 2'-position of the ribose ring is substituted with a methoxyethoxy group, and the base moiety is selected from adenine, cytosine, guanine, and thymine. In addition, unless otherwise specified, LNA has a sugar moiety in which the oxygen atom at the 2'-position of the ribose ring and the carbon atom at the 4'-position are cross-linked by a methylene group, and the base moiety is selected from adenine, 5-methylcytosine, guanine, and thymine.
[0032] <Measurement of the amount of target oligonucleotide (FLP), by-products, and impurities (degradation products)> Crude solutions of each oligonucleotide produced in the following examples were measured by ultra-high performance liquid chromatography (UHPLC) under the following conditions, and the proportions of target oligonucleotide (FLP), by-products (Unilinker conjugates), and impurities (degradation products) (target peak area / total area (%) of all peaks) were calculated. Note that impurities include degradants generated during cleavage of the oligonucleotide as well as degradants generated during the preparation of the solid phase support to which the oligonucleotide is bound. Measurement conditions: UHPLC apparatus: ACQUITY® UPLC®, column: Waters ACQUITY UPLC Oligonucleotide BEH C18, 130 Å, 1.7 μm, 2.1 mm × 100 mm, UV detection: 260 nm, Buffer A: 100 mM HFIP, 8 mM TEA in H ２ O:MeCN=98:2, BufferB;H ２ O:MeCN=50:50, temperature: 60°C. Furthermore, the absorbance of each sample solution of crude oligonucleotide prepared in the Examples was measured using a spectrophotometer, and the yield (the ratio of the OD value actually measured to the theoretically achievable maximum OD value) was calculated from the obtained OD value.
[0033] Example 1 Using an OligoPilot (registered trademark) plus 100 nucleic acid synthesizer, a reaction was carried out to synthesize an oligonucleotide (FLP) having the base sequence of SEQ ID NO: 1 by sequentially condensing phosphoramidites with nucleosides bound to a solid support via a linker. Next, the solid support after the reaction was immersed and shaken at 25°C for 30 minutes in a 40 wt% aqueous methylamine solution / THF (volume ratio 3:7) mixed solution in an amount of 100 mL per 1 mmol of linker supported on the solid support, to cleave the bond between the solid support and the cleavable linker.
[0034] The solution after cleavage was filtered to remove the solid support, and the solid support was washed with a mixed solution of 40% by weight of methylamine aqueous solution / THF (volume ratio 3:7). 150 mM of sodium acetate was added to the combined wash solution and filtrate, and the mixture was shaken at 35° C. for 6 hours to synthesize the amino group (—NH ２ The protecting groups (acetyl group (cytosine), benzoyl group (adenine), and isobutyryl group (guanine)) of the target oligonucleotide were deprotected, and the linker was removed. Dimethyl sulfoxide and triethylamine hydrogen trifluoride were then added to this solution, and the mixture was shaken at 55°C for 1 hour to deprotect the protecting group of the 2'-hydroxyl group of the target oligonucleotide.
[0035] Acetonitrile was added to the deprotected solution to precipitate the target oligonucleotide, and the resulting precipitate was then recovered and dissolved in water to obtain a crude solution of the target oligonucleotide. The solid phase support, linker, and target oligonucleotide mentioned above are as follows: Solid phase support, linker: NittoPhase (registered trademark) HL Unilinker 250 (Kinovate) Target oligonucleotide (FLP): 5'-acuguuauacucagaaucgcg-3' (SEQ ID NO: 1: 21mer, each nucleoside unit indicated in lowercase represents the same structural unit as RNA.)
[0036] Examples 2 to 4 and Comparative Examples 1 and 2 Crude solutions of oligonucleotides of Examples 2 to 4 and Comparative Examples 1 and 2 were obtained by carrying out the same operations under the same conditions as in Example 1, except that the sodium acetate used in Example 1 was replaced with one of the metal salts of an organic acid listed in Table 1. The amounts of the target oligonucleotide (FLP) and by-products for the crude solutions of each oligonucleotide produced in Examples 1 to 4 and Comparative Examples 1 and 2 were measured, and the results are shown in Table 1.
[0037]
[0038] As shown in Table 1, in Examples 1 to 4, the target oligonucleotide (FLP) could be cleaved without much by-product (Unilinker conjugate) or decomposition product. As can be seen from Examples 1 to 4 and Comparative Examples 1 and 2, alkali metal salts (sodium salt, lithium salt, potassium salt) and magnesium salts are preferred because they enable efficient cleavage of oligonucleotides, and alkali metal salts (sodium salt, lithium salt, potassium salt) are even more preferred because they enable oligonucleotides to be obtained with high purity and yield.
[0039] Examples 5 to 12 The solid-phase support to which the target oligonucleotide synthesized in Example 1 was bound (before the cleavage and deprotection steps) was immersed and shaken for 4 hours at 45°C in a solution containing 100 mL of a 40 wt% aqueous methylamine solution / any of the solvents listed in Table 2 (volume ratio 6:4) per 1 mmol of solid-phase support, to which 150 mM of a metal salt of any of the organic acids listed in Table 2 was added. This cleavage of the target oligonucleotide from the solid-phase support and deprotection of the base protecting group were carried out. The solid-phase support was then removed by filtration and washed with dimethyl sulfoxide. Triethylamine hydrogen trifluoride was added to the combined wash solution and filtrate, and the mixture was shaken at 55°C for 1 hour to deprotect the 2'-hydroxyl group of the target oligonucleotide. Acetonitrile was added to this solution to precipitate the target oligonucleotide, and the resulting precipitate was dissolved in water to obtain a crude solution of the target oligonucleotide. The amounts of target oligonucleotide (FLP) and by-products measured for the crude solutions of each oligonucleotide produced in Examples 5 to 12 are shown in Table 2.
[0040]
[0041] As shown in Table 2, in Examples 5 to 12, the amount of by-products (Unilinker conjugates) and decomposition products was low, and the target oligonucleotide (FLP) could be cleaved. It was found that all of the organic acid salts used in Examples 5 to 12 were preferable. It was found that all of the organic solvents used in Examples 5 to 12 were preferable, with THF, N-methylpyrrolidone, and N,N-dimethylimidazolidinone being more preferable.
[0042] Examples 13 to 16: The solid support to which the target oligonucleotide synthesized in Example 1 was bound (before the cleavage and deprotection steps) was immersed in a 40 wt% methylamine aqueous solution / THF mixed solution containing sodium 3-mercapto-1-propanesulfonate at a salt concentration as shown in Table 3, with the amount of solution and volume % THF per mmol of solid support shown in Table 3. The solution was then shaken at the temperature and time shown in Table 3 to cleave the target oligonucleotide from the solid support and deprotect the protecting group of the base moiety. The solid support was then removed by filtration and washed with dimethyl sulfoxide. Triethylamine hydrogen trifluoride was added to the combined wash solution and filtrate, and the solution was shaken at 55°C for 1 hour to deprotect the 2'-hydroxyl group of the target oligonucleotide. Acetonitrile was added to this solution to precipitate the target oligonucleotide, and the resulting precipitate was dissolved in water to obtain a crude solution of the target oligonucleotide. The amounts of the target oligonucleotide (FLP) and by-products were measured for the crude solutions of each oligonucleotide produced in Examples 13 to 16, and the results are shown in Table 3.
[0043]
[0044] The reaction rate changes depending on the composition of the reaction solution (type and volume % of organic solvent, type and concentration of metal salt of organic acid, etc.). As can be seen from Table 3, the reaction rate could be increased by extending the reaction time or increasing the reaction temperature.
[0045] Example 17 The target oligonucleotide (FLP) synthesized in Example 1 was changed to the oligonucleotide "5'-aC(F)uG(F)uU(O)auA(O)cU(F)caG(O)aA(F)ucgC(O)g-3' (SEQ ID NO: 2: 21mer, each nucleoside unit with (F) written to the right of the base has the same structure as 2'-F, and each nucleoside unit with (O) written to the right of the base has the same structure as 2'-OMe, and other nucleoside units written in lowercase letters have the same structural unit as RNA)," and the same procedures were carried out under the same conditions as in Example 1, except that "shaking at 35°C for 6 hours" was changed to "shaking at 35°C for 2 hours," to obtain a crude solution of the target oligonucleotide. The crude solutions of each oligonucleotide produced in Example 17 were analyzed for the amounts of target oligonucleotide (FLP), by-products, and 2'-F-derived decomposition products. The results are shown in Table 4.
[0046]
[0047] Under harsh cleavage conditions, 2'-F is prone to producing characteristic decomposition products in which F is converted to a hydroxyl group, etc. However, as shown in Table 4, even with oligonucleotides containing 2'-F and 2'-OMe, the production of by-products and decomposition products was suppressed, and the oligonucleotides could be efficiently cleaved, as in Example 1, and oligonucleotides could be obtained with high purity and yield.
[0048] Example 18 In the same manner as in Example 1, an oligonucleotide "5'-G^A(L)C(F)U(F)G(O)U(F)U(F)A(L)U(F)A(M)C(F)U(F)C(F)A(M)G(O)A(M)A(M)U(F)C(F)G(O)C(F)G(O)-3' (SEQ ID NO: 3: 22mer, each nucleoside unit with (F) written to the right of the base has the same structure as 2'-F, each nucleoside unit with (O) written to the right of the base has the same structure as 2'-OMe, and each nucleoside unit with (M) written to the right of the base has the same structure as 2'-MOE, Each nucleoside unit with (L) written to the right of the base has the same structure as LNA, and other nucleoside units show the same structural unit as DNA, and ^ shows a thiophosphate bond.)) was synthesized, and the solid phase support to which the synthesized target oligonucleotide was bound was immersed and shaken for 4 hours at 21°C in a solution prepared by adding 150 mM sodium acetate to a 40 wt% aqueous methylamine solution / THF (volume ratio 75 / 25) mixed solution in a liquid volume of 200 mL per 1 mmol of the solid phase support, to thereby cleave the target oligonucleotide from the solid phase support and to depolymerize the amino group (-NH ２ The protecting groups (acetyl group (cytosine), benzoyl group (adenine), and isobutyryl group (guanine)) of the target oligonucleotide were deprotected, and the linker was removed. The solid phase support was then removed by filtration and washed with water to obtain a crude solution of the target oligonucleotide. The solid phase support and linker used were the same as those used in Example 1. The amounts of the target oligonucleotide (FLP) and by-products in the crude solution of each oligonucleotide produced in Example 18 were measured, and the results are shown in Table 5.
[0049] Even in a sequence containing 50% 2'-F, the production of by-products and degradation products was suppressed, and the oligonucleotide could be efficiently excised, allowing the oligonucleotide to be obtained with high purity and yield.
[0050] Comparative Example 3 A crude solution of oligonucleotide of Comparative Example 3 was obtained by carrying out the same procedure under the same conditions as in Example 1, except that the organic solvent used in Example 1 was changed to ethanol.
[0051] Comparative Example 4 An experiment was conducted according to Example 1 of Patent Document 1 using the solid-phase support to which the target oligonucleotide synthesized in Example 1 was bound (before the cleavage step and deprotection step). The solid-phase support to which the target oligonucleotide synthesized in Example 1 was bound was immersed and shaken at 25°C for 30 minutes in 100 mL of a 40 wt% aqueous methylamine / 2-propanol (1:1) mixed solution per 1 mmol of solid-phase support, and the bond between the solid-phase support and the cleavable linker was cleaved. The solid-phase support was then removed by filtration and washed with a 40 wt% aqueous methylamine / 2-propanol (1:1 volume ratio) mixed solution. 20 mM sodium bromide was added to the combined wash solution and filtrate, and the mixture was shaken at 45°C for 4 hours to deprotect the protecting group at the base moiety of the target oligonucleotide and remove the linker. Dimethyl sulfoxide and triethylamine hydrogen trifluoride were added to this solution, and the mixture was shaken at 55°C for 1 hour to deprotect the 2'-hydroxyl group of the target oligonucleotide. Acetonitrile was added to this solution to precipitate the target oligonucleotide, and the resulting precipitate was then dissolved in water to obtain a crude solution of the oligonucleotide of Comparative Example 4.
[0052] Comparative Example 5 An experiment was carried out according to Example 21 of Patent Document 1 using the solid phase support (before the cleavage step and deprotection step) to which the target oligonucleotide synthesized in Example 1 was bound. Specifically, the same operation was carried out under the same conditions as in Comparative Example 4, except that the volume percent of the solvent used in Comparative Example 4 was changed to 20 volume percent, the reaction temperature was changed to 25°C, and the reaction time was changed to 21 hours, to obtain a crude solution of the oligonucleotide of Comparative Example 5. The results of measuring the amounts of the target oligonucleotide (FLP), by-products, decomposition products, etc. for the crude solutions of each oligonucleotide produced in Comparative Examples 3 to 5 are shown in Table 6, along with the measurement results of Example 1 above.
[0053]
[0054] As shown in Table 6, in Comparative Example 3, where the organic solvent was ethanol, the amount of target oligonucleotide (FLP) decreased despite the reaction being carried out under the same reaction conditions as in Example 1, where the organic solvent was THF. This is because the deprotection reaction rate of the base protecting group (isobutyryl protecting group of the guanine base) was slow in the ethanol solvent, resulting in a significant amount of undeprotected oligonucleotide remaining. If the reaction conditions were made more severe by increasing the reaction temperature or extending the reaction time to promote deprotection, FLP would likely decompose and increase the amount of by-products. Furthermore, in Comparative Example 4 (Example 1 of Patent Document 1), compared to Example 1 of the present invention, there were more by-products (Unilinker conjugates) and more decomposition products, resulting in a decreased amount of target oligonucleotide (FLP). In Comparative Example 5 (Example 21 of Patent Document 1), there were significantly more decomposition products and the amount of target oligonucleotide (FLP) decreased compared to Example 1 of the present invention. As described above, the present invention differs from Patent Document 1 in that an organic solvent selected from ethers, cyclic amides, and cyclic ureas is used, and a metal salt of an organic acid is further added. This makes it possible to suppress the generation of by-products, decomposition products, etc., to efficiently cleave oligonucleotides, and to obtain oligonucleotides with high purity and yield.
[0055] The embodiments disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is indicated by the entire description of the specification, including the above-mentioned description of the embodiments, and the scope of the claims, and is intended to include all modifications within the meaning and scope of the claims.
[0056] According to the production method of the present invention, it is possible to cleave an oligonucleotide with high purity and yield from a complex comprising an oligonucleotide, a cleavable linker, and a support, while suppressing the production of by-products and decomposition products to which the cleavable linker is attached.
Claims
1. A method for producing an oligonucleotide, comprising the steps of cleaving the oligonucleotide from a complex comprising the oligonucleotide, a cleavable linker, and a support in a solution containing an alkylamine and an organic solvent, and optionally deprotecting the protecting group of the oligonucleotide, The aforementioned cleavage linker has a cyclic vicinaldiol structure, A method for producing the organic solvent, wherein the organic solvent is selected from ethers, cyclic amides, and cyclic ureas.
2. The manufacturing method according to claim 1, wherein the solution further contains a metal salt of an organic acid.
3. The production method according to claim 2, wherein the organic acid is selected from substituted or unsubstituted alkyl sulfonic acid, substituted or unsubstituted aryl sulfonic acid, substituted or unsubstituted alkanoic acid, and substituted or unsubstituted alkanediic acid.
4. The manufacturing method according to claim 2 or 3, wherein the metal salt is an alkali metal salt.
5. The manufacturing method according to claim 2 or 3, wherein the metal salt is a lithium salt, a sodium salt, a potassium salt, or a magnesium salt.
6. The manufacturing method according to claim 1 or 2, wherein the cleavable linker is a cleavable linker having bicyclo[2.2.1]heptane in which methylene is substituted with an oxygen atom, a sulfur atom, or an imino group, a cleavable linker having bicyclo[3.2.1]octane in which methylene is substituted with an oxygen atom, a sulfur atom, or an imino group, or a linker represented by the following formula (C). 【Chemistry 1】 [In the formula, X is an oxygen atom or NH. Z is an alkyl, alkoxy, aryl, or acyl.]
7. The manufacturing method according to claim 1 or 2, wherein the organic solvent is at least one selected from the group consisting of tetrahydrofuran (THF), tetrahydropyran, 2-methyltetrahydrofuran, 4-methyltetrahydropyran, 1,4-dioxane, diethyl ether, diisopropyl ether, dibutyl ether, dibenzyl ether, methyl-tert-butyl ether (MTBE), ethyl-tert-butyl ether (ETBE), methyl-tert-amyl ether (MTAE), ethyl-tert-amyl ether (ETAE), dimethoxyethane, dipropylene glycol dimethyl ether, N-methylpyrrolidone, 1,3-dimethyl-2-imidazolidinone, and N,N'-dimethylpropylene urea.
8. A method for producing an oligonucleotide, comprising the steps of cleaving the oligonucleotide from a complex comprising the oligonucleotide, a cleavable linker, and a support in a solution containing an alkylamine, and optionally deprotecting the protecting group of the oligonucleotide, The aforementioned cleavage linker has a cyclic vicinaldiol structure, A method for producing the aforementioned solution, wherein the solution further contains a metal salt of an organic acid.
9. The production method according to claim 8, wherein the organic acid is selected from substituted or unsubstituted alkyl sulfonic acid, substituted or unsubstituted aryl sulfonic acid, substituted or unsubstituted alkanoic acid, and substituted or unsubstituted alkanediic acid.
10. The manufacturing method according to claim 8 or 9, wherein the metal salt is an alkali metal salt.
11. The manufacturing method according to claim 8 or 9, wherein the metal salt is a lithium salt, a sodium salt, a potassium salt, or a magnesium salt.
12. The manufacturing method according to claim 8 or 9, wherein the cleavable linker is a cleavable linker having bicyclo[2.2.1]heptane in which methylene is substituted with an oxygen atom, a sulfur atom, or an imino group, a cleavable linker having bicyclo[3.2.1]octane in which methylene is substituted with an oxygen atom, a sulfur atom, or an imino group, or a linker represented by the following formula (C). 【Chemistry 2】 [In the formula, X is an oxygen atom or NH. Z is an alkyl, alkoxy, aryl, or acyl.]