Improved method for oligonucleotide synthesis

Abstract

The present invention relates to an improved method for oligonucleotide synthesis. In the method for oligonucleotide synthesis according to the present invention, a synthesis cycle is performed at a temperature lower than room temperature, thereby suppressing side reactions occurring during the synthesis process and stably synthesizing oligonucleotides with higher purity.

Description

Improved methods for oligonucleotide synthesis

[0001] The present invention relates to an improved method for oligonucleotide synthesis.

[0002] The synthesis of oligonucleotides has become an essential technology in life sciences and medicine. In particular, oligonucleotides are widely used in genetic research, diagnostics, therapeutic development, and genetic manipulation, requiring high levels of synthetic accuracy and purity. Poor quality of synthesized oligonucleotides can lead to errors in research and diagnostic results, and, especially when used for pharmaceutical purposes, can result in reduced efficacy or adverse effects. For these reasons, the development of high-purity, high-efficiency oligonucleotide synthesis methods is emerging as a critical challenge.

[0003] Since its introduction in the 1980s, the phosphoramidite synthesis method has become the standard for oligonucleotide synthesis. This method involves stepwise linking of nucleoside precursors, boasting high precision in ensuring precise nucleotide sequences at each step. Phosphoramidite synthesis involves protecting the 5' end of the nucleoside while inducing linkage on a solid support using a phosphoramidite group at the 3' end. This method enables large-scale synthesis and precise sequence control using automated equipment, and is widely utilized in both research and industrial settings.

[0004] However, existing phosphoramidite synthesis methods have limited efficiency and can produce byproducts due to incomplete bonding or side reactions, making high-purity synthesis difficult. Therefore, there is a growing need for methods that minimize side reactions and ensure stable synthesis to increase oligonucleotide purity and yield.

[0005] The inventors of this application have continuously conducted in-depth research to improve the efficiency and purity of oligonucleotide synthesis, and have explored various approaches to overcome the limitations of existing synthetic methods. As a result, the inventors of this application have developed the present invention, which overcomes the limitations of existing technologies, suppresses side reactions occurring during the synthesis process, and enables the stable synthesis of high-purity oligonucleotides.

[0006] One aspect of the present invention provides a method for synthesizing an oligonucleotide on a solid support comprising the following steps (i) to (v), wherein at least one of the steps is performed at a temperature below room temperature (RT):

[0007] (i) a step of removing a protecting group from a nucleoside or nucleotide having a protecting group at a hydroxyl group, thiol group or amino group at the 5' position or the 3' position, thereby forming a nucleoside or nucleotide having a deprotected hydroxyl group, thiol group or amino group at the 5' position or the 3' position;

[0008] (ii) a step of coupling a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 5' or 3' position to a deprotected hydroxyl group, thiol group or amino group at the 5' or 3' position;

[0009] (iii) a step of forming a phosphate ester group by oxidation, a phosphorothioate ester group by thiolation, or a phosphoramidate group by imination of a phosphite ester group generated through coupling;

[0010] (iv) a step of removing a protecting group of a hydroxyl group, thiol group or amino group at the 5' or 3' position of a coupled nucleoside phosphoramidite or nucleotide phosphoramidite, thereby forming a deprotected hydroxyl group, thiol group or amino group at the 5' or 3' position;

[0011] (v) A step of synthesizing the desired oligonucleotide by repeating steps (ii) to (iv) at least once.

[0012] One aspect is to provide a method for synthesizing an oligonucleotide, wherein the nucleoside or nucleotide having a protecting group at the hydroxyl group, thiol group or amino group at the 3' position is an abasic nucleoside or nucleotide.

[0013] One aspect is to provide a method for synthesizing an oligonucleotide, wherein the temperature below room temperature (RT) is less than 22°C.

[0014] One aspect of the present invention provides a method for synthesizing an oligonucleotide on a solid support comprising the following steps (i) to (v), wherein at least one of the steps is performed at a temperature below room temperature (RT):

[0015] (i) a step of removing a protecting group from a nucleoside or nucleotide having a protecting group at a hydroxyl group, thiol group or amino group at the 5' position or the 3' position, thereby forming a nucleoside or nucleotide having a deprotected hydroxyl group, thiol group or amino group at the 5' position or the 3' position;

[0016] (ii) a step of coupling a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 5' or 3' position to a deprotected hydroxyl group, thiol group or amino group at the 5' or 3' position;

[0017] (iii) a step of forming a phosphate ester group by oxidation, a phosphorothioate ester group by thiolation, or a phosphoramidate group by imination of a phosphite ester group generated through coupling;

[0018] (iv) a step of removing a protecting group of a hydroxyl group, thiol group or amino group at the 5' or 3' position of a coupled nucleoside phosphoramidite or nucleotide phosphoramidite, thereby forming a deprotected hydroxyl group, thiol group or amino group at the 5' or 3' position;

[0019] (v) A step of synthesizing the desired oligonucleotide by repeating steps (ii) to (iv) at least once.

[0020] The method for synthesizing an oligonucleotide according to the present invention may additionally include a step of capping a deprotected hydroxyl group, thiol group, or amino group that has not undergone a coupling reaction.

[0021] In one specific example, the capping step may be additionally included in the oligonucleotide synthesis method according to the present invention when the step (iii) is a step of forming a phosphate ester group by oxidizing a phosphite ester group generated through coupling.

[0022] In the oligonucleotide synthesis method according to the present invention, when steps (ii) to (iv) are repeated one or more times, in one or more of the repetitions, one or more of steps (ii) to (iv) may be performed at a temperature below room temperature. Specifically, one or more of the repetitions includes all possible repetitions such as one or more, two or more, three or more, etc., and may include discontinuously selected repetitions. In addition, specifically, in one or more of the repetitions, step (ii), step (iii), step (iv), steps (ii) and (iii), steps (ii) and (iv), steps (iii) and (iv), or steps (ii), (iii) and (iv) may be performed at a temperature below room temperature.

[0023] In the oligonucleotide synthesis method according to the present invention, the last step of the last repetition may be step (iii) or (iv).

[0024] The method for synthesizing an oligonucleotide according to the present invention may additionally include a step of removing a phosphorus protecting group after step (v).

[0025]

[0026] In the oligonucleotide synthesis method according to the present invention, the nucleoside may refer to a compound containing a sugar group covalently bonded to a nucleobase. The term "nucleobase" is used interchangeably with "base." Each of the nucleosides may contain a nucleobase.

[0027] As used herein, the term “nucleobase” or “base” refers to purines and pyrimidines, such as adenine, thymine, cytosine, and guanine, which are nucleobases of DNA, adenine, uracil, cytosine, and guanine, which are nucleobases of RNA, and non-DNA / RNA nucleobases. And, the non-DNA / RNA nucleobase can be a nitrogenous base, including, for example, 5-methylcytosine (MeC), isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propynyl-6-fluorouracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine, 7-propyn-7-diazaadenine, 7-propyn-7-diazaguanine, and 2-chloro-6-aminopurine.

[0028] A nucleoside according to one embodiment may be a naturally occurring nucleoside, wherein the sugar group is deoxyribose or ribose, and the nucleobase is adenine, thymine, uracil, cytosine, or guanine, or may be a modified nucleoside.

[0029] The modified nucleoside may have a modified sugar group. Specifically, the modification of the sugar group may include replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring, replacing the D-ribose or D-deoxyribose ring of a naturally occurring nucleic acid with an L-ribose or L-deoxyribose ring, or replacing the β-anomer of a naturally occurring nucleic acid with an α-anomer.

[0030] The modified nucleoside may be a modified nucleobase. The modified nucleobase may be a non-DNA / RNA nucleobase.

[0031] In one embodiment, the modified nucleoside may be a 2'-modified nucleoside or a 2'-substituted nucleoside. Specifically, the modified nucleoside may be a nucleoside modified or substituted with 2'-O-alkyl, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, 2'-arabino-fluoro, 2'-O-aryl, 2'-O-benzyl, 2'-O-allyl, 2'-acetyl, 2'-aminomethyl, 2'-thio, or 2'-O-methyl-4-pyridine.

[0032] In one specific example, the modified nucleoside may have the hydroxyl group (-OH) at the 5' position or the 3' position replaced with a thiol group (-SH) or an amino group (-NH2).

[0033] In one specific example, the modified nucleoside may be an abasic nucleoside. An abasic nucleoside refers to a nucleoside without a nucleobase attached. More specifically, it refers to a sugar group or sugar moiety of a nucleoside without a nucleobase attached.

[0034] In one specific example, the modified nucleoside may be an inverted nucleoside. An inverted nucleoside refers to a nucleoside having a structure in which the bonding direction is opposite to that of a normal nucleoside. For example, in a method for synthesizing an oligonucleotide on a solid support, a normal nucleoside on the solid support is linked to the next unit through a functional group at the 5' position, but an inverted nucleoside is linked to the next unit through a functional group at the 3' position.

[0035] In one specific example, the modified nucleoside may be an inverted abasic nucleoside.

[0036]

[0037] In the oligonucleotide synthesis method according to the present invention, the nucleotide may refer to a compound (e.g., nucleic acid, DNA, RNA, or an analog thereof) in which a sugar group, a nucleobase, and a phosphate group are bonded. The term "nucleobase" is used interchangeably with "base." Each of the nucleotides may include a nucleobase. The nucleobase or base is as described above.

[0038] In one specific example, the nucleotide may be a naturally occurring nucleotide in which the sugar group is deoxyribose or ribose and the nucleobase is adenine, thymine, uracil, cytosine, or guanine, for example, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, thymidine triphosphate, thymidine diphosphate, thymidine monophosphate, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, cytidine triphosphate, cytidine diphosphate, cytidine monophosphate, uridine triphosphate, uridine diphosphate, uridine monophosphate, etc.

[0039] Additionally, the nucleotide may be a modified nucleotide.

[0040] The above modified nucleotide may have a modified sugar group. The modification of the sugar group is as described above.

[0041] The above modified nucleotide may have a modified nucleobase. The modified nucleobase is as described above.

[0042] The above modified nucleotide may have a modified phosphate group. The modified phosphate group may be phosphorothioate, phosphorodithioate, methylphosphonate, phosphatemethyl, or phosphoramidate.

[0043] The modified nucleotide may have a protecting group attached to the phosphorus residue. The phosphorus protecting group may be a methyl group or a cyanoethyl group.

[0044] In one embodiment, the modified nucleotide may be a 2'-modified nucleotide or a 2'-substituted nucleotide. Specifically, the modified nucleotide may be a nucleotide modified or substituted with 2'-O-alkyl, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, 2'-arabino-fluoro, 2'-O-aryl, 2'-O-benzyl, 2'-O-allyl, 2'-acetyl, 2'-aminomethyl, 2'-thio, or 2'-O-methyl-4-pyridine.

[0045] In one specific example, the modified nucleotide may have a hydroxyl group (-OH) at the 5' position or the 3' position replaced with a thiol group (-SH) or an amino group (-NH2).

[0046] In one specific example, the modified nucleotide may be an abasic nucleotide. An abasic nucleotide refers to a nucleotide without a nucleobase attached. More specifically, it refers to a sugar group and a phosphate group of a nucleotide, or a sugar and a phosphate moiety of a nucleoside, without a nucleobase attached.

[0047] In one specific example, the modified nucleotide may be an inverted nucleotide. An inverted nucleotide refers to a nucleotide having a structure in which the bonding direction is opposite to that of a normal nucleotide. For example, in a method for synthesizing an oligonucleotide on a solid support, a normal nucleotide is linked to the next unit through a functional group at the 5' position, whereas an inverted nucleotide is linked to the next unit through a functional group at the 3' position.

[0048] In one specific example, the modified nucleotide may be an inverted abasic nucleotide.

[0049] In one specific embodiment, the nucleotide may be a nucleotide, a di-nucleotide, a tri-nucleotide, or a nucleotide of a higher oligomer.

[0050]

[0051] In the oligonucleotide synthesis method according to the present invention, the nucleoside or nucleotide having a protecting group at the hydroxyl group, thiol group or amino group at the 5' position or 3' position may be fixed to a solid support.

[0052] In one embodiment, the solid support may be, but is not limited to, resin, glass, cellulose, nylon, acrylamide gel, dextran, CPG (Controlled Pore Glass), polystyrene, TentaGel, silica-based support, trityl resin, amphorite resin, cellulose-based support, Glycidyl Methacrylate (GMA)-based support, or PEG-modified support.

[0053] In one specific example, the nucleoside or nucleotide having a protecting group on the hydroxyl group, thiol group or amino group at the 5' position may be fixed to a solid support via the 3' position or the 2' position, and the nucleoside or nucleotide having a protecting group on the hydroxyl group, thiol group or amino group at the 3' position may be fixed to the solid support via the 5' position or the 2' position. The nucleoside or nucleotide of the nucleoside or nucleotide having a protecting group on the hydroxyl group, thiol group or amino group at the 3' position may be an abasic nucleoside or nucleotide, or an inverted abasic nucleoside or nucleotide.

[0054]

[0055] In the oligonucleotide synthesis method according to the present invention, the protecting group may be a known protecting group that protects a hydroxyl group, thiol group, or amino group at the 5' position or the 3' position. For example, the protecting group is trityl (Trt), dimethoxytrityl (DMT), monomethoxytrityl (MMT), tert-Butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), t-butoxycarbonyl (Boc), methyl phosphodiester, benzoyl (Bz), trifluoroacetyl (TFA), benzyl (Bn), p-methoxytriphenylmethyl (p-Methoxytrityl, PMT), mono-methoxyphosphotriester (MMP), fluorenylmethoxycarbonyl (Fmoc), tetrahydrofuran (THP), acetyl (Ac), mercaptotrityl (Mercapto-trityl, MTT), 4-methoxytrityl (Mmt), thiopyridyl, benzylthio, fluorenylmethoxycarbonyl (Fmoc), carbamate (Cbz), trifluoroacetyl (TFA), or isobutyryl (iBu), phenoxyacetyl (PAC), or dimethylformamidine (dmf) protecting groups.

[0056] In the present invention, the term, protecting group removal, is used interchangeably with deprotection or protecting group elimination.

[0057] In one specific example, the removal of the protecting group is possible under appropriate acidic or basic conditions. Methods for removing the protecting group include general, optimized protocols for removing the protecting group for each protecting group, which are known in the art.

[0058] That is, by removing a protecting group from a nucleoside or nucleotide having a hydroxyl group, thiol group or amino group at the 5' position or the 3' position according to one specific example, a deprotected nucleoside or nucleotide having a hydroxyl group, thiol group or amino group at the 5' position or the 3' position can be formed.

[0059]

[0060] In the oligonucleotide synthesis method according to the present invention, through the coupling, the deprotected hydroxyl group, thiol group or amino group at the 5' position or the 3' position and a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 5' position or the 3' position can be connected.

[0061] In the oligonucleotide synthesis method according to the present invention, the oxidation can increase stability by converting the phosphite ester group generated through coupling into a phosphate ester group. This oxidation can be achieved using a protocol known in the art, and for example, an iodine (I₂) solution or tert-butyl hydroperoxide (t-BuOOH) can be used.

[0062] In the oligonucleotide synthesis method according to the present invention, the sulfation can increase stability by converting the phosphite ester group generated through coupling into a phosphorothioate ester group. This sulfation can be achieved using a protocol known in the art, and for example, phenylacetyl disulfide (PADS), bis(oxadiazolyl) sulfide (BOBS), disulfide tetrazole (DDTT), or xanthan hydride can be used.

[0063] In the oligonucleotide synthesis method according to the present invention, the iminization can increase stability by converting the phosphite ester group generated through coupling into a phosphoramidate group. This iminization can be achieved using a protocol known in the art.

[0064] In the oligonucleotide synthesis method according to the present invention, the capping may serve to deactivate the deprotected 5'-position or 3'-position hydroxyl group, thiol group, or amino group that has not undergone a coupling reaction so that it does not participate in an additional oligonucleotide synthesis reaction. Such capping may be achieved using a protocol known in the art, for example, a hydroxyl group may be deactivated through an acetylation reaction using acetic anhydride (Ac₂O) and N-methylimidazole (NMI), a thiol group may be deactivated into a thioester or methyl thio form using methyl iodide (MeI) or acetyl disulfide, and an amino group may be deactivated through an acetylation or carbonylation reaction using acetic anhydride (Ac₂O) or t-butoxycarbonyl (t-Boc).

[0065] In the oligonucleotide synthesis method according to the present invention, the phosphorus protecting group removal may be the removal of the phosphorus protecting group of the backbone of the oligonucleotide. In one specific embodiment, the phosphorus protecting group may be a methyl group or a cyanoethyl group. The phosphorus protecting group removal can be achieved using a protocol known in the art, and the methyl group can be removed using, for example, thiophenol or disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate, and the cyanoethyl group can be removed using, for example, a non-nucleophilic or hindered amine, such as diethylamine, tert-butylamine, or 1,8-Diazabicycloundec-7-ene (DBU).

[0066]

[0067] In the oligonucleotide synthesis method according to the present invention, the oligonucleotide may refer to a compound having a structure in which nucleoside or nucleotide units are linked via phosphate ester, phosphorothioate ester, or phosphoramidate bonds. The nucleosides and nucleotides are as described above.

[0068] In one specific example, the synthesized oligonucleotide may be 2 to 100 oligomers (2-100 mer), or more.

[0069]

[0070] The method for synthesizing an oligonucleotide according to the present invention may further include, prior to step (i), a step of preparing a nucleoside or nucleotide that is fixed to the surface of a solid support and has a protecting group at a hydroxyl group, thiol group, or amino group at the 5' position or the 3' position.

[0071] In one specific example, the preparing step may include a step of fixing a nucleoside or nucleotide having a protecting group at a hydroxyl group, thiol group, or amino group at the 5' position or the 3' position to a solid support.

[0072] In one specific example, the fixation may be by directly linking the nucleoside or nucleotide to the solid support, or by linking it via a linker.

[0073] In one embodiment, the linker can be designed to attach a nucleoside or nucleotide to a solid support via various chemical bonds. The linker may be, for example, a universal linker, a UnyLinker™, a diacyl moiety, an aminoalkyl moiety, an alkyl moiety, an alkenyl moiety, a phosphate moiety, a succinyl moiety, an oxalyl moiety, a malonyl moiety, a glutaryl moiety, a trityl moiety, a polyethylene glycol (PEG) moiety, or -OC(O)CH2CH2C(O)O-, -NHC(O)CH2CH2C(O)NH-, -NHC(O)CH2CH2C(O)O-, -OC(O)CH2CH2C(O)NH-, -OC(O)CH2CH2C(O)S-, -NHC(O)CH2CH2C(O)S-, -OC(O)CH2C(O)O-, -NHC(O)CH2C(O)NH-, -NHC(O)CH2C(O)O-, -OC(O)CH2C(O)NH-, -OC(O)CH2C(O)S-, -NHC(O)CH2C(O)SH-, -OC(O)C(O)O-, -NHC(O) C(O)NH-, -NHC(O) C(O)O-, -OC(O)C(O)NH-, -OC(O)C(O)S-, -NHC(O) C(O)S-, -C(O)O-, -C(O)NH-, -C(O)S- -NH-, -O-, -S-, -SS-, -C(N)HO-, -NHC(O)O-, It may be a linker comprising a moiety of -NHC(O)NH-, -OC(O)O-, -OPO3-, or a combination thereof, but is not limited thereto.

[0074] The right end of the above moiety may represent a linkage site with a nucleoside or nucleotide. The above moiety may include a spacer at the left end. The spacer is C 1-30 Alkyl, C 2-30Various spacers used in the art for oligonucleotide synthesis, such as alkenyl and polyethylene glycol (PEG), can be applied.

[0075] The above linker can secure a physical distance between a solid support and a nucleoside or nucleotide, including a spacer.

[0076] The above fixation method can be selectively adjusted depending on specific synthetic conditions or goals, and may include various binding methods that stabilize or functionalize the bond between the solid support and the nucleotide. However, the fixation method is not limited thereto.

[0077]

[0078] The method for synthesizing an oligonucleotide according to the present invention may additionally include a step of recovering the synthesized oligonucleotide.

[0079] In one specific example, the recovery step may refer to detaching, releasing, separating, cleaving, or cleaving the desired oligonucleotide from the solid support after it has been synthesized. Additionally, the recovery may also refer to removing a protecting group. The protecting group may refer to a cyanoethyl protecting group of a phosphoramidite group and / or a protecting group of a base.

[0080] Specifically, the above recovery step may mean removing the cyanoethyl protecting group of the phosphoramidite group of the synthesized oligonucleotide after the desired oligonucleotide has been synthesized, detaching the synthesized oligonucleotide from the solid support, and removing the protecting group of the base.

[0081] This recovery step can be accomplished using protocols known in the art. The recovery can be accomplished using reagents such as diethylamine, triethylamine, ammonium hydroxide, and methylamine. Specifically, diethylamine and triethylamine can be primarily used to remove the cyanoethyl protecting group from the phosphoramidite group, while ammonium hydroxide, methylamine, or a combination thereof can be primarily used to desorb the synthesized oligonucleotide from the solid support and remove the protecting group from the base.

[0082]

[0083] In the method for synthesizing an oligonucleotide according to the present invention, in step (i),

[0084] A nucleoside or nucleotide having a protecting group at the hydroxyl, thiol or amino group at the 5' position is fixed to the surface of a solid support via the 3' position or the 2' position;

[0085] A nucleoside or nucleotide having a protecting group at the hydroxyl, thiol or amino group at the 3' position may be fixed to the surface of a solid support via the 5' position or the 2' position.

[0086] In one specific example, the nucleoside or nucleotide having a protecting group at the hydroxyl group, thiol group, or amino group at the 3' position in step (i) may be an abasic nucleoside or nucleotide. The abasic nucleoside or nucleotide may be an inverted abasic nucleoside or nucleotide.

[0087] In one specific example, the abasic nucleoside may be a compound represented by the following formula (I):

[0088] [Chemical Formula I]

[0089]

[0090] In the above chemical formula I, A1 is -H, -OH, 2'-O-alkyl, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, 2'-arabino-fluoro, 2'-O-aryl, 2'-O-benzyl, 2'-O-allyl, 2'-acetyl, 2'-aminomethyl, 2'-thio, or 2'-O-methyl-4-pyridine,

[0091] A 2 is -OH, -NH2, or -SH, and

[0092] A 3 is a -OH, -NH2, -SH, -OPO3, -O(PO3)2, -O(PO3)3, or phosphoramidite functional group.

[0093] The above phosphoramidite functional group is -OP(OR 1 )NR 2 It is indicated as 2. In one specific example, the R 1 and R 2 are phosphoramidite substituents, each independently -H, C 1-7 Alkyl group, C 5-20 or C 5-20 It may be an aryl group. In one specific example, the phosphoramidite functional group may be -OP(OCH2CH3)N(CH3)2, -OP(OCH2CH3)N(i-Pr)2, or -OP(OCH2CH2CN)N(i-Pr)2, but is not limited thereto.

[0094] In a method for synthesizing an oligonucleotide according to the present invention,

[0095] The above abasic nucleoside may be an inverted abasic nucleoside represented by the following formula I, which is fixed to the surface of a solid support via a linker:

[0096] [Formula I]

[0097] .

[0098] In the above formula, P is a protecting group, L is a linker,

[0099] A 1is -H, -OH, 2'-O-alkyl, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, 2'-arabino-fluoro, 2'-O-aryl, 2'-O-benzyl, 2'-O-allyl, 2'-acetyl, 2'-aminomethyl, 2'-thio, or 2'-O-methyl-4-pyridine,

[0100] A 2 is -O-, -NH-, or -S-.

[0101] The above protecting group P may be a known protecting group that protects a hydroxyl group, a thiol group, or an amino group. For example, the protecting group is trityl (Trt), dimethoxytrityl (DMT), monomethoxytrityl (MMT), tert-Butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), t-butoxycarbonyl (Boc), methyl phosphodiester, benzoyl (Bz), trifluoroacetyl (TFA), benzyl (Bn), p-methoxytriphenylmethyl (p-Methoxytrityl, PMT), mono-methoxyphosphotriester (MMP), fluorenylmethoxycarbonyl (Fmoc), tetrahydrofuran (THP), acetyl (Ac), mercaptotrityl (Mercapto-trityl, MTT), 4-methoxytrityl (Mmt), thiopyridyl, benzylthio, fluorenylmethoxycarbonyl (Fmoc), carbamate (Cbz), trifluoroacetyl (TFA), or isobutyryl (iBu), phenoxyacetyl (PAC), or dimethylformamidine (dmf) protecting groups.

[0102] The above linker can be designed to attach the compound of formula I to a solid support via various chemical bonds. The linker may be, for example, a universal linker, a UnyLinker™, a diacyl moiety, an aminoalkyl moiety, an alkyl moiety, an alkenyl moiety, a phosphate moiety, a succinyl moiety, an oxalyl moiety, a malonyl moiety, a glutaryl moiety, a trityl moiety, a polyethylene glycol (PEG) moiety, or -OC(O)CH2CH2C(O)O-, -NHC(O)CH2CH2C(O)NH-, -NHC(O)CH2CH2C(O)O-, -OC(O)CH2CH2C(O)NH-, -OC(O)CH2CH2C(O)S-, -NHC(O)CH2CH2C(O)S-, -OC(O)CH2C(O)O-, -NHC(O)CH2C(O)NH-, -NHC(O)CH2C(O)O-, -OC(O)CH2C(O)NH-, -OC(O)CH2C(O)S-, -NHC(O)CH2C(O)SH-, -OC(O)C(O)O-, -NHC(O)C(O)NH-, -NHC(O)C(O)O-, -OC(O)C(O)NH-, -OC(O)C(O)S-, -NHC(O)C(O)S-, -C(O)O-, -C(O)NH-, -C(O)S- -NH-, -O-, -S-, -SS-, -C(N)HO-, -NHC(O)O-, It may be a linker comprising a moiety of -NHC(O)NH-, -OC(O)O-, -OPO3-, or a linker comprising a combination thereof, but is not limited thereto.

[0103] The right end of the above moiety may represent a linkage site with the compound of formula I. The above moiety may include a spacer at the left end. The spacer may be C 1-30 Alkyl, C 2-30Various spacers used in the art for oligonucleotide synthesis, such as alkenyl and polyethylene glycol (PEG), can be applied.

[0104] The above linker can secure a physical distance between the solid support and the compound of formula I, including a spacer.

[0105] The above fixation method can be selectively adjusted depending on specific synthetic conditions or goals, and can include various bonding methods that stabilize or functionalize the bond between the solid support and the compound of formula I. However, the fixation method is not limited thereto.

[0106]

[0107] In the oligonucleotide synthesis method according to the present invention, the nucleoside phosphoramidite or nucleotide phosphoramidite may refer to a nucleoside or nucleotide derivatized with amidite. The nucleoside or nucleotide is as described above.

[0108] The above nucleoside phosphoramidite or nucleotide phosphoramidite may be a modified nucleoside phosphoramidite or a modified nucleotide phosphoramidite.

[0109] The above modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may have a modified sugar group. The modification of the sugar group is as described above.

[0110] The modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may have a modified nucleobase. The modified nucleobase is as described above.

[0111] The modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may have a modified phosphate group. The modified phosphate group may be a phosphoramidite group derivatized with amidite. In addition, the modified phosphate group may include a phosphate ester group, a phosphorothioate ester group, or a phosphoramidate group formed by oxidation, sulfation, or imination of a phosphoramidite group.

[0112] The above modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may have a protecting group bonded to the phosphorus moiety. The phosphorus protecting group may be a methyl group or a cyanoethyl group.

[0113] In one embodiment, the modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may be a 2'-modified nucleoside phosphoramidite or nucleotide phosphoramidite, or a 2'-substituted nucleoside phosphoramidite or nucleotide phosphoramidite. Specifically, the modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may be a nucleoside phosphoramidite or nucleotide phosphoramidite modified or substituted with 2'-O-alkyl, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, 2'-arabino-fluoro, 2'-O-aryl, 2'-O-benzyl, 2'-O-allyl, 2'-acetyl, 2'-aminomethyl, 2'-thio, or 2'-O-methyl-4-pyridine.

[0114] In one specific example, the modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may have a hydroxyl group (-OH) at the 5' position or the 3' position substituted with a thiol group (-SH) or an amino group (-NH2).

[0115] In one specific example, the modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may be an abasic nucleoside phosphoramidite or an abasic nucleotide phosphoramidite. An abasic nucleoside phosphoramidite or an abasic nucleotide phosphoramidite means a nucleoside phosphoramidite or a nucleotide phosphoramidite without a nucleobase attached.

[0116] In one specific example, the modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may be an inverted nucleoside phosphoramidite or an inverted nucleotide phosphoramidite. An inverted nucleoside phosphoramidite or an inverted nucleotide phosphoramidite means a nucleoside phosphoramidite or a nucleotide phosphoramidite having a structure in which the bonding direction is opposite to that of a general nucleoside phosphoramidite or nucleotide phosphoramidite. For example, in a method for synthesizing an oligonucleotide on a solid support, a general nucleoside phosphoramidite or nucleotide phosphoramidite is linked to the next unit through the functional group at the 5' position, but an inverted nucleoside phosphoramidite or inverted nucleotide phosphoramidite is linked to the next unit through the functional group at the 3' position.

[0117] In one specific example, the modified nucleoside phosphoramidite or modified nucleotide phosphoramidite may be an inverted abasic nucleoside phosphoramidite or an inverted abasic nucleotide phosphoramidite.

[0118] In one specific example, the nucleotide phosphoramidite may be a nucleotide phosphoramidite, a di-nucleotide phosphoramidite, a tri-nucleotide phosphoramidite, or a nucleotide phosphoramidite of a higher oligomer.

[0119]

[0120] In the oligonucleotide synthesis method according to the present invention, the nucleoside phosphoramidite or nucleotide phosphoramidite may have a phosphoramidite group at the 3' position or the 5' position by derivatizing one of the hydroxyl group, thiol group, or amino group at the 3' position, or the hydroxyl group, thiol group, or amino group at the 5' position to an amidite. When the nucleoside phosphoramidite or nucleotide phosphoramidite has a phosphoramidite group at the 3' position, a protecting group may be bonded at the 5' position, and when the nucleoside phosphoramidite or nucleotide phosphoramidite has a phosphoramidite group at the 5' position, a protecting group may be bonded at the 3' position. The 5' position, the 3' position, and the protecting group are as described above.

[0121] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite may be a monomer, dimer, trimer or higher oligomer.

[0122]

[0123] In the oligonucleotide synthesis method according to the present invention, step (ii)

[0124] The deprotected hydroxyl, thiol or amino group at the 5' position is linked to the 3' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 5' position; or

[0125] The deprotected hydroxyl, thiol or amino group at the 5' position is linked to the 5' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 3' position; or

[0126] The deprotected hydroxyl, thiol or amino group at the 3' position is linked to the 3' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 5' position; or

[0127] The deprotected hydroxyl, thiol or amino group at the 3' position may be linked to the 5' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 3' position.

[0128] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 5' position or the 3' position may be an abasic nucleoside phosphoramidite or an abasic nucleotide phosphoramidite.

[0129] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group on the hydroxyl group, thiol group or amino group at the 3' position may mean an inverted nucleoside phosphoramidite or an inverted nucleotide phosphoramidite.

[0130] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 3' position may be an inverted abasic nucleoside phosphoramidite or an inverted abasic nucleotide phosphoramidite.

[0131]

[0132] In the oligonucleotide synthesis method according to the present invention, step (ii) of the last repetition may be a step of coupling the 5' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group on the 3' position of a hydroxyl group, thiol group or amino group to the deprotected 5' position or 3' position of a hydroxyl group, thiol group or amino group.

[0133] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 3' position may be an abasic nucleoside phosphoramidite or an abasic nucleotide phosphoramidite.

[0134] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group on the hydroxyl group, thiol group or amino group at the 3' position may mean an inverted nucleoside phosphoramidite or an inverted nucleotide phosphoramidite.

[0135] In one specific example, the nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 3' position may be an inverted abasic nucleoside phosphoramidite or an inverted abasic nucleotide phosphoramidite.

[0136]

[0137] In the oligonucleotide synthesis method according to the present invention, step (vi) may be a step of synthesizing a desired oligonucleotide by repeating steps (ii) to (v) 1 to 100 times, 1 to 75 times, or 1 to 50 times.

[0138] In one specific example, the last step of the last iteration may be step (iii) or (iv).

[0139]

[0140] In the oligonucleotide synthesis method according to the present invention, at least one of steps (i) to (v) may be performed at a temperature below room temperature (RT). Specifically, step (i), step (ii), step (iii), step (iv), at least one of steps (i) to (v), at least one of steps (i) to (iv), at least one of steps (ii) to (iii), steps (i), (ii) and (iv), steps (i), (iii) and (iv), steps (i) and (iv), steps (i), (ii), (iii) and (iv), steps (i), (ii) and (iv), or steps (ii) and (iii) may be performed at a temperature below room temperature. More specifically, step (ii), steps (i), (ii) and (iv), or steps (i) to (iv) may be performed at a temperature below room temperature. In addition, the step of capping the deprotected hydroxyl group, thiol group or amino group that has not undergone the coupling reaction may be performed at a temperature below room temperature.

[0141] In one specific example, the temperature may mean an inlet temperature.

[0142] In the oligonucleotide synthesis method according to the present invention, the temperature below room temperature (RT) may be below 22°C. Specifically, temperatures below room temperature (RT) are less than 22℃, less than 21℃, less than 20℃, 0℃ or more but less than 22℃, 5℃ or more but less than 22℃, 10℃ or more but less than 22℃, 12℃ or more but less than 22℃, 13℃ or more but less than 22℃, 15℃ or more but less than 22℃, 0℃ or more but less than 20℃, 5℃ or more but less than 20℃, 10℃ or more but less than 20℃, 12℃ or more but less than 20℃, 13℃ or more but less than 20℃, 15℃ or more but less than 20℃, 0℃ or more but less than 19℃, 5℃ or more but less than 19℃, 10℃ or more but less than 19℃, 12℃ or more but less than 19℃, 13℃ or more It can be 19℃ or less, 15℃ or more but 19℃ or less, 0℃ or more but 18℃ or less, 5℃ or more but 18℃ or less, 10℃ or more but 18℃ or less, 12℃ or more but 18℃ or less, 13℃ or more but 18℃ or less, or 15℃ or more but 18℃ or less.

[0143] That is, the oligonucleotide synthesis method according to the present invention comprises steps (i), (ii), (iii), (iv), at least one of steps (i) to (v), at least one of steps (i) to (iv), at least one of steps (ii) to (iii), steps (i), (ii) and (iv), steps (i), (iii) and (iv), steps (i) and (iv), steps (i), (ii), (iii) and (iv), steps (i), (ii) and (iv), or steps (ii) and (iii), specifically, steps (ii), (ii) and (iv), or steps (i) to (iv), are performed at a temperature of less than 22°C, less than 21°C, less than 20°C, 0°C or more but less than 22°C, 5°C or more but less than 22°C, 10°C or more but less than 22°C, 12°C or more but less than 22°C, 13 ℃ or higher but less than 22 ℃, 15 ℃ or higher but less than 22 ℃, 0 ℃ or higher but less than 20 ℃, 5 ℃ or higher but less than 20 ℃, 10 ℃ or higher but less than 20 ℃, 12 ℃ or higher but less than 20 ℃, 13 ℃ or higher but less than 20 ℃, 15 ℃ or higher but less than 20 ℃, 0 ℃ or higher but less than 19 ℃, 5 ℃ or higher but less than 19 ℃, 10 ℃ or higher but less than 19 ℃, 12 ℃ or higher but less than 19 ℃, 13 ℃ or higher but less than 19 ℃, 15 ℃ or higher but less than 19 ℃, 0 ℃ or higher but less than 18 ℃, 5 ℃ or higher but less than 18 ℃, 10 ℃ or higher but less than 18 ℃, 12 ℃ or higher but less than 18 ℃, 13 ℃ or higher but less than 18 ℃, or 15 ℃ or higher It may be performed at a temperature below 18 ℃.

[0144] The oligonucleotide synthesis method according to the present invention can suppress side reactions occurring during the synthesis process and stably synthesize oligonucleotides having higher purity by carrying out the synthesis cycle at a temperature below room temperature.

[0145] Figure 1 is a schematic diagram of an oligonucleotide synthesis method.

[0146] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0147] Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0148] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0149] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0150]

[0151] Example 1. Synthesis of oligonucleotides

[0152] Oligonucleotides were synthesized on a 1-3 mmol scale using a solid support with inverted abasic nucleosides (manufacturer: Kinovate, loading capacity: 300 μmol / g) and an AKTA oligopilot 100 solid-phase synthesizer.

[0153] The structure of the solid support to which the above-mentioned inverted abasic nucleoside is attached is as shown in the following chemical formula II.

[0154] [Chemical Formula II]

[0155] .

[0156]

[0157] Specifically, the method of synthesizing an oligonucleotide by linking each nucleoside according to the desired sequence consists of the following four-step synthetic cycle (Figure 1):

[0158] 1) Detritylation

[0159] 2) Coupling

[0160] 3) Oxidation / Thiolation

[0161] 4) Capping.

[0162]

[0163] Nucleoside phosphoramidites, all other synthetic reagents, and solvents were obtained from commercially available sources and were prepared and used according to the reagent composition without any purification or treatment. For example, nucleoside phosphoramidites can be obtained from Hongene Biotech, Thermo, and Sigma; dichloroacetic acid from CABB; and acetonitrile and toluene from SK chemicals.

[0164]

[0165] Detritylation step was performed using 2.5-10% dichloroacetic acid in toluene as reagent.

[0166] The coupling step was performed by circulating a solution of nucleoside-phosphoramidite (0.2 M in acetonitrile) and 5-Ethylthio-1H-Tetrazole (0.5-1.0 M in acetonitrile) corresponding to the desired oligonucleotide sequence through a solid support.

[0167] Oxidiation step was performed using 0.05 M iodine in pyridine and water. Thiolation step was performed using 0.2 M 3-amino-1, 2, 4-dithiazole-5-thione (XH) in pyridine.

[0168] The capping step was performed using a mixture of acetic anhydride, 2,6-lutidine, and N-methylimidazole in acetonitrile.

[0169] Deprotection (removal of cyanoethyl protecting group) of cyanoethyl phosphodiester was performed using a 20% diethylamine (DEA) solution in acetonitrile (AN).

[0170] After the solid-phase oligonucleotide synthesis was completed, detachment and deprotection (removal of the base protecting group) of the oligonucleotide from the solid support was performed by treating with 28-30% ammonium hydroxide at 40-60°C for 6-20 hours.

[0171] The synthesis parameters are summarized in Table 1 below.

[0172]

[0173] Processing step Variable reaction - Cycle unit Synthesis support Support Solid support with inverted abasic nucleoside - Loading 300-350 μmol / g Detritylation reagent 2.5 - 10% DCA in Toluene Deblocking volume 4.3 - 11.7 CV Reaction time 4.5 - 12.3 min Coupling Nucleoside phosphoramidite equivalent 1.5 - 1.7 eq Nucleoside phosphoramidite concentration 0.2 M Activator : Nucleoside phosphoramidite ratio 3 : 2 - Activator 0.5 - 1.0 M ETT - Recirculation flow 100-200 cm / hr Recirculation time 7.0 min Oxidation reagent 0.05 M I2 in PYD / H2O - Loading volume 2.1 CV Reaction time 4.3 min Sulfation reagent 0.1 M XH in PYD / AN - filling volume 2.2CV Reaction time 4.6 min Capping reagent Cap A, 2:8 [NMI / AN] Cap B 2:3:5 [Ac2O / 2, 6-lutidine / AN] - capping filling volume 0.5CV Reaction time 1.3 min Cyanoethyl phosphodiester deprotecting reagent 20% DEA in AN - filling volume 4.8CV Reaction time 10 min

[0174]

[0175] The oligonucleotides of the following manufacturing examples and comparative manufacturing examples were synthesized using this synthetic method.

[0176]

[0177] In the oligonucleotide sequences of the following synthesized Manufacturing Examples 1 to 7 and Comparative Manufacturing Examples 1 to 16, the meanings of invAb, s, fa, fu, fc, and fg are as follows.

[0178] “invAb” is an inverted abasic nucleotide (nucleoside).

[0179] “s” is a phosphorothioate internucleoside linkage.

[0180] “fa, fu, fc, fg” are 2'-fluoro modified nucleotides (nucleosides).

[0181] “a, c, g” are nucleotides (nucleosides) modified with 2'-OMe.

[0182]

[0183] Experimental Example 1. Purity and Yield Analysis of Synthesized Oligonucleotides

[0184] The purity and yield of the oligonucleotide products of the synthesized manufacturing examples and comparative manufacturing examples were analyzed.

[0185] Specifically, purity was measured by LC-UV as UV purity (%), and yield (%) was calculated by measuring OD concentration using a UV spectrometer.

[0186] Specifically, UV purity (%) was measured by measuring the purity according to the peak area of ​​the UV signal within the retention time range of 5 to 30 minutes using an HPLC (Manufacturer: Agilent, Model: 1290 infinity (1260 DAD)) instrument. OD concentration was measured by measuring the absorbance at a wavelength of 260 nm using a UV / Vis Spectrophotometer (Manufacturer: Agilent, Model: Cary-3500).

[0187]

[0188] Manufacturing example 1.

[0189] According to the method of Example 1, a 23-mer oligonucleotide of Preparation Example 1 represented by the following sequence was prepared. The average temperature of the four-step synthetic cycle process of Preparation Example 1 is 17.0°C.

[0190] Sequence of Manufacturing Example 1 (SEQ ID NO: 1): 5'-as guggcuua fu fc fc uauuucauc us invAb-3'.

[0191] The UV purity of Manufacturing Example 1 was found to be quite high at 90.7%, and the yield was found to be 80.1%.

[0192]

[0193] Comparative manufacturing example 1.

[0194] According to the method of Example 1, a 23-mer oligonucleotide of Comparative Manufacturing Example 1 represented by the following sequence was manufactured. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 1 is 23.2°C.

[0195] Sequence of comparative manufacturing example 1 (SEQ ID NO: 1): 5'-as guggcuua fu fc fc uauuucauc us invAb-3'.

[0196] The UV purity of Comparative Manufacturing Example 1 was 88.5%, and the yield was 79.8%.

[0197]

[0198] Manufacturing example 2.

[0199] According to the method of Example 1, a 24-mer oligonucleotide of Preparation Example 2 represented by the following sequence was prepared. The average temperature of the four-step synthetic cycle process of Preparation Example 2 is 16.5°C.

[0200] Sequence of Manufacturing Example 2 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0201] The UV purity of Manufacturing Example 2 was found to be quite high at 91.4%, and the yield was found to be 74.6%.

[0202]

[0203] Comparative manufacturing example 2.

[0204] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 2 represented by the following sequence was manufactured. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 2 is 23.1°C.

[0205] Sequence of comparative manufacturing example 2 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0206] The UV purity of Comparative Manufacturing Example 2 was 88.2%, and the yield was 72.9%.

[0207]

[0208] Comparative manufacturing example 3.

[0209] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 3 represented by the following sequence was manufactured. The average temperature of the first cycle process among the four-step synthetic cycle processes of Comparative Manufacturing Example 3 was 17.3°C, and the average temperature of the second to last cycles was 23.6°C.

[0210] Sequence of comparative manufacturing example 3 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0211] The UV purity of Comparative Manufacturing Example 3 was 88.5%, and the yield was 69.2%.

[0212]

[0213] Comparative manufacturing example 4.

[0214] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 4 represented by the following sequence was manufactured. The average temperature of the first to 13th cycle processes of the four-step synthetic cycle process of Comparative Manufacturing Example 4 was 23.6°C, and the average temperature of the 14th to the last cycle processes was 17.1°C.

[0215] Sequence of comparative manufacturing example 4 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0216] The UV purity of Comparative Manufacturing Example 4 was 89.7%, and the yield was 70.1%.

[0217]

[0218] Manufacturing example 3.

[0219] According to the method of Example 1, a 24-mer oligonucleotide of Preparation Example 3 represented by the following sequence was prepared. The average temperature of the four-step synthetic cycle process of Preparation Example 3 is 17.1°C.

[0220] Sequence of Manufacturing Example 3 (SEQ ID NO: 3): 5'-invAbs agu invAb gcuua fu fc fc uauuucauc us invAb-3'.

[0221] The UV purity of Manufacturing Example 3 was found to be quite high at 90.9%, and the yield was found to be 70.1%.

[0222]

[0223] Comparative manufacturing example 5.

[0224] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 5 represented by the following sequence was manufactured. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 5 is 23.4°C.

[0225] Sequence of comparative manufacturing example 5 (SEQ ID NO: 3): 5'-invAbs agu invAb gcuua fu fc fc uauuucauc us invAb-3'.

[0226] The UV purity of Comparative Manufacturing Example 5 was 88.3%, and the yield was 68.8%.

[0227]

[0228] Comparative manufacturing example 6.

[0229] According to the method of Example 1, a 23-mer oligonucleotide of Comparative Manufacturing Example 6 represented by the following sequence was manufactured. A solid support (manufacturer: Kinovate, loading capacity: 350 μmol / g) to which 2'-OMe Adenosine (Bz, with a benzoyl protecting group) was attached was used. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 6 was 17.2°C.

[0230] Sequence of comparative manufacturing example 6 (SEQ ID NO: 4): 5'-as guggcuua fu fc fc uauuucauc us a-3'.

[0231] The UV purity of Comparative Manufacturing Example 6 was found to be 85.6%, and the yield was found to be 75.8%.

[0232]

[0233] Comparative manufacturing example 7.

[0234] According to the method of Example 1, a 23-mer oligonucleotide of Comparative Manufacturing Example 7 represented by the following sequence was manufactured. A solid support with 2'-O-Me Adenosine attached (manufacturer: Kinovate, loading capacity: 350 μmol / g) was used as the solid support. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 7 was 15.9°C.

[0235] Sequence of comparative manufacturing example 7 (SEQ ID NO: 4): 5'-as guggcuua fu fc fc uauuucauc us a-3'.

[0236] The UV purity of Comparative Manufacturing Example 7 was 83.8%, and the yield was 74.6%.

[0237]

[0238] Comparative manufacturing example 8.

[0239] According to the method of Example 1, a 23-mer oligonucleotide of Comparative Manufacturing Example 8 represented by the following sequence was manufactured. A solid support with 2'-O-Me Adenosine attached (manufacturer: Kinovate, loading capacity: 350 μmol / g) was used as the solid support. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 8 was 22.7°C.

[0240] Sequence of comparative manufacturing example 8 (SEQ ID NO: 4): 5'-as guggcuua fu fc fc uauuucauc us a-3'.

[0241] The UV purity of Comparative Manufacturing Example 8 was found to be 85.6%, and the yield was found to be 75.8%.

[0242]

[0243] Comparative manufacturing example 9.

[0244] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 9 represented by the following sequence was manufactured. A solid support (CPG) with IdT (Inverted deoxy thymidine) attached was used as the solid support. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 9 was 17.5°C.

[0245] Sequence of comparative manufacturing example 9 (SEQ ID NO: 5): 5'-invAbs aguggcuua fu fc fc uauuucauc us IdT-3'.

[0246] The UV purity of Comparative Manufacturing Example 9 was found to be 81.6%, and the yield was found to be 54.9%.

[0247]

[0248] Comparative manufacturing example 10.

[0249] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 10 represented by the following sequence was manufactured. A solid support (CPG) with IdT (Inverted deoxy thymidine) attached was used as the solid support. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 10 was 23.8°C.

[0250] Sequence of comparative manufacturing example 10 (SEQ ID NO: 5): 5'-invAbs aguggcuua fu fc fc uauuucauc us IdT-3'.

[0251] The UV purity of Comparative Manufacturing Example 10 was 82.8%, and the yield was 55.7%.

[0252]

[0253] Comparative manufacturing example 11.

[0254] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 11 represented by the following sequence was manufactured. A solid support with 2'-OMe Uridine attached (manufacturer: Kinovate, loading capacity: 350 μmol / g) was used as the solid support. The average temperature of the coupling step in the four-step synthetic cycle process of Comparative Manufacturing Example 11 was 17.6°C, and the average temperatures of the other three steps were 23.1°C.

[0255] Sequence of comparative manufacturing example 11 (SEQ ID NO: 6): 5'-invAbs aguggcuua fu fc fc uauuucauc invAbs u-3'.

[0256] The UV purity of Comparative Manufacturing Example 11 was 83.6%, and the yield was 69.0%.

[0257]

[0258] Comparative manufacturing example 12.

[0259] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 12 represented by the following sequence was manufactured. A solid support with 2'-OMe Uridine attached (manufacturer: Kinovate, loading capacity: 350 μmol / g) was used as the solid support. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 12 was 23.1°C.

[0260] Sequence of comparative manufacturing example 12 (SEQ ID NO: 6): 5'-invAbs aguggcuua fu fc fc uauuucauc invAbs u-3'.

[0261] The UV purity of Comparative Manufacturing Example 12 was 85.3%, and the yield was 68.1%.

[0262]

[0263] Comparative manufacturing example 13.

[0264] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 13 represented by the following sequence was manufactured. The average temperature of the four-step synthetic cycle process of Comparative Manufacturing Example 13 is 22.9°C.

[0265] Sequence of comparative manufacturing example 13 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0266] The UV purity of Comparative Manufacturing Example 13 was 86.0%, and the yield was 66.2%.

[0267]

[0268] Comparative manufacturing example 14.

[0269] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 14 represented by the following sequence was manufactured. The average temperature of the oxidation / sulfuration step in the four-step synthetic cycle process of Comparative Manufacturing Example 14 is 16.5 °C / 17.1 °C, and the average temperature of the other three steps is 22.9 °C.

[0270] Sequence of comparative manufacturing example 14 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0271] The UV purity of Comparative Manufacturing Example 14 was 85.6%, and the yield was 64.3%.

[0272]

[0273] Comparative manufacturing example 15.

[0274] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 15 represented by the following sequence was manufactured. The average temperatures of the detritylation and coupling steps in the four-step synthetic cycle process of Comparative Manufacturing Example 15 are 17.6°C and 18.5°C, and the average temperatures of the other two steps are 22.9°C.

[0275] Sequence of comparative manufacturing example 15 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0276] The UV purity of Comparative Manufacturing Example 15 was 89.1%, and the yield was 68.1%.

[0277]

[0278] Manufacturing example 4.

[0279] According to the method of Example 1, a 24-mer oligonucleotide of Preparation Example 4 represented by the following sequence was prepared. The average temperature of the coupling step in the four-step synthetic cycle process of Preparation Example 4 was 17.4°C, and the average temperatures of the other three steps were 22.9°C.

[0280] Sequence of Manufacturing Example 4 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0281] The UV purity of Manufacturing Example 4 was found to be quite high at 90.8%, and the yield was found to be 66.0%.

[0282]

[0283] Manufacturing Example 5.

[0284] According to the method of Example 1, a 24-mer oligonucleotide of Preparation Example 5 represented by the following sequence was prepared. The average temperature of the coupling step in the four-step synthetic cycle process of Preparation Example 5 was 19.0°C, and the average temperatures of the other three steps were 22.9°C.

[0285] Sequence of Manufacturing Example 5 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0286] The UV purity of Manufacturing Example 5 was found to be quite high at 90.1%, and the yield was found to be 66.8%.

[0287]

[0288] Manufacturing example 6.

[0289] According to the method of Example 1, a 24-mer oligonucleotide of Preparation Example 6 represented by the following sequence was prepared. The average temperature of the coupling step in the four-step synthetic cycle process of Preparation Example 6 was 15.1°C, and the average temperatures of the other three steps were 22.9°C.

[0290] Sequence of Manufacturing Example 6 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0291] The UV purity of Manufacturing Example 6 was found to be quite high at 90.7%, and the yield was found to be 66.0%.

[0292]

[0293] Manufacturing example 7.

[0294] According to the method of Example 1, a 24-mer oligonucleotide of Preparation Example 7 represented by the following sequence was prepared. The average temperature of the coupling step in the four-step synthetic cycle process of Preparation Example 7 was 13.0°C, and the average temperatures of the other three steps were 22.9°C.

[0295] Sequence of Manufacturing Example 7 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0296] The UV purity of Manufacturing Example 7 was found to be quite high at 90.6%, and the yield was found to be 67.5%.

[0297]

[0298] Comparative manufacturing example 16.

[0299] According to the method of Example 1, a 24-mer oligonucleotide of Comparative Manufacturing Example 16 represented by the following sequence was manufactured. The average temperature of the coupling step in the four-step synthetic cycle process of Comparative Manufacturing Example 16 is 10.9°C, and the average temperatures of the other three steps are 22.9°C.

[0300] Sequence of comparative manufacturing example 16 (SEQ ID NO: 2): 5'-invAbs aguggcuua fu fc fc uauuucauc us invAb-3'.

[0301] The UV purity of Comparative Manufacturing Example 16 was found to be 86.9%, and the yield was found to be 65.6%.

[0302]

[0303] Comparison of purity and yield of synthesized oligonucleotides

[0304] The process temperature, UV purity, and yield of the oligonucleotides of Manufacturing Examples 1 to 7 and Comparative Manufacturing Examples 1 to 16 are compared and shown in Table 2 below.

[0305]

[0306] Control stepAve temp (℃)UV Purity (%)Yield (%)Manufacturing example 1Pre-process 17.090.780.1Comparative manufacturing example 1Pre-process 23.288.579.8Manufacturing example 2Pre-process 16.591.474.6Comparative manufacturing example 2Pre-process 23.188.272.9Comparative manufacturing example 3Cycle 1 st / cycle 2 nd - end17.3 / 23.688.569.2 Comparison Manufacturing Example 4 Cycle 1 st - 13 th / cycle 14 th- end23.6 / 17.189.770.1 Manufacturing example 3 Pre-process 17.190.970.1 Comparative Manufacturing example 5 Pre-process 23.488.368.8 Comparative Manufacturing example 6 Pre-process 17.285.675.8 Comparative Manufacturing example 7 Pre-process 15.983.874.6 Comparative Manufacturing example 8 Pre-process 22.785.675.8 Comparative Manufacturing example 9 Pre-process 17.581.654.9 Comparative Manufacturing example 10 Pre-process 23.882.855.7 Comparative Manufacturing example 11 Coupling / remaining 17.6 / 23.183.669.0 Comparative Manufacturing example 12 Pre-process 23.185.368.1 Comparative Manufacturing example 13 Pre-process 22.986.066.2 Comparative Manufacturing example 14 Oxidation, Sulfation / remainder 16.5, 17.1 / 22.985.664.3 Comparative Manufacturing Example 15 Detritylation, coupling / remainder 17.6, 18.5 / 22.989.168.1 Manufacturing Example 4 Coupling / remainder 17.4 / 22.990.866.0 Manufacturing Example 5 Coupling / remainder 19.0 / 22.990.166.8 Manufacturing Example 6 Coupling / remainder 15.1 / 22.990.766.0 Manufacturing Example 7 Coupling / remainder 13.0 / 22.990.667.5 Comparative Manufacturing Example 16 Coupling / remainder 10.9 / 22.986.965.6

[0307]

[0308] As shown in Table 2 above, it was confirmed that oligonucleotides were synthesized at an average temperature lower than 22°C, resulting in higher synthetic purity. This result was also observed when the sequence of the oligonucleotide residues was changed using the same synthetic method. It was confirmed that a solid support to which an inverted abasic nucleoside was attached had a higher synthetic purity. It was confirmed that a higher synthetic purity was obtained by controlling the average temperature of the entire synthetic process rather than controlling the temperature for each cycle. In addition, it was confirmed that a higher synthetic purity was obtained when the average temperature of the coupling step process among the four reaction steps of the synthetic cycle was 22°C or lower.

[0309]

[0310] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the experimental examples and embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A method for synthesizing an oligonucleotide on a solid support comprising steps (i) to (v), wherein at least one of the steps is performed at a temperature below room temperature (RT): (i) a step of removing a protecting group from a nucleoside or nucleotide having a protecting group at a hydroxyl group, thiol group or amino group at the 5' position or the 3' position, thereby forming a nucleoside or nucleotide having a deprotected hydroxyl group, thiol group or amino group at the 5' position or the 3' position; (ii) a step of coupling a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl group, thiol group or amino group at the 5' or 3' position to a deprotected hydroxyl group, thiol group or amino group at the 5' or 3' position; (iii) a step of forming a phosphate ester group by oxidation, a phosphorothioate ester group by thiolation, or a phosphoramidate group by imination of a phosphite ester group generated through coupling; (iv) a step of removing a protecting group of a hydroxyl group, thiol group or amino group at the 5' or 3' position of a coupled nucleoside phosphoramidite or nucleotide phosphoramidite, thereby forming a deprotected hydroxyl group, thiol group or amino group at the 5' or 3' position; (v) A step of synthesizing the desired oligonucleotide by repeating steps (ii) to (iv) at least once.

2. A method for synthesizing an oligonucleotide, further comprising a step of capping a deprotected hydroxyl group, thiol group, or amino group that has not undergone a coupling reaction in the first paragraph.

3. A method for synthesizing an oligonucleotide, wherein, in the first paragraph, when steps (ii) to (iv) are repeated one or more times, in one or more of the repetitions, at least one of steps (ii) to (iv) is performed at a temperature below room temperature.

4. A method for synthesizing an oligonucleotide in the first paragraph, wherein the last step of the last repetition is step (iii) or (iv).

5. A method for synthesizing an oligonucleotide, further comprising a step of removing a protecting group after step (v) in the first paragraph.

6. In paragraph 1, before step (i), A method for synthesizing an oligonucleotide, further comprising the step of preparing a nucleoside or nucleotide that is fixed to the surface of a solid support and has a protecting group at a hydroxyl group, thiol group, or amino group at the 5' position or the 3' position.

7. In paragraph 1, A method for synthesizing an oligonucleotide, further comprising the step of recovering the synthesized oligonucleotide.

8. In the first paragraph, the protecting group is trityl (Trt), dimethoxytrityl (DMT), monomethoxytrityl (MMT), tert-Butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), t-butoxycarbonyl (Boc), methyl phosphodiester, benzoyl (Bz), trifluoroacetyl (TFA), benzyl (Bn), p-methoxytriphenylmethyl (p-Methoxytrityl, PMT), mono-methoxyphosphotriester (MMP), fluorenylmethoxycarbonyl (Fmoc), tetrahydrofuran (THP), acetyl (Ac), mercaptotrityl A method for synthesizing an oligonucleotide, wherein the protecting group is Mercapto-trityl (MTT), 4-methoxytrityl (Mmt), Thiopyridyl, Benzylthio, Fluorenylmethoxycarbonyl (Fmoc), Carbamate (Cbz), Trifluoroacetyl (TFA), Isobutyryl (iBu), Phenoxyacetyl (PAC), or Dimethylformamidine (dmf).

9. In paragraph 1, in step (i) A nucleoside or nucleotide having a protecting group at the hydroxyl, thiol or amino group at the 5' position is fixed to the surface of a solid support via the 3' position or the 2' position; A method for synthesizing an oligonucleotide, wherein a nucleoside or nucleotide having a protecting group at a hydroxyl group, thiol group, or amino group at the 3' position is fixed to the surface of a solid support via the 5' position or the 2' position.

10. In paragraph 1, (i) A method for synthesizing an oligonucleotide, wherein the nucleoside or nucleotide having a protecting group at the hydroxyl group, thiol group or amino group at the 3' position in step is an abasic nucleoside or nucleotide.

11. In paragraph 10, A method for synthesizing an oligonucleotide, wherein the above-mentioned abasic nucleoside is an inverted abasic nucleoside represented by the following formula I, which is fixed to the surface of a solid support via a linker: [Formula I] In the above formula, P is a protecting group, L is a linker, A 1 is -H, -OH, 2'-O-alkyl, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, 2'-arabino-fluoro, 2'-O-aryl, 2'-O-benzyl, 2'-O-allyl, 2'-acetyl, 2'-aminomethyl, 2'-thio, or 2'-O-methyl-4-pyridine, and A 2 is -O-, -NH-, or -S-.

12. In paragraph 1, step (ii) The deprotected hydroxyl, thiol or amino group at the 5' position is linked to the 3' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 5' position; or The deprotected hydroxyl, thiol or amino group at the 5' position is linked to the 5' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 3' position; or The deprotected hydroxyl, thiol or amino group at the 3' position is linked to the 3' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 5' position; or The deprotected hydroxyl, thiol or amino group at the 3' position is linked to the 5' position of a nucleoside phosphoramidite or nucleotide phosphoramidite having a protecting group at the hydroxyl, thiol or amino group at the 3' position. A method for synthesizing oligonucleotides.

13. In paragraph 1, (ii) step; (i), (ii) and (iv); or a method for synthesizing an oligonucleotide, wherein steps (i) to (iv) are performed at a temperature below room temperature.

14. In any one of paragraphs 1 to 13, A method for synthesizing an oligonucleotide, wherein the temperature below room temperature (RT) is less than 22°C.

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