Compositions and Methods for Liquid Phase Oligonucleotide Synthesis

Abstract

Embodiments of the present application relate to polymers used as polymeric multivalent hubs for liquid-phase oligonucleotide synthesis. Also provided is a method for making oligonucleotides by liquid-phase oligonucleotide synthesis using this polymer. The present disclosure relates to methods and compositions for liquid-phase oligonucleotide synthesis using the use of polymers having pendant poly(ethylene glycol) arms. Further embodiments of the present application relate to oligonucleotides prepared by any of the methods described herein.

Description

Technical Field

[0001] Background Field The present disclosure relates to methods and compositions for liquid-phase oligonucleotide synthesis using the use of polymers having pendant poly(ethylene glycol) arms.

Background Art

[0002] Description of Related Art Oligonucleotide-based drugs have become powerful and typical drugs having the ability to treat various diseases. Currently, the demand for oligonucleotides can be met by conventional solid-phase oligonucleotide synthesis (SPOS). SPOS has certain advantages such as easy isolation of products and use of an anhydrous synthesis environment. However, SPOS generally has a low overall yield after multiple steps for the oligo sequence and high costs for reagents, solid supports, and waste management. In addition, SPOS can result in mismatched oligo sequences that make purification difficult. The increasing demand for metric-ton quantities of oligonucleotides far exceeds the production capacity of solid-phase oligonucleotide synthesis.

[0003] Liquid-phase oligonucleotide synthesis (LPOS) is a technology having the potential to provide the required production capacity. One of the main advantages of LPOS over SPOS is the absence of the heterogeneous nature of the process, i.e., the absence of an insoluble solid support. The use of soluble scaffolds or supports used in LPOS allows each step of the synthesis to be carried out in a liquid phase with improved reaction rates. Polyethylene glycol (PEG) is one of the most widely used soluble polymer supports and has received considerable attention. Current Protocol in Nucleic Acid Chemistry (2019) 77, e82. First, the cost of the PEG process is low. Second, the PEG platform is highly beneficial for large-scale production due to its high coupling efficiency and the possibility of using convergent synthesis. Therefore, further exploration of PEG-based platforms is quite promising for LPOS development.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0005] Summary Some aspects of the present disclosure relate to polymers for liquid-phase oligonucleotide synthesis having the structure of formula (I)

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[0006] In some embodiments of the polymer of formula (I), R is -CH3. In some other embodiments, R is H. In some embodiments, W is -CH2CH2NH-. In other embodiments, W is -CH2CH2O-. In some embodiments, W is C2-C6 alkylene, such as -CH2CH2-. In another embodiment, W is a bond.

[0007] In some embodiments of the polymer of formula (I), L 1 Is C2-C6 alkylene. In other embodiments, L 1is a 3- to 12-membered or 3- to 6-membered heteroalkylene containing 1, 2 or 3 heteroatoms selected from N, O or S. In a further embodiment, the heteroalkylene contains 1 or 2 nitrogen atoms.

[0008] In some embodiments of the polymer of formula (I), R 1 is -OH or a protected hydroxy. In other embodiments, R 1 is -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3 (-NHAc), or a protected amino. In still other embodiments, R 1 is

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[0009] In some embodiments of the polymer of formula (I), Q is

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[0010] In some embodiments of the polymer of formula (I), each R 3 is independently H or a hydroxy protecting group, and each R 4a and R 4b are independently H, optionally substituted C1-C6 alkyl, -C(=O)CH3, or an amino protecting group. In some other embodiments, each R 4a is H, and each R 4b is independently H, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group, or

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[0011] In some further embodiments of the polymer of formula (I) or (I'), Q is

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[0012] In some embodiments of the polymer of formula (I) or any substructure described herein, L 2a and L 2b each independently is C1 - C 10is alkylene or 2- to 10-membered heteroalkylene, and R 5a , R 5b , R 6a and R 6b each independently is H, -OR 7 or -NR 8a R 8b , and each of R 7 independently is H, C1-C6 alkyl, a hydroxy protecting group, or

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[0013] In some embodiments, the polymer of formula (I) has the structure of formula (Ia).

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[0014] In other embodiments, the polymer of formula (I) or (Ia) has the structure of formula (Ib), (Ib-1), (Ic), or (Ic-1).

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[0015] In some embodiments, the polymer of formula (I) has the structure of formula (Id), (Ie), or (If).

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[0016] In some embodiments of the polymer of formula (I) or any of its partial structures, j is an integer from about 50 to 1000, about 100 to about 800, about 200 to about 700, about 300 to about 600, or about 400 to about 500. In some embodiments, the polymer has an average molecular weight of about 5 kDa to about 100 kDa, about 10 kDa to about 75 kDa, about 12.5 kDa to about 50 kDa, or about 15 kDa to about 30 kDa. In some specific embodiments, the polymer has a molecular weight of about 20 kDa.

[0017] Another aspect of the present disclosure relates to a polymer for liquid-phase oligonucleotide synthesis having the structure of formula (II).

Chemical formula

[0018] In some embodiments of the polymer of formula (II), s + t is 4. In some specific embodiments, s is 2 and t is 2. In some embodiments, the polymer of formula (II) has the structure of (IIa). [Chemical formula]

[0019] In some embodiments, A is a carbon atom or phenylene. In some specific embodiments, A is a carbon atom.

[0020] In some other embodiments of the polymer of formula (II), A is C6-C 12 alkylene. In such some embodiments, s + t is 8. In some specific embodiments, each of s and t is 4. In other embodiments, s is 5 and t is 3, or s is 3 and t is 5. In some embodiments, the polymer of formula (II) has the structure of formula (IIb). [Chemical formula]

[0021] In some embodiments of the polymers of formula (II) (including formulas (IIa) and (IIb)), each q 1 is, independently, from about 20 to about 150, from about 40 to about 100, or from about 50 to about 75. In some embodiments, each q 2 is, independently, from about 30 to about 150, from about 40 to about 100, or from about 50 to about 75. In some embodiments, the average molecular weight of the polymer is from about 2 kDa to about 60 kDa, from about 5 kDa to about 50 kDa, from about 10 kDa to about 50 kDa, from about 15 kDa to about 30 kDa, or about 20 kDa.

[0022] Another aspect of the present disclosure is a method for preparing an oligonucleotide by liquid-phase oligonucleotide synthesis, comprising: dissolving a polymer described herein in a first solvent to form a reaction matrix; reacting the polymer with one or more nucleoside analogs to form a first bioconjugate comprising a structure of formula (III)

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[0023] In some embodiments of the methods described herein, the structure of formula (III) is also represented by formula (IIIa).

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[0024] In some embodiments, B 1 is independently, optionally protected adenine, optionally protected deazaadenine, optionally protected cytosine, optionally protected guanine, optionally protected deazaguanine, optionally protected thymine or optionally protected uracil. In some embodiments, B 1 is

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[0025] In some embodiments of the methods described herein, the polymer has an average molecular weight of about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

[0026] In some embodiments, the methods described herein include a 5'-hydroxy blocking group (G 1Removing [[ID=]] to form a first bioconjugate with an unblocked 5'-end, and isolating the first bioconjugate with an unblocked 5'-end. In some embodiments, the isolation of the first bioconjugate with an unblocked 5'-end is achieved by precipitation, dialysis, or filtration. In some specific embodiments, the isolation of the first bioconjugate with an unblocked 5'-end is achieved by precipitation. In some such specific embodiments, the precipitation is achieved in diethyl ether. In some other such specific embodiments, the precipitation is achieved in isopropanol.

[0027] In some embodiments, the method described herein (a) reacting a first bioconjugate with an unblocked 5'-end with one or more nucleoside phosphoramidite analogs in a second solvent to form a second bioconjugate having the structure of formula (IV); [Chemical formula] [wherein, G 2 is a 5'-hydroxy blocking group, B 2 is a nitrogenous base, R e is a phosphite protecting group], (b) oxidizing the phosphite moiety of formula (IV); and (c) removing the 5'-blocking group G 2 to form a second bioconjugate with an unblocked 5'-end comprising the structure of formula (IV'); [Chemical formula] [wherein Z is O or S], (d) isolating the second bioconjugate with an unblocked 5'-end. and further comprises.

[0028] In some embodiments, the structure of formula (IV) is also represented by (IVa), and formula (IV') is also represented by formula (IV'a).

Chemical formula

[0029] In some embodiments of the methods described herein, the methods described herein further comprise, prior to step (b), blocking the unreacted 5'-hydroxy group of the first bioconjugate with an unblocked 5'. In some embodiments, B 2 is independently, optionally protected adenine, optionally protected deazaadenine, optionally protected cytosine, optionally protected guanine, optionally protected deazaguanine, optionally protected thymine, or optionally protected uracil. In some embodiments, B 2 is

Chemical formula

[0030] In some embodiments, the isolation of the second bioconjugate with an unblocked 5' end is achieved by precipitation, filtration, or dialysis. In some preferred embodiments, the isolation of the second bioconjugate with an unblocked 5' end is achieved by precipitation. In some preferred embodiments, the precipitation is carried out in diethyl ether. In other preferred embodiments, the precipitation is carried out in isopropanol.

[0031] In some embodiments of the methods described herein, steps (a)-(d) are repeated in multiple cycles until oligonucleotides of one or more desired lengths are synthesized. In some embodiments, steps (a)-(d) of the methods described herein are repeated at least about 10 cycles. In some embodiments, the methods described herein further comprise the step of removing oligonucleotides from the polymer.

[0032] In some embodiments, the first solvent and the second solvent comprise one or more aprotic polar solvents, or combinations thereof. In some embodiments, the one or more aprotic polar solvents comprise acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or combinations thereof. In some particular embodiments, the one or more aprotic polar solvents are acetonitrile.

[0033] A further aspect of the disclosure relates to oligonucleotides prepared by the methods described herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0049] Detailed Description Solid-phase oligonucleotide synthesis enables oligonucleotide synthesis at the solid support-liquid interface. The solid support is insoluble in a liquid medium (e.g., an organic solvent). Examples of solid supports include controlled pore glass (CPG) and particles of porous cross-linked polystyrene. In contrast, liquid-phase oligonucleotide synthesis (LPOS) relies on a soluble organic compound as a support (hub) to perform oligonucleotide synthesis in solution. Conventional LPOS typically utilizes a soluble support that conjugates to the oligonucleotide and has one or several functional groups as an anchor for synthesizing the oligonucleotide. Embodiments of the present disclosure relate to methods for liquid-phase oligonucleotide synthesis by using a soluble polymer having a plurality of functional groups as an anchor for oligonucleotide synthesis. For example, the polymers described herein can contain, for example, reactive amino groups that enable efficient conjugation with nucleosides or nucleotide analogs with improved yields compared to known liquid-phase and solid-phase oligonucleotide synthesis. The polymers described herein contain one or more polyethylene groups, the length of which can be controlled to efficiently reduce the capture of impurities and non-specific adsorption to the polymer, thereby resulting in improved performance when using these polymers for liquid-phase oligonucleotide synthesis. The methods described herein are suitable for synthesizing oligonucleotides in multi-kilogram amounts, achieving good loading capacity and oligonucleotide yields. Definitions

[0050] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications referred to herein are incorporated by reference in their entirety unless otherwise noted. In the case of multiple definitions for terms herein, the definitions in this section shall control unless otherwise noted. The singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. Unless otherwise specified, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used. The use of "or" or "and" means "and / or" unless otherwise stated. Further, the terms "including" and other forms such as "include", "includes", and "included" are not limiting. As used herein, whether in the transitional phrase of a claim or in the body thereof, the terms "comprise" and "comprising" are to be interpreted in an open-ended sense. That is, these terms are to be construed as synonyms of the phrase "at least having" or "at least including". When used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. "Consisting of" means including and limited to whatever follows the phrase "consisting of". "Consisting essentially of" means including any elements recited after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements.

[0052] As used herein, the term "average molecular weight" is the weight average molecular weight (Mw) of a population of samples made from polymer species having molecular weight diversity. This quantity is given by the equation [Number] defined by, where n i is the number of molecules of species i and M i is the molecular weight of the i-th species. As used herein, the term "molecular weight" refers to the weight average molecular weight unless otherwise specified.

[0053] As used herein, the term "polymer", used herein in its conventional meaning, is a large molecule composed of smaller monomer or oligomer subunits that are linked together by covalent bonds to form chains. A "homopolymer" is a polymer made from only the repeating units of one type of monomer. A "copolymer" refers to a polymer made from the repeating units of two or more types of monomers. A linear polymer is composed of monomer subunits that are linked together in a continuous length to form a polymer chain. A branched polymer is similar to a linear polymer but has side chains that project from various branching points along the main polymer. A star polymer is similar to a branched polymer except that multiple branches radiate laterally from a single branching site, giving it the appearance of a star or a wheel and spokes.

[0054] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that includes a fully saturated (no double or triple bonds) hydrocarbon group. An alkyl group can have from 1 to 20 carbon atoms (whenever it appears herein, a numerical range, such as "1 to 20", refers to each integer within the given range; for example, "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but the definition of the present invention also encompasses occurrences of the term "alkyl" where no numerical range is specified). The alkyl group can also be a medium-sized alkyl having 1 to 10 carbon atoms. The alkyl group can also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of a compound can be designated as "C1-C4 alkyl" or a similar nomenclature. By way of mere example, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight or branched), and hexyl (straight or branched). The alkyl group can be substituted or unsubstituted.

[0055] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can have 2 to 20 carbon atoms. By way of example only, "C2-C6 alkenyl" indicates that there are 2 to 6 carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl. An alkenyl group can be substituted or unsubstituted.

[0056] As used herein, "alkynyl" refers to a straight-chain or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group can have 2 to 20 carbon atoms. By way of example only, "C2-C4 alkynyl" indicates that there are 2 to 6 carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. An alkynyl group can be substituted or unsubstituted.

[0057] As used herein, "cycloalkyl" refers to a completely saturated (no double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, these rings may be connected together in a fused, bridged or spiro fashion. As used herein, the term "fused" refers to two rings sharing two atoms and one bond. As used herein, the term "bridged cycloalkyl" refers to a compound in which the cycloalkyl contains one or more linkages of atoms that connect non-adjacent atoms. As used herein, the term "spiro" refers to two rings sharing one atom and not being connected by a bridge. A cycloalkyl group can contain 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s), or 3 to 6 atoms in the ring(s). A cycloalkyl group can be unsubstituted or substituted. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Examples of bicyclic fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl and tetradecahydroanthracenyl, examples of bicyclic bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, adamantanyl and norbornyl, and examples of bicyclic spirocycloalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.

[0058] As used herein, "carbocyclic" refers to a non-aromatic monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings may be linked together in a fused, bridged or spiro fashion as described herein. The carbocyclic group can contain 3 to 30 atoms within the ring(s), 3 to 20 atoms within the ring(s), 3 to 10 atoms within the ring(s), 3 to 8 atoms within the ring(s) or 3 to 6 atoms within the ring(s). The carbocyclic group can be unsubstituted or substituted. Examples of carbocyclic groups include cycloalkyl groups as defined herein, and the non-aromatic portions of 1,2,3,4-tetrahydronaphthalene, 2,3-dihydro-1H-indene, 5,6,7,8-tetrahydroquinoline and 6,7-dihydro-5H-cyclopenta[b]pyridine, but are not limited thereto.

[0059] As used herein, "aryl" refers to a carbocyclic (all-carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems in which two carbocyclic rings share a chemical bond) having a π-electron system that is completely delocalized throughout all of the rings. The number of carbon atoms in the aryl group can vary. For example, the aryl group can be a C6 aryl group, or a C 10 aryl group. Examples of aryl groups include benzene and naphthalene, but are not limited thereto. The aryl group can be substituted or unsubstituted.

[0060] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system (a ring system having a fully delocalized π - electron system) containing one or more heteroatoms (e.g., 1, 2, or 3 heteroatoms), i.e., elements other than carbon including, but not limited to, nitrogen, oxygen, and sulfur. The number of atoms in the ring(s) of the heteroaryl group can vary. For example, the heteroaryl group can contain 5 - 10 atoms in the ring(s), 6 - 10 atoms in the ring(s), or 5 - 6 atoms in the ring(s), such as 9 carbon atoms and 1 heteroatom, 8 carbon atoms and 2 heteroatoms, 7 carbon atoms and 3 heteroatoms, 8 carbon atoms and 1 heteroatom, 7 carbon atoms and 2 heteroatoms, 6 carbon atoms and 3 heteroatoms, 5 carbon atoms and 4 heteroatoms, 5 carbon atoms and 1 heteroatom, 4 carbon atoms and 2 heteroatoms, 3 carbon atoms and 3 heteroatoms, 4 carbon atoms and 1 heteroatom, 3 carbon atoms and 2 heteroatoms, or 2 carbon atoms and 3 heteroatoms. Further, the term "heteroaryl" includes fused ring systems in which two rings, e.g., at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3 - oxadiazole, 1,2,4 - oxadiazole, thiazole, 1,2,3 - thiadiazole, 1,2,4 - thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. The heteroaryl group can be substituted or unsubstituted.

[0061] As used herein, "heterocyclyl" refers to 3-, 4-, 5-, 6-, 7-, 8-, 9-, and 10-membered monocyclic, bicyclic, and tricyclic ring systems in which carbon atoms together with 1 to 5 heteroatoms form a ring system. However, the heterocycle may optionally contain one or more unsaturated bonds positioned in such a way that a completely delocalized π - electron system does not occur through all of the rings (i.e., the heterocyclyl group is not aromatic). The heteroatom(s), which are not limited, are elements other than carbon, including oxygen, sulfur, and nitrogen. The heterocycle can further contain one or more carbonyl functional groups such that its definition includes oxo - containing systems, such as lactams, lactones, and cyclic carbamates. When composed of two or more rings, these rings may be connected together in a fused, bridged, or spiro fashion. As used herein, the term "fused" refers to two rings sharing two atoms and one bond. As used herein, the term "bridged heterocyclyl" refers to a compound in which the heterocyclyl contains a connection of one or more atoms that link non - adjacent atoms. As used herein, the term "spiro" refers to two rings sharing one atom and not being connected by a bridge. The heterocyclyl group can contain 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s), 3 to 6 atoms in the ring(s), or 5 to 6 atoms in the ring(s). For example, 5 carbon atoms and 1 heteroatom, 4 carbon atoms and 2 heteroatoms, 3 carbon atoms and 3 heteroatoms, 4 carbon atoms and 1 heteroatom, 3 carbon atoms and 2 heteroatoms, 2 carbon atoms and 3 heteroatoms, 1 carbon atom and 4 heteroatoms, 3 carbon atoms and 1 heteroatom, or 2 carbon atoms and 1 heteroatom. In addition, any nitrogen in the heterocyclyl group can be quaternized. The heterocyclyl group can be linked to the remainder of the molecule through a carbon atom of the heterocyclyl group (C - linked), or by a heteroatom of the heterocyclyl group, such as a nitrogen atom (N - linked). The heterocyclyl group can be unsubstituted or substituted.Examples of such "heterocyclyl" groups include, but are not limited to, aziridine, oxirane, thiirane, azetidine, oxetane, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, azepane, pyrrolidone, pyrrolidinone, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline and / or 3,4-methylenedioxyphenyl). Examples of spiroheterocyclyl groups include 2-azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-oxaspiro[3.4]octane and 2-azaspiro[3.4]octane.

[0062] As used herein, "alkylene" refers to a branched or straight-chain, fully saturated diradical chemical group containing only carbon and hydrogen that is attached to the remainder of the molecule through two attachment points. By way of example only, "C1-C 10"Alkylene" indicates that there are 1 to 10 carbon atoms in the alkylene chain. Non-limiting examples include ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), and pentylene (-CH2CH2CH2CH2CH2-).

[0063] As used herein, "alkenylene" refers to a linear or branched diradical chemical group that contains only carbon and hydrogen, contains at least one carbon-carbon double bond, and is bonded to the remainder of the molecule through two attachment points. The alkenylene group may be designated as "C2~C 10 alkenylene" or a similar nomenclature. By way of example only, "C2~C 10 alkenylene" indicates that there are 2 to 10 carbon atoms in the alkenylene chain.

[0064] As used herein, "alkynylene" refers to a linear or branched diradical chemical group that contains only carbon and hydrogen, contains at least one carbon-carbon triple bond, and is bonded to the remainder of the molecule through two attachment points. The alkynylene group may be designated as "C2~C 10 alkenylene" or a similar nomenclature. By way of example only, "C2~C 10 alkynylene" indicates that there are 2 to 10 carbon atoms in the alkynylene chain.

[0065] As used herein, "heteroalkylene" refers to an alkylene group as defined herein (i.e., an alkylene group in which one or more carbon atoms are replaced by a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom) that contains one or more heteroatoms in its carbon backbone. For example, -CH2- may be replaced by -O-, -S-, or -NH-, or -CH(R)- may be replaced by -N(R)-. Heteroalkylene groups include, but are not limited to, ether, thioether, amino-alkylene, and alkylene-amino-alkylene moieties. In some embodiments, heteroalkylene may include 1, 2, 3, 4, or 5 -CH2CH2O- units. Alternatively and / or additionally, one or more carbon atoms (e.g., -CH2-) may also be replaced by oxo (=O) to form carbonyl -C(=O)-, or by (=S) to form thiocarbonyl -C(=S)-.

[0066] As used herein, "aralkyl" and "(aryl)alkyl" refer to an aryl group as defined above that is linked as a substituent via the aforementioned alkylene group. The alkylene and aryl groups of aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl. In some embodiments, the alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0067] As used herein, "heteroalkyl" and "(heteroaryl)alkyl" refer to a heteroaryl group as defined above that is linked as a substituent via an alkylene group as defined above. The alkylene and heteroaryl groups of heteroalkyl can be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and their benzo-fused analogs. In some embodiments, the alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0068] As used herein, "(heterocyclyl)alkyl" refers to a heterocyclic or heterocyclyl group as defined above that is linked as a substituent via an alkylene group as defined above. The alkylene and heterocyclyl groups of (heterocyclyl)alkyl can be substituted or unsubstituted. Examples include, but are not limited to, (tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and (1,3-thiazinan-4-yl)methyl. In some embodiments, the alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0069] As used herein, "cycloalkylalkyl" and "(cycloalkyl)alkyl" refer to a cycloalkyl group (as defined herein) that is linked as a substituent via an alkylene group. Examples include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, and cyclohexylpropyl. In some embodiments, the alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0070] As used herein, "alkoxy" refers to the formula -OR, where R is alkyl as defined above, and includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, etc. "C 1~9 alkoxy", etc.

[0071] As used herein, "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen (e.g., mono-haloalkyl, di-haloalkyl, and tri-haloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl and 1-chloro-2-fluoromethyl, 2-fluoroisobutyl. Haloalkyl can be substituted or unsubstituted.

[0072] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by halogen (e.g., mono-haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy and 1-chloro-2-fluoromethoxy, 2-fluoroisobutoxy. Haloalkoxy can be substituted or unsubstituted.

[0073] As used herein, "amino" refers to the -NH2 group. The term "monosubstituted amino group" as used herein refers to an amino (-NH2) group in which one hydrogen atom is replaced by a substituent. The term "disubstituted amino group" as used herein refers to an amino (-NH2) group in which each of the two hydrogen atoms is replaced by a substituent. The term "optionally substituted amino" as used herein refers to the -NR A R B group, where R A and R Bis, independently, hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, aralkyl, or heterocyclyl(alkyl) as defined herein.

[0074] As used herein, "alkylamino" or "(alkyl)amino" refers to an -NR A R B group, where R A and R B are hydrogen or alkyl as defined above, and at least one of R A and R B is alkyl. The alkyl portion of the (alkyl)amine includes, for example, a C1-C6 alkyl group.

[0075] As used herein, "aminoalkyl" or "(amino)alkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by an amino group or "-NR A R B " group as defined herein. The alkyl portion of aminoalkyl includes, for example, C1-C6 alkyl.

[0076] The term "halogen atom" or "halogen", as used herein, means any one of the atoms in Group 7 of the Periodic Table of the Elements that are stable to radiation, for example, fluorine, chlorine, bromine, and iodine.

[0077] As used herein, "alkoxyalkyl" or "(alkoxy)alkyl" refers to an alkoxy group linked through an alkylene group, for example C2-C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, for example, -(CH2) 1~3 -OCH3.

[0078] As used herein, "-O-alkoxyalkyl" or "-O-(alkoxy)alkyl" refers to an alkoxy group linked through an -O-(alkylene) group, for example -O-(C1-C6 alkoxy)C1-C6 alkyl, for example, -O-(CH2)1~3 Refers to -OCH3.

[0079] As used herein, "aryloxy" and "arylthio" refer to RO- and RS-, where R is aryl as defined above, such as phenyl, but not limited thereto. Aryloxy and arylthio can both be substituted or unsubstituted.

[0080] The "sulfenyl" group refers to the "-SR" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic(alkyl) as defined above. Sulfenyl can be substituted or unsubstituted.

[0081] The "sulfinyl" group refers to the "-S(=O)-R" group, where R can be the same as defined for sulfenyl. Sulfinyl can be substituted or unsubstituted.

[0082] The "sulfonyl" group refers to the "SO2R" group, where R can be the same as defined for sulfenyl. Sulfonyl can be substituted or unsubstituted.

[0083] The "O-carboxy" group refers to the "RC(=O)O-" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic(alkyl) as defined herein. O-carboxy can be substituted or unsubstituted.

[0084] The terms "ester" and "C-carboxy" refer to the "-C(=O)OR" group, where R can be the same as defined for O-carboxy. Ester or C-carboxy can be substituted or unsubstituted.

[0085] The "trihalomethanesulfonyl" group refers to the "X3CSO2-" group, where X is a halogen.

[0086] The "trihalomethanesulfonamide" group refers to the "X3CS(O)2N(R)-" group, where X is a halogen and R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic(alkyl) as defined herein.

[0087] The "mercapto" group refers to the "-SH" group.

[0088] The "S-sulfonamide" group refers to the "-SO2N(R A R B )" group, where R A and R B are independently hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic(alkyl) as defined herein. S-sulfonamide can be substituted or unsubstituted.

[0089] The "N-sulfonamide" group refers to the "RSO2N(R A )-" group, where R and R A are independently hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic(alkyl) as defined herein. N-sulfonamide can be substituted or unsubstituted.

[0090] The "O-carbamyl" group refers to the "-OC(=O)N(R A R B )" group, where R A and R B can be the same as those defined for S-sulfonamide. O-carbamyl can be substituted or unsubstituted.

[0091] The "N-carbamoyl" group refers to the "ROC(=O)N(R A )-" group, where R and R A can be the same as those defined for N-sulfonamides. N-carbamoyl can be substituted or unsubstituted.

[0092] The "O-thiocarbamoyl" group refers to the "-OC(=S)-N(R A R B )" group, where R A and R B can be the same as those defined for S-sulfonamides. O-thiocarbamoyl can be substituted or unsubstituted.

[0093] The "N-thiocarbamoyl" group refers to the "ROC(=S)N(R A )-" group, where R and R A can be the same as those defined for N-sulfonamides. N-thiocarbamoyl can be substituted or unsubstituted.

[0094] The "C-amide" group refers to the "-C(=O)N(R A R B )" group, where R A and R B can be the same as those defined for S-sulfonamides. C-amide can be substituted or unsubstituted.

[0095] The "N-amide" group refers to the "RC(=O)N(R A )-" group, where R and R A can be the same as those defined for N-sulfonamides. N-amide can be substituted or unsubstituted.

[0096] When the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may contain one or more of the same or different halogens.

[0097] In any of the compounds described herein having one or more chiral centers, if the absolute stereochemistry is not explicitly indicated, each center is understood to be independently either in the R-configuration, the S-configuration, or a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, enantiomerically enriched, or a mixture of stereoisomers, including all diastereomeric forms and enantiomeric forms. Additionally, in any of the compounds described herein having one or more double bonds that can give rise to geometric isomers defined as E or Z, each double bond is understood to be independently either E or Z, or a mixture thereof. If desired, stereoisomers can be obtained by methods such as stereoselective synthesis and / or separation of stereoisomers by chiral chromatography columns. Similarly, it is understood that all tautomeric forms are intended to be included in any of the compounds described.

[0098] Whenever a substituent is shown as a diradical (i.e., having two attachment points to the remainder of the molecule), the substituent should be understood to be attached in either orientation of stereochemistry, unless otherwise specified. Thus, for example, substituents shown as -AE- or

Chem.

Chem.

Chem.

[0099] When the compounds disclosed in this specification have unsatisfied valences, it should be understood that these valences will be satisfied by hydrogen and / or deuterium.

[0100] The compounds described herein can be understood to be labeled isotopically or by other means including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels. Substitution with isotopes, such as deuterium, can result in certain therapeutic advantages arising from higher metabolic stability, such as an extended in vivo half-life or a reduced required dose. Each chemical element represented in a compound structure can include any isotope of said element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed as being present in the compound or may be so understood. At any position in a compound where a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen including, but not limited to, hydrogen-1 (protium), hydrogen-2 (deuterium), and hydrogen-3 (tritium). Accordingly, references to compounds herein include all potentially possible isotopic forms unless otherwise explicitly indicated by the context.

[0101] The methods and formulations described herein are understood to include the crystalline forms, amorphous phases, and / or pharmaceutically acceptable salts, solvates, hydrates, and conformational isomers of the compounds of the preferred embodiments, as well as the metabolites and active metabolites of these compounds having the same type of activity. Conformational isomers are structures that are stereoisomers. Conformational isomerism is a phenomenon of molecules where the structural formula is the same, but the conformation (conformational isomer) of the atoms around the rotating bond is different. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain a stoichiometric or non-stoichiometric amount of the solvent and can be formed with pharmaceutically acceptable solvents, such as water, ethanol, etc., during the crystallization process. When the solvent is water, a hydrate is formed, or when the solvent is alcohol, an alcoholate is formed. Additionally, the compounds provided herein can exist in either unsolvated or solvated forms. Generally, for the purposes of the compounds and methods provided herein, solvated forms are considered equivalent to unsolvated forms. Other forms that can provide the compounds of the preferred embodiments include amorphous forms, micronized forms, and nanosized particulate forms.

[0102] Similarly, the compounds described herein, such as the compounds of the preferred embodiments, are understood to include any of the compounds in the forms described herein (e.g., pharmaceutically acceptable salts, crystalline forms, amorphous forms, solvated forms, enantiomeric forms, tautomeric forms, etc.).

[0103] As used herein, the abbreviations for any protecting groups, amino acids, and other compounds follow their general usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 11:942 - 944 (1972)) unless otherwise specified.

[0104] The term "protecting group" (singular or plural), as used herein, refers to any atom or group of atoms that is added to a molecule to prevent an existing group in the molecule from undergoing an unwanted chemical reaction. Examples of protecting group moieties are described in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999 and J.F.W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, which are hereby incorporated by reference herein for the limited purpose of disclosing suitable protecting groups. The protecting group moiety can be selected such that it is stable to certain reaction conditions and can be readily removed at a convenient stage using methods known in the art.Non-limiting lists of protecting groups include benzyl (Bn), substituted benzyl, alkylcarbonyl (e.g., t-butoxycarbonyl (BOC), acetyl (i.e., -C(=O)CH3 or Ac), or isobutyryl (iBu)), arylalkylcarbonyl (e.g., benzyloxycarbonyl or benzoyl (i.e., -C(=O)Ph or Bz)), substituted methyl ether (e.g., methoxymethyl ether (MOM)), substituted ethyl ether (e.g., methoxyethyl ether (MOE), substituted benzyl ether, tetrahydropyranyl ether, silyl ether (e.g., trimethylsilyl (TMS), triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), or t-butyldiphenylsilyl), ester (e.g., benzoic acid ester), carbonate (e.g., methoxymethyl carbonate), sulfonate (e.g., tosylate or mesylate), acyclic ketal (e.g., dimethyl acetal), cyclic ketal (e.g., 1,3-dioxane or 1,3-dioxolane), acyclic acetal, cyclic acetal, acyclic hemiacetal, cyclic hemiacetal, cyclic dithioketal (e.g., 1,3-dithiane or 1,3-dithiolane), and triarylmethyl groups (e.g., trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), or 4,4',4''-trimethoxytrityl (TMTr)).

[0105] As used herein, the term "leaving group" refers to any atom or moiety that can be replaced by another atom or moiety in a chemical reaction. More specifically, in some embodiments, a "leaving group" refers to an atom or moiety that is replaced in a nucleophilic substitution reaction. In some embodiments, a "leaving group" is any atom or moiety that is the conjugate base of a strong acid. Examples of suitable leaving groups include, but are not limited to, tosylate and halogen. Non-limiting characteristics and examples of leaving groups can be found, for example, in Organic Chemistry, 2d ed., Francis Carey (1992), pages 328-331, Introduction to Organic Chemistry, 2d ed., Andrew Streitwieser and Clayton Heathcock (1981), pages 169-171, and Organic Chemistry, 5 th ed., John McMurry (2000), pages 398 and 408, all of which are hereby incorporated by reference herein for the limited purpose of disclosing characteristics and examples of leaving groups.

[0106] The term "pharmaceutically acceptable salt", as used herein, is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art (and should not be limited to a special or specialized meaning), and refers, without limitation, to salts of a compound that do not cause significant irritation to the organism to which they are administered and do not inactivate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutically acceptable salts can be obtained by reacting the compound with an inorganic acid, such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid. Pharmaceutically acceptable salts can also be obtained by reacting the compound with an organic acid, such as an aliphatic or aromatic carboxylic acid or sulfonic acid, such as formic acid, acetic acid (AcOH), propionic acid, glycolic acid, pyruvic acid, malonic acid, maleic acid, fumaric acid, trifluoroacetic acid (TFA), benzoic acid, cinnamic acid, mandelic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, or naphthalenesulfonic acid. Pharmaceutically acceptable salts can also be obtained by reacting the compound with a base to form a salt, such as an ammonium salt, an alkali metal salt, such as a lithium, sodium or potassium salt, an alkaline earth metal salt, such as a calcium, magnesium or aluminum salt, an organic base, such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, (C1-C7 alkyl)amine, cyclohexylamine, dicyclohexylamine, triethanolamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine salts, as well as salts with amino acids, such as arginine and lysine, or inorganic bases, such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0107] As used herein, "nucleotide" includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, while in DNA it is deoxyribose, a sugar lacking the hydroxy group present in ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as their modified derivatives or analogs, such as deazapurine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine.

[0108] As used herein, "nucleoside" is structurally similar to a nucleotide but lacks the phosphate moiety. Examples of nucleoside analogs are those in which a label is linked to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" is used herein in its ordinary meaning as understood by one of ordinary skill in the art. By way of example, but not limitation, ribonucleosides containing a ribose moiety and deoxyribonucleosides containing a deoxyribose moiety are included. Modified pentose moieties are pentose moieties in which an oxygen atom is replaced by carbon and / or carbon is replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that can have a substituted base and / or sugar moiety. In addition, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0109] The term "purine base" is used herein in its ordinary meaning as understood by one of ordinary skill in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary meaning as understood by one of ordinary skill in the art and includes its tautomers. A non-limiting list of purine bases optionally substituted includes purine, deazapurine, 7-deazapurine, adenine, 7-deazaadenine, guanine, 7-deazaguanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0110] As used herein, the terms "derivative" or "analog" mean synthetic nucleoside or nucleotide derivatives having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are described, for example, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also include modified phosphodiester linkages including phosphorothioates, phosphorodithioates, alkyl-phosphonates, phosphororanilidates, phosphoramidites, and phosphoramidate linkages. The terms "derivative" and "analog" can be used interchangeably as used herein and are encompassed by the terms "nucleotide" and "nucleoside" as defined herein.

[0111] As used herein, the term "phosphate" is used in its ordinary meaning as understood by one of ordinary skill in the art and includes its protonated form (e.g.,

Chemical formula

[0112] As used herein, "loading capacity" or "loading" is expressed in mmol or μmol of nucleoside bound to the polymer described herein per gram of polymer (i.e., mmol / g). Polymers for liquid-phase oligonucleotide synthesis

[0113] Some aspects of the present application relate to polymers for liquid-phase synthesis. In some embodiments, liquid-phase synthesis includes liquid-phase oligonucleotide synthesis, liquid-phase peptide synthesis, liquid-phase polynucleotide (i.e., nucleic acid) synthesis, or liquid-phase small molecule synthesis. In some embodiments, the polymer includes or is a polymer for liquid-phase oligonucleotide synthesis. The polymer may include poly(ethylene glycol) (PEG) pendant arms having reactive groups that enable reaction with nucleosides or nucleotide analogs, including, but not limited to, amine, alcohol, azide, or alkyne groups, or combinations thereof. The average molecular weight of the polymer can be controlled by controlling the length of the pendant PEG arms. The length of the PEG arms can be adjusted in a manner that reduces the capture of impurities and enables improved performance of these structures for liquid-phase oligonucleotide synthesis. Polymers of formula (I) or (I')[

[0114] Some embodiments of the present disclosure relate to polymers for liquid-phase oligonucleotide synthesis having the structure of formula (I). [Chem.] [wherein, R is H, or unsubstituted or substituted C1-C6 alkyl, W is C1-C 20 alkylene, 2- to 20-membered heteroalkylene, or a bond, Q is

Chemical formula

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Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0115] In some embodiments of the polymer of formula (I), R is unsubstituted C1-C6 alkyl (e.g., -CH3). In other embodiments, R is substituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl independently substituted with an azide or alkyne group). In some other embodiments, R is H. In some embodiments, W is -CH2CH2NH-. In other embodiments, W is -CH2CH2O-. In some embodiments, W is C2-C6 alkylene, e.g., -CH2CH2-. In another embodiment, W is a bond.

[0116] In some embodiments of the polymers described herein, the polymer of formula (I) can also be represented by the structure of formula (I’) [Chemical formula] [wherein, R is C1-C6 alkyl, W is -NH- or -O-, Q is [Chemical formula] and L 1 is a C1-C 10 alkylene linker or a 2- to 10-membered heteroalkylene linker, R 1 and R 2 each independently is -OR 3 or -NR 4a R 4b and R 3 is H, C1-C6 alkyl, a hydroxy protecting group, or [Chemical formula] and R 4a and R 4b each independently is H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group, or [Chemical formula] or R 4a and R 4b together form a divalent amino protecting group, L 2a and L 2b each independently is a C1-C 10 alkylene linker or a 2- to 10-membered heteroalkylene linker, R 5a R 5b R 6a and R 6b each independently is hydrogen, -OR 7 or -NR 8a R 8b and R 7 Each of them is independently H, C1-C6 alkyl, a hydroxy protecting group, or

Chem.

Chem.

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

[0117] In some embodiments of the polymer of formula (I) or (I’), L 1 is C2-C6 alkylene. In some embodiments of the polymer of formula (I), L 1 is 3- to 12-membered, 3- to 8-membered, 3- to 6-membered, or 4- to 6-membered heteroalkylene containing 1, 2, or 3 heteroatoms selected from N, O, S, C(=O), or C(=S). In further embodiments, the heteroalkylene contains 1 or 2 nitrogen atoms.

[0118] In further embodiments of the polymer of formula (I) or (I’), Q is

Chemical formula

Chemical formula

[0119] In a further embodiment of a part of the polymer of formula (I) or (I'), Q is, [Chem.] [Chem.] selected from the group consisting of. In such some embodiments, each R 3 is independently H or a hydroxy protecting group. In some other embodiments, each R 3 is, [Chem.] is. In still other embodiments, one of R 3 is H and the other R 3 is, [Chem.] is. In some further embodiments, m2 is 1. In other embodiments, m2 is 0.

[0120] In some embodiments of the polymer of formula (I) or (I’), L 2a and L 2b each is, independently, a C1-C 10 alkylene linker or a 2- to 10-membered heteroalkylene linker. In some other embodiments, L 2a and L 2b each is, independently, a 3- to 12-membered, 3- to 8-membered, 3- to 6-membered, or 4- to 6-membered heteroalkylene containing 1, 2, or 3 heteroatoms selected from N, O, S, C(=O), or C(=S). In further embodiments, the heteroalkylene contains 1 or 2 nitrogen atoms. In other embodiments of the polymer of formula (I), L 2a and L 2b each is, independently, phenylene optionally substituted, or a C2-C6 alkylene or 3- to 12-membered heteroalkylene replaced by phenylene in which one methylene unit is optionally substituted.

[0121] In some embodiments of the polymer of formula (I), R 5a , R 5b , R 6a and R 6b each is, independently, hydrogen, -OH, protected hydroxy, -NH2, -NH(C1-C6 alkyl optionally substituted), -NHC(=O)CH3, or protected amino. In such some embodiments, at least one of R 5a , R 5b , R 6a and R 6b is H. In some embodiments of the polymer of formula (I’), R 5a , R 5b , R 6a and R 6b each is, independently, -OH, protected hydroxy, -NH2, -NH(C1-C6 alkyl optionally substituted), -NHC(=O)CH3, or protected amino (e.g., -NHAc). In some embodiments of the polymer of formula (I) or (I’), R 5a , R 5b , R 6aand R 6b Each of them is hydroxy or protected hydroxy. In other embodiments, R 5a , R 5b , R 6a and R 6b Each of them is -NH2, -NH(optionally substituted C1-C6 alkyl), or protected amino (e.g., -NHAc). In other embodiments of the polymer of formula (I), at least one of R 7 is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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Chemical formula

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[0122] In some embodiments, the polymer of formula (I) or (I') has the structure of formula (Ia).

Chemical formula

[0123] In some embodiments, the polymer of formula (I) or (I') has the structure of formula (Ia').

Chemical formula

[0124] In some other embodiments, the polymer of formula (I) or (I') has the structure of formula (Ib), (Ib-1), (Ic) or (Ic-1).

Chemical formula

[0125] In some embodiments, the polymer of formula (I) has the structure

Chemical formula

[0126] In some embodiments, the polymer of formula (I) has the structure

Chemical formula

[0127] In such some embodiments, L 2b is C2-C6 alkylene. In other embodiments, L 2b is a 3- to 12-membered or 4- to 8-membered heteroalkylene containing one or more oxygen or nitrogen atoms. In other embodiments, L 2b is a phenylene optionally substituted. In other embodiments, L 2b is C2-C6 alkylene, wherein one methylene unit is replaced by a ring structure (e.g., a phenylene optionally substituted) as described herein. In still other embodiments, L 2b is a 3- to 12-membered or 4- to 8-membered heteroalkylene containing one or more oxygen or nitrogen atoms, wherein the methylene units are replaced by a ring structure (e.g., a phenylene optionally substituted) as described herein. In such some embodiments, each of R 5b and R 6b is independently -NH2 or -NHAc. In such other embodiments, each of R 5b and R 6b is -OH or protected hydroxy. In further embodiments, at least one of R 5b and R 6b is -OR 7 , and R 7 is

Chemical formula

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Chemical formula

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Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0128] In some embodiments of the polymer of formula (I), (I'), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il), it is about 50 to about 1000, about 200 to about 800, or about 300 to about 600. In some embodiments, j is about 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, or within a range defined by any of the foregoing values. In some embodiments, the polymer of formula (I), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il) has an average molecular weight of about 2 kDa to about 60 kDa. For example, the polymer of formula (I), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il) can have an average molecular weight of about 2 kDa, 5 kDa, 10 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 32 kDa, 34 kDa, 36 kDa, 38 kDa, 40 kDa, 42 kDa, 44 kDa, 46 kDa, 48 kDa, 50 kDa, 52 kDa, 54 kDa, 58 kDa, or 60 kDa, or within a range defined by any of the foregoing values.For example, the polymers of formula (I), (Ia), (Ia’), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il) have an average molecular weight of about 2 to about 60 kDa, about 5 to about 50 kDa, about 5 to about 30 kDa, about 10 to about 40 kDa, about 15 to 30 kDa, or about 15 to 25 kDa. The polymer of formula (II)

[0129] In some embodiments of the polymers described herein, the polymer is a compound having the structure of formula (II)

Chemical formula

Chemical formula

[0130] In some embodiments of the polymer of formula (II), s + t is 4. In other embodiments, s + t is 8. In some embodiments, s + t is 5, 6, or 7. In some specific embodiments, s is 2 and t is 2, or one of s and t is 1 and the other of s and t is 3. In certain other specific embodiments, s is 4 and t is 4. In still certain other specific embodiments, one of s and t is 5 and the other of s and t is 3. In one embodiment, s is 5 and t is 3. In another embodiment, s is 3 and t is 1. In another embodiment, s is 3 and t is 3. In yet another embodiment, s is 3 and t is 5. In a further embodiment, s is 2 and t is 4, or s is 4 and t is 2. In still further embodiments, s is 2 and t is 5, or s is 5 and t is 2, or s is 3 and t is 4, or s is 4 and t is 3.

[0131] In some embodiments, the polymer of formula (II) has the structure of formula (IIa).

Chemical formula

Chemical formula

[0132] In some embodiments, the polymer of formula (II) has the structure of formula (IIa’).

Chemical formula

Chemical formula

[0133] Alternative embodiments of the polymers of formula (IIa'), (IIa'-1) and (IIa'-2) can include those having one or three free amino groups and three or one protected amino group (e.g., acetyl or Ac group). In addition to the Ac group, other common amino protecting groups, such as Bz, can also be used. In some other embodiments, A is C2-C 20 alkylene, and the polymer of formula (II) has a structure of formula (IIb') having eight PEG pendant arms.

Chemical formula

[0134] In some embodiments of the polymers of formula (II), (IIa), (IIa’), (IIa-1), (IIa-2), (IIa’-1), (IIa’-2), (IIb), or (IIb’), the number q of polyethylene glycol units (PEG) 1 and q 2 can be controlled to adjust the properties of the polymer. In some embodiments, q 1 and q 2 each independently is an integer from 10 to 500. For example, in some embodiments, q 1 and q 2 each independently is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500, or can be within a range defined by any two of the foregoing values. For example, in some embodiments, each q 1 independently is from about 30 to about 150, from about 40 to about 100, or from about 50 to about 75. In some embodiments, each q 2 independently is from about 30 to about 150, from about 40 to about 100, or from about 50 to about 75. In some embodiments, q 1 and q 2 may have the same value. In other embodiments, q 1 and q 2 may have different values. In some embodiments, it is preferred to control the lengths of q 1 and q 2 such that the average molecular weight of the polymer is about 2 kDa to about 60 kDa, about 5 kDa to about 50 kDa, about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

[0135] In any embodiment of the polymers described herein, when the polymer contains two or more PEG pendant arms, at least one of the PEG pendant arms has a reactive end group (e.g., -NH2 or -OH) that can be used as an anchor for liquid phase synthesis. In a further embodiment, when the polymer contains two, three or more PEG pendant arms, at least one of the PEG pendant arms has a non-reactive end group (e.g., a protected amino or protected hydroxy group), and thus that pendant arm cannot be used as an anchor for liquid phase synthesis. Method for preparing oligonucleotides by liquid phase oligonucleotide synthesis (LPOS)

[0136] Another aspect of the present application relates to a method for making a compound by liquid phase synthesis. The compound can be an oligonucleotide, a peptide, a polynucleotide (i.e., a nucleic acid), or a small molecule. In certain embodiments, the method is a method for making an oligonucleotide by liquid phase oligonucleotide synthesis.

[0137] In some embodiments of the methods described herein, the method comprises dissolving a polymer described herein in a first solvent to form a reaction matrix, and contacting or otherwise reacting the polymer with one or more nucleoside analogs to form a first bioconjugate comprising the structure of formula (III) and [Chemistry] [wherein B 1 is a nitrogenous base, G 1 is a 5'-hydroxy blocking group, X is O or NR 20 and R a is -H, -OH, halogen, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), or -OY, where Y is a 2'-hydroxy protecting group, and L 5is a cleavable heteroalkylene linker, where one or more carbon atoms are replaced by O, S, N, C(=O) or C(=S)]. In some such embodiments, the nitrogenous base includes a purine base, a deazapurine base, or a pyrimidine base. In some embodiments, the structure of formula (III) is also represented by formula (IIIa) or (IIIb).

Chemical formula

[0138] In some embodiments, X is O. In other embodiments, X is NR 20 and R 20 is H. In some embodiments, the amide bond -NR 20 -C(=O)- can be formed from the terminal amino group of a polymer that reacts with a first nucleoside analog containing 3'-succinate (containing a free carboxy group). In other embodiments, the amide bond -NR 20 -C(=O)- can be formed from a linker attached to the 3'-position of the first nucleoside analog. Other alternative linkers can include hydroquinone -O,O'-diacetic acid (HQDA or Q-linker).

[0139] In some embodiments of the methods described herein, B 1 is independently adenine optionally protected, deazaguanine optionally protected, cytosine optionally protected, guanine optionally protected, deazaguanine optionally protected, thymine optionally protected or uracil optionally protected. In some embodiments, B 1 is

Chemical formula

[0140] In some embodiments, the polymer has an average molecular weight of about 2 kDa to about 1000 kDa, about 5 kDa to about 1000 kDa, or about 10 kDa to about 1000 kDa, or about 20 kDa to about 500 kDa, or about 30 kDa to about 100 kDa, or about 15 kDa to about 30 kDa. In some embodiments, the polymer has an average molecular weight of about 10 kDa to about 100 kDa. Without being bound by any particular theory, a polymer having an average molecular weight of about 20 kDa may provide an optimal balance of reaction yield and product purity.

[0141] In some embodiments of the methods described herein, the method further comprises removing the 5'-hydroxy blocking group (G 1 ) to form a first bioconjugate with an unblocked 5', and isolating the first bioconjugate with an unblocked 5'. In such some embodiments, the isolation of the first bioconjugate with an unblocked 5' is achieved by precipitation, dialysis, or filtration.

[0142] In some embodiments, isolation is achieved by precipitation. In some embodiments, precipitation of the first bioconjugate with an unblocked 5′ is effected by adding a solution containing the bioconjugate to a solvent comprising pentane, hexane, heptane, a dialkyl ether (e.g., diethyl ether, t-butyl methyl ether, etc.), toluene, isopropyl acetate, dichloromethane, dimethyl sulfoxide, ethyl acetate, an alkanol (e.g., methanol, ethanol, isopropanol) or alkenol, or combinations thereof. In some such embodiments, the solvent is diethyl ether. In other such embodiments, the solvent is isopropanol. In other embodiments, isolation is achieved by a filtration step. The filtration step can include dialysis, filtration, nanofiltration, ultrafiltration, or any known filtration technique suitable for use herein, and combinations thereof. In some embodiments, the filtration step includes dialysis or filtration. In further embodiments, the filtration step includes the use of a membrane. The membrane can include cellulose acetate, glass fiber, carbon-based polymers, regenerated cellulose, and combinations thereof. In certain embodiments, the regenerated cellulose has an electrostatic charge. In some embodiments, the regenerated cellulose membrane is negatively charged. In some embodiments, the regenerated cellulose has a structure

Chemical formula

[0143] In some embodiments of the methods described herein, the method (a) reacting a first bioconjugate with an unblocked 5' end in a second solvent with one or more nucleoside phosphoramidite analogs to form a second bioconjugate having the structure of formula (IV);

Chemical formula

Chemical formula

Chemical formula

[0144] In some embodiments of the methods described herein, B 2 is independently optionally protected adenine, optionally protected deazaadenine, optionally protected cytosine, optionally protected guanine, optionally protected deazaguanine, optionally protected thymine, or optionally protected uracil. In some embodiments, B 2 is

Chemical formula

[0145] In some embodiments of the methods described herein, the method further includes blocking the unreacted 5'-hydroxy group of the first bioconjugate that is not 5'-blocked prior to step (b). In such some embodiments, the blocking is effected by reacting the 5'-hydroxy group with acetic anhydride (Ac2O).

[0146] In some embodiments of the methods described herein, the isolation or purification of the second bioconjugate that is not 5'-blocked is achieved by precipitation, filtration, or dialysis. In some embodiments, the isolation is achieved by precipitation. In some embodiments, the precipitation of the second bioconjugate that is not 5'-blocked is effected by adding a solution containing the bioconjugate to a solvent comprising pentane, hexane, heptane, diethyl ether, t-butyl methyl ether, toluene, isopropyl acetate, dichloromethane, dimethyl sulfoxide, ethyl acetate, methanol, ethanol, isopropanol, or a combination thereof. In such some embodiments, the solvent is diethyl ether. In such other embodiments, the solvent is isopropanol. In other embodiments, the isolation / purification uses a regenerated cellulose membrane having a molecular weight cut-off (MWCO) of about 5 kDa to about 50 kDa, about 6 kDa to about 40 kDa, about 7 kDa to about 30 kDa, or about 8 kDa to about 12 kDa. In some further embodiments, steps (a)-(d) are repeated in multiple cycles until an oligonucleotide of the desired length is synthesized.

[0147] In some embodiments of the methods described herein, steps (a)-(d) are repeated for a plurality of cycles until oligonucleotides of one or more desired lengths are synthesized. In such some embodiments, steps (a)-(d) are repeated for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 cycles. In such some embodiments, the synthesized oligonucleotides can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases.

[0148] In some embodiments, the loading capacity of the polymers described herein is within the range defined by about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 μmol of nucleotides per gram of polymer, or any range defined by any two of the foregoing loading capacities.

[0149] In some embodiments of the methods described herein, the method further comprises the step of removing oligonucleotides from the polymer. In such some embodiments, the removing step comprises the step of cleaving a covalent chemical bond. In some embodiments, the removing step comprises hydrolysis. In certain embodiments, the removing step comprises hydrolysis at a temperature of about 0°C to about 80°C, or about 10°C to about 60°C, or about 15°C to about 30°C. In further embodiments, when the first nucleoside is covalently bound to the polymer by reaction of 3'-succinic acid that reacts with the amino group of the polymer, the amide bond formed between the first nucleoside and the polymer can be cleaved by hydrolysis.

[0150] In some embodiments of the methods described herein, each of the first solvent and the second solvent comprises one or more aprotic polar solvents, or combinations thereof. In some embodiments, the one or more aprotic polar solvents comprise acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or combinations thereof. In one embodiment, the first solvent and / or the second solvent comprises acetonitrile. In another embodiment, the first solvent and / or the second solvent comprises a mixture of acetonitrile and sulfolane.

[0151] Further embodiments of the present application relate to oligonucleotides prepared by any of the methods described herein.

Examples

[0152] Some aspects of the embodiments discussed above are disclosed in more detail in the following examples, which are in no way intended to limit the scope of the present disclosure. Those skilled in the art will recognize that many other embodiments are also included within the scope of the compositions, kits and methods of the present application as described above in this specification and the claims. General Procedure for the Measurement of DMT Loading

[0153] UV / Vis spectrophotometry is used to determine the DMT loading (μmol / g) of 5'-DMT protected nucleotides conjugated onto a polyvalent hub (PVH). The sample is dissolved in acetonitrile (AcN) containing p-toluenesulfonic acid (TSA). The acid cleaves the DMT protecting group and its loading is determined quantitatively. An Agilent 8453 UV-Visible spectrophotometer, Agilent UV-Visible ChemStation software Rev. A.10.0, and an Agilent UV-Visible rectangular cell 10 mm, 3.5 mL (P / N 5061-3387) are used for the measurement. · Prepare the master batch of the TSA solution in AcN by dissolving 8.0 g of TSA in 500 mL of HPLC-grade AcN. The resulting solution is stable for 4 weeks at ambient temperature. · Start the Agilent UV-Visible ChemStation software and select a fixed wavelength (multiple available) of 498 nm. · Obtain the blank spectrum using the TSA / AcN solution. · Accurately weigh 20.0 - 26.0 mg of the nucleotide conjugate polymer sample and transfer it to 100 mL of the TSA / AcN solution. · Vortex for 2 minutes and allow any insoluble material to sediment to the bottom in 10 - 15 minutes. · Transfer the supernatant to a dry cuvette (Agilent UV-Visible rectangular cell, 10 mm, 3.5 mL). · Scan the sample solution to obtain the absorbance value at 498 nm. Determine the DMT loading on the CPG support using the following equation.

Equation

Chemical Structure

[0154] A certain amount of 2 g of commercially available MeO-PEG-amine (MW = 20 kDa, Nanosoft Polymer Inc.), 178 mg of Fmoc-Lys(Fmoc)-OH (AAPPTec), 126 mg of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (AK Scientific) and 56 mg of diisopropylethylamine (TCI Chemicals) were dissolved in 10 mL of dry acetonitrile (Sigma Aldrich). The reaction mixture was stirred for 2 hours. Next, the mixture was added to diethyl ether under stirring to precipitate the PEG modified with Fmoc-Lys(Fmoc)-OH. To the dry precipitate, 20 mL of 20% piperidine in dimethylformamide (DMF) was added and stirred for 1 hour to remove the Fmoc group to obtain the polymer (1a), which was isolated by precipitation in diethyl ether to obtain 1.88 g of the polymer (1a) (yield: 93.5%). (Example 2) Synthesis of Polymer (1a)-DMT-dT-3’-succinate Conjugate [Chemical formula]

[0155] Polymer (1a) (1.88 g) was dissolved in 6 mL of dry acetonitrile. Separately, 298 mg of DMT-dT-3'-succinate TEA salt (Hongene Biotech), 168 mg of HBTU and 74.2 mg of diisopropylethylamine were dissolved in 5 mL of dry acetonitrile, and the resulting solution was allowed to stand at room temperature for 15 minutes. The polymer solution and the DMT-dT-3'-succinate solution were combined and stirred overnight at room temperature. The mixture was precipitated with diethyl ether to obtain the first dT conjugate with DMT. To the dried precipitate, 1.356 mL of trichloroacetic acid (TCA) (600 mg / mL in DCM) and 324 mg of triethylsilane (TES) were added with stirring. After approximately 10 minutes, the reaction mixture was precipitated with diethyl ether to obtain 2.04 g of polymer (1a)-dT conjugate (yield: 96%). (Example 3) Synthesis of Polymer (2)-DMT-dT-3'-succinate Conjugate

Chemical formula

[0156] Commercially available PEG(2g) with four pendants, where each pendant-type arm has an average molecular weight of approximately 5 kDa, was dissolved in 6 mL of anhydrous acetonitrile. In a separate container, 149 mg of DMT-dT-3'-succinate TEA salt, 168 mg of HBTU, and 74.2 mg of diisopropylethylamine were combined in 5 mL of anhydrous acetonitrile, mixed, and allowed to stand for 15 minutes. Next, the solution of PEG with four pendants and the solution of DMT-dT-3'-succinate were combined and stirred overnight at room temperature. The mixture was poured into diethyl ether with stirring to precipitate the first dT conjugate with DMT. The dried precipitate was added to 5 mL of a nucleotide capping solution (capping reagent A, Sigma Aldrich and 1-methylimidazole / tetrahydrofuran, applied biosystem in a 1:1 ratio), and the mixture was stirred for 1 hour. The reaction mixture was poured into diethyl ether with stirring to precipitate the polymer(2)-dT conjugate with 5'DMT. To the aforementioned precipitate, 1.356 mL of trichloroacetic acid (TCA) (600 mg / mL in DCM) and 324 mg of triethylsilane (TES) were added while continuously stirring over 10 minutes. The reaction mixture was poured into stirred diethyl ether and dried under reduced pressure to precipitate 2.03 g of the polymer(2)-dT conjugate (yield 98.5%). (Example 4) Direct conjugation of eight DMT-dT-3'-succinate units onto polymer(1a) [Chemical formula]

[0157] 1.02 g of a polymer(1a)-dT conjugate in which the polymer(1a) has an average molecular weight of approximately 20 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM in 4.76 mL of anhydrous acetonitrile, Sigma Aldrich), and 248 mg of DMT-dT phosphoramidite (Hongene Biotech) in a 100 mL round-bottom flask. After continuously magnetic stirring for 60 minutes, 158 mg of m-CPBA (3-chloroperbenzoic acid, Sigma Aldrich) was added as a powder. After continuously stirring for 10 minutes, 2.04 mL of trichloroacetic acid (TCA, 600 mg / mL in DCM) and 0.706 mL of TES were added. After continuously stirring for 10 minutes, the reaction mixture was poured into isopropanol while stirring. The precipitate was washed twice with diethyl ether to obtain 1.024 g of polymer(1a)-2dT (yield: 97.4%). The above procedure was repeated 6 more times to obtain polymer(1a)-8dT, which showed a complete product purity (FLP) of 82.9% after nucleotide cleavage (yield: 77.2%). The HPLC results are provided in Figure 1, showing the relative amounts of oligonucleotides after cleavage from the polymer support. (Example 5) Direct conjugation of eight DMT-dT-3'-succinate units onto polymer(2)

Chemical Structure

[0158] 2.03 g of polymer (2)-dT conjugate was dissolved in an ETT activator (250 mM in 9.52 mL of anhydrous acetonitrile), and 496 mg of DMT-dT phosphoramidite was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. After continuously magnetic stirring for 60 minutes, 317 mg of mCPBA was added as a powder. After continuously magnetic stirring for 10 minutes, 4.08 mL of TCA (600 mg / mL in DCM) and 1.417 mL of TES were added. After continuously magnetic stirring for 10 minutes, the reaction mixture was poured into isopropanol while stirring. The precipitate was washed twice with diethyl ether to obtain 1.93 g of polymer (2)-2dT (yield: 90.8%). The above procedure was repeated 6 more times to obtain polymer (2)-8dT with 81.6% FLP after nucleotide cleavage (yield: 82.9%). The HPLC results are provided in Figure 2, showing the relative amounts of oligonucleotides after cleavage from the polymer support. (Example 6) Complete product purity (FLP%) of mononucleotides and oligonucleotides

[0159] The full-length product purity (FLP) of dT after cleavage from the polymer support can be found in Table 1. As can be seen, both polymers (1a) and (2) show efficient nucleotide synthesis efficiency with a high FLP exceeding 80% as long as the nucleotide length is 8. However, since small impurities can be trapped during precipitation, FLP cannot be used, especially, to accurately calculate the coupling efficiency with the synthesized long dT sequences.

Table 1

Chemical formula

[0160] A polymer (1a)-dT conjugate (0.85 g) having an average molecular weight of approximately 20 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-dT phosphoramidite (Hongene Biotech) in a manner similar to that described in Example 4 to obtain polymer (1a)-2dT. This procedure was repeated 6 more times with other nucleotides to prepare the polymer (1a)-dT-dT-dA-dA-dG-dG-dC-dC-OH sequence, which showed a complete product purity (FLP) of 82% after nucleotide cleavage (yield: 88.9%). The HPLC results are provided in Figure 3 and show the relative amounts of oligonucleotides after cleavage from the polymer support. (Example 8) Direct conjugation of eight DMT-dT-3'-succinate units to polymer (1b) [Chemical formula]

[0161] A polymer (1b)-dT conjugate having an average molecular weight of approximately 10 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM in 4.76 mL of anhydrous acetonitrile, Sigma Aldrich), and 248 mg of DMT-dT phosphoramidite (Hongene Biotech) in a 100 mL round-bottom flask. After continuously magnetic stirring for 60 minutes, 158 mg of m-CPBA (3-chloroperbenzoic acid, Sigma Aldrich) was added as a powder. After continuously stirring for 10 minutes, 2.04 mL of trichloroacetic acid (TCA, 600 mg / mL in DCM) and 0.706 mL of TES were added. After continuously stirring for 10 minutes, the reaction mixture was poured into isopropanol while stirring. The precipitate was washed twice with diethyl ether to obtain 1.024 g of polymer (1b)-2dT. The above procedure was repeated 6 more times to obtain polymer (1b)-8dT, which showed a full-length product purity of 98.3% after nucleotide cleavage (yield: 92.8%). The HPLC results are provided in Figure 4, showing the relative amounts of oligonucleotides after cleavage from the polymer support. (Example 9) Direct conjugation of DMT-nucleotide-3'-succinate units to polymer (1c) [Chemical formula]

[0162] A polymer (1c)-dU conjugate having an average molecular weight of approximately 5 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and a DMT-mU (i.e., DNT-2'-OMe-U) or DMT-mA (i.e., DMT-2'-OMe-A) phosphoramidite (Hongene Biotech) in a manner similar to the method described in Example 4 to obtain polymer (1c)-2mU or polymer (1c)-2mUmA. This procedure was repeated with other nucleotides (e.g., 2'-OMe-U (mU), 2'-fluoro-U (fU), 2'-OCH2CH2OCH3-T (moeT)) as needed to prepare a polymer (1c)-mU-mU-mU-mU-mU-mU-mU-mU sequence (showing 93.6% full product purity (FLP) after nucleotide cleavage), a polymer (1c)-mU-mU-mU-mU-mU-mU-mU-mU-fU-moeT sequence (showing 90.4% full-length product (FLP) purity after nucleotide cleavage), and a polymer (1c)-mU-mA-mA-mA-mA-mA sequence (showing 92.8% full-length product purity after nucleotide cleavage). (Example 10) General procedure for the synthesis of polymer (3a) [Chemical formula]

[0163] A certain amount of polymer (1b), Fmoc-Lys(Fmoc)-OH, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylethylamine were dissolved in dry acetonitrile. The reaction mixture was stirred for 2 hours. Next, the mixture was added to stirred diethyl ether to precipitate the intermediate product modified with Lys. To the dry precipitate, 20 mL of 20% piperidine in dimethylformamide (DMF) was added and stirred for 1 hour to remove the Fmoc group to obtain polymer (3a), which was isolated by precipitation in diethyl ether. Polymer (3b) was prepared from polymer (1c) having a molecular weight of approximately 5 kDa according to a similar procedure. (Example 11) Direct conjugation of DMT-nucleotide-3'-succinate units to polymer (3a) [Chemical formula]

[0164] A polymer(3a)-dT conjugate having an average molecular weight of approximately 10 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM in 4.76 mL of anhydrous acetonitrile, Sigma Aldrich), and DMT-mU (i.e., DMT-2'-OMe-U) or DMT-U phosphoramidite (Hongene Biotech) in a 100 mL round-bottom flask. After continuously stirring magnetically for 60 minutes, 158 mg of m-CPBA (3-chloroperbenzoic acid, Sigma Aldrich) was added as a powder. After continuously stirring for 10 minutes, 2.04 mL of trichloroacetic acid (TCA, 600 mg / mL in DCM) and 0.706 mL of TES were added. After continuously stirring for 10 minutes, the reaction mixture was poured into isopropanol with stirring. The precipitate was washed twice with diethyl ether to obtain polymer(3a)-dT-mU-mU-mU-mU-mU-mU-mU-mU (showing 94% full product purity (FLP) after nucleotide cleavage as shown in Figure 5) and polymer(3a)-dT-mU-mU-mU-mU-mU-mU-mU-mU-mU-mU-mU-mU (showing 88 - 90% full-length product purity (FLP) after nucleotide cleavage). (Example 12) Elongation of the nucleotide chain polymer(3b)-9dT [Chemical formula]

[0165] A polymer (3b)-9dT conjugate (Figure 6A) having an average molecular weight of approximately 5 kDa PEG and a complete product purity (FLP) of 76.1%, prepared using the methods described in Examples 4 and 11, was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-dT phosphoramidite (Hongene Biotech) in a manner similar to that described in Examples 4 and 11 to obtain polymer (3b)-10dT. This procedure was repeated 13 more times with other nucleotides to prepare the polymer (3b)-23dT sequence, which showed a complete product purity (FLP) of 63.2% after nucleotide cleavage (Figure 6B). In the coupling reaction, an ACN / DCM mixture was utilized to improve polymer solubility. (Example 13) Preparation of Sulfurized Polymer (3b)-dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT

Chemical formula

[0166] A polymer (3b)-dT conjugate (223 mg) having an average molecular weight of approximately 5 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole, anhydrous acetonitrile, 250 mM in 2.05 mL, Sigma Aldrich) and 168 mg of DMT-dT phosphoramidite (Hongene Biotech). After continuously shaking for 90 minutes, 37 μL of isopropanol was added. After continuously shaking for 60 minutes, excess xanthine hydride (45 mg) was added and the mixture was continuously shaken for an additional 60 minutes. Excess trichloroacetic acid (600 mg / mL in DCM, 1.53 mL) and TES (527 μL) were added. After continuously shaking for 10 minutes, the reaction mixture was poured into isopropanol with stirring. The precipitate was washed twice with diethyl ether to obtain polymer (3b)-dT(S)dT. (S) indicates a thiophosphate bond (P=S) in the oligonucleotide backbone. The above procedure was repeated 6 more times to obtain polymer (3b)-dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT, which showed a complete product purity (FLP) of 97.0% after nucleotide cleavage (Figure 7). (Example 14) Preparation of polymer (1b)-dTdTdT(S)dT(S)dT(S)dT(S)dT(S)dT [Chemical formula]

[0167] A polymer (1b)-3dT conjugate (154 mg) having an average molecular weight of approximately 10 kDa, prepared according to the method described in Example 4, was treated in a manner similar to that described in Example 13 to obtain polymer (1b)-dTdTdT(S)dT. This procedure was repeated 4 more times to obtain polymer (1b)-dTdTdT(S)dT(S)dT(S)dT(S)dT(S)dT, which showed a complete product purity (FLP) of 96.3% after nucleotide cleavage (Figure 8). (Example 15) Mixed backbone oligonucleotide research [Chemistry]

[0168] A polymer (3b)-8dT(S) conjugate (FLP 97.0%) having a molecular weight of approximately 5 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-dT phosphoramidite (Hongene Biotech) in a manner similar to that described in Example 4. However, the reaction was carried out using either m-chloroperoxybenzoic acid (m-CPBA) or tBuOOH as the oxidizing agent to obtain polymer (3b)-7dT(S)dT(O)dT-OH. The HPLC results are provided in Figures 9A and 9B respectively, which showed that the m-CPBA acid induced obvious desulfurization, while no obvious desulfurization was observed when tBuOOH was used as the oxidizing agent. (Example 16) Synthesis of polymer (1b)-dT-8mU(VP)mU [Chemistry]

[0169] A polymer (1b)-dT-8mU conjugate having a molecular weight of approximately 10 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and 3 equivalents of 5’-POM-VP-mU (i.e., 5’-[O,O-bis(pivaloyloxymethyl)-vinylphosphonate, 2’-OMe-U phosphoramidite) (Hongene Biotech) in a manner similar to that described in Example 4 without a deblocking step to obtain polymer (1b)-dT-8mU-VPmU, which showed a complete product purity (FLP) of 83.0% after nucleotide cleavage (Figure 10). MS: 1599[M-2H / 2]-2; 1066[M-3H / 3]-3. (Example 17) Synthesis of polymer (1c)-8dT-(VP)mA [Chemistry]

[0170] A polymer (1)-8dT conjugate having a molecular weight of approximately 5 kDa was dissolved in a mixture of an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and 3 equivalents of 5'-POM-VP-mA (Hongene Biotech) in a manner similar to that described in Example 4 without a deblocking step to obtain polymer (1c)-8dT-VPmA, which showed a complete product purity (FLP) of 82.0% after nucleotide cleavage (Figure 11). The results were confirmed by HPLC and LC / MS. (Example 18) Synthesis of polymer (1c)-dTdT(S)dT [Chemical formula]

[0171] A polymer (1c)-dT conjugate having a molecular weight of approximately 5 kDa was dissolved in a mixture of tBuOOH as an oxidizing agent, an ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile, 319 μL), and 1.03 equivalents of DMT-dT(S)dT-phosphoramidite (Hongene Biotech) in a manner similar to that described in Example 15 to obtain polymer (1c)-dTdT(S)dT, which showed a complete product purity (FLP) of 91.9% after nucleotide cleavage (Figure 12). Even when using a slightly excessive 1 molar equivalent of DMT-dT(P=S)dT-phosphoramidite, the conversion rate was up to 99.2%. (Example 19) Synthesis of DPEG-OH anchor for oligonucleotide synthesis [Chemical formula]

[0172] Synthesis of Polymer (4): Commercially available PEG with an amine terminus (600 mg, 0.12 mmol) was stirred in 5 mL of ACN with 3-bromo-1-propanol (133.4 mg, 0.96 mmol) and potassium carbonate (132.7 mg, 0.96 mmol) at 83 °C for a duration of 3 days. The completion of the reaction was confirmed using the ninhydrin test. The reaction mixture was filtered and the reaction mixture was subjected to 3 rounds of MTBE precipitation to obtain Polymer (4) by using DCM as the resuspension solvent.

[0173] Synthesis of Polymer (5): Commercially available PEG with an amine terminus (5 g, 1 mmol) was stirred in 50 mL of DMF with 3-bromo-2-(bromomethyl)propionic acid (737.7 mg, 3.0 mmol), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, 1.25 g, 3.3 mmol), and N,N-diisopropylethylamine (DIPEA, 775.4 mg, 6.0 mmol) at 33 °C overnight. The completion of the reaction was confirmed using the ninhydrin test. The reaction mixture was poured into cooled MBTE and the resulting solid was dried under vacuum to obtain the intermediate product. Hydrolysis of the halide was achieved in a basic aqueous solution (pH = 10) at 90 °C for 3 days. After hydrolysis, water was removed by lyophilization. The remaining solid was resuspended in DCM and then the supernatant was obtained by filtration. Polymer (5) was obtained by MTBE precipitation.

[0174] Synthesis of Polymer (6): Commercially available PEG with an amine terminus (5 g, 1 mmol) was stirred in 50 mL of DMF with 2,4-dibromobutyryl chloride (881 mg, 3 mmol) and DIPEA (775.4 mg, 6.0 mmol) at room temperature overnight. The reaction mixture was poured into cooled MBTE and the resulting solid was dried under vacuum to obtain the intermediate product. Hydrolysis of the halide was achieved in a basic aqueous solution (pH = 10) at 90 °C for 3 days. After hydrolysis, water was removed by lyophilization. The remaining solid was resuspended in DCM and then the supernatant was obtained by filtration. Polymer (6) was obtained by MTBE precipitation. (Example 20) Conjugation of Succinate dT to DPEG-OH Anchor and Extension of Nucleotide Chain

[0175] Polymer (4), (5), or (6) and 5'-O-(4,4'-Dimethoxytrityl)-thymidine-3'-O-succinate, triethylamine salt (305.6 mg, 0.41 mmol) were dissolved in 3 mL of anhydrous DCM under argon together with N-methylimidazole (NMI, 67.28 mg, 0.82 mmol). To this solution, 2,6-dichlorobenzoyl chloride (DcbCl, 85.81 mg, 0.41 mmol) was added and the mixture was stirred for 20 minutes to activate the succinate. Next, the activated succinate solution was slowly added dropwise to polymer 4, 5, or 6 (0.2 mmol) over 40 minutes and the reaction was continued overnight at room temperature. Next, to deblock the trityl group on the base, trichloroacetic acid (TCA, 600 mg / mL in DCM, 7.7 mmol) was added to the solution, followed by triethylsilane (TES, 4.6 mmol) as a scavenger reagent. The product was obtained by 2 rounds of IPA precipitation and MBTE washing, followed by vacuum drying. Further conjugation of additional bases to the polymer (4), (5), or (6)-dT anchor was performed according to a procedure similar to that described in Example 4. Oligonucleotides composed of eight thymidines were synthesized using polymer (4), (5), or (6) as the starting material. The products obtained from polymers (4) and (6) were characterized by HPLC (Figure 13A and Figure 13B, respectively). The yields and full product purities of the 8dT oligonucleotides after cleavage are provided in Table 2 below.

Table 2

Chemical Structure

[0176] First, 4-acetoxybenzoic acid (216.2 mg, 1.2 mmol) was pre-activated with 1-hydroxybenzotriazole (HOBt, 162.4 mg, 1.2 mmol) and N,N'-diisopropylcarbodiimide (DIC, 166.58 mg, 1.32 mmol) in 5 mL of DMF for 10 minutes. Next, this solution was added to the DPEG-diamine polymer (1c) (600 mg, 0.24 mmol), and the mixture was stirred overnight at room temperature. The reaction was stopped by precipitation with IPA followed by washing with MTBE. To obtain the final product, 5 mL of a 10% piperidine / DMF solution was added to the precipitate and stirred at room temperature for 1 hour. The polymer (7) was obtained by two rounds of MTBE precipitation followed by vacuum drying. (Example 22) Conjugation of succinate dT to DPEG-OH anchor (7) and extension of the nucleotide chain

[0177] To conjugate the first thymidine base to the polymer (7), 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-succinate, triethylamine salt (305.6 mg, 0.41 mmol) was dissolved in 3 mL of anhydrous DCM under argon together with N-methylimidazole (NMI, 67.28 mg, 0.82 mmol). To this solution, 2,6-dichlorobenzoyl chloride (DcbCl, 85.81 mg, 0.41 mmol) was added, and the mixture was stirred for 20 minutes to activate the succinate. Next, the activated succinate solution was slowly added dropwise to the polymer 7 (0.2 mmol) over 40 minutes, and the reaction was continued overnight at room temperature. Next, to deblock the trityl group on the base, trichloroacetic acid (TCA, 600 mg / mL in DCM, 7.7 mmol) was added to the solution, followed by triethylsilane (TES, 4.6 mmol) as a scavenger reagent. The product was obtained by two rounds of IPA precipitation and MBTE washing followed by vacuum drying.

[0178] Further conjugation of additional bases to the polymer(7)-dT anchor was carried out according to a procedure similar to that described in Example 4. An oligonucleotide composed of eight thymidines was synthesized using polymer(7) as the starting material. The final oligonucleotide product was cleaved from the anchor using a 1:1 v / v mixture of NH4OH and methylamine and characterized by HPLC (Figure 13C). The product was recovered in 81.8% overall yield and showed 93.2% full product purity.

[0179] It was observed that LOPS polymer anchors with terminal hydroxy groups had equivalent and / or higher loading, FLP, and yields compared to LPOS polymer anchors with terminal -NH2 groups. Additionally, asymmetric polymer anchors, such as polymers(4) and (7), resulted in lower impurity levels based on HPLC results and precipitation during oligo synthesis was relatively easier.

Claims

1. A polymer for liquid-phase oligonucleotide synthesis having the structure of formula (I) 【Chemical 162】 [wherein, R is H, or unsubstituted or substituted C １ ~C ６ alkyl, and W is C １ ~C ２０ an alkylene, a 2- to 20-membered heteroalkylene, or a bond, Q is, 【Chemical 163】 and is, L １ is a C １ -C ２０ alkylene, 2-20 membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene, or optionally substituted C ３ -C １０ cycloalkylene, or one or more methylene repeating units are each independently replaced by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted C ３ -C １０ cycloalkylene, -C(=O)-, -CH=CH-, and -C≡C-, and is a C １ -C ２０ alkylene or 2-20 membered heteroalkylene, R １ and R ２ each of which is independently, -OR ３ or -N Ｒ４ａ R ４ｂ wherein R ３ is H, C １ ~C ６ alkyl, a hydroxy protecting group, or 【Chemical 164】 and is, Each R ４ａ and R ４ｂ is independently H, C optionally substituted １ to C ６ alkyl, -C(=O)(C １ to C ６ alkyl), -C(=O)phenyl, an amino protecting group, or 【Chemical 165】 or R ４ａ and R ４ｂ together form a divalent amino protecting group, L ２ａ and L ２ｂ each of which is independently C １ to C ２０ alkylene, 2- to 20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 3- to 10-membered heterocyclylene, or optionally substituted C ３ to C １０ cycloalkylene, or one or more methylene repeating units are each independently replaced by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 3- to 10-membered heterocyclylene, optionally substituted C ３ to C １０ cycloalkylene, -C(=O)-, -CH=CH-, and -C≡C-, and is C １ to C ２０ alkylene or 2- to 20-membered heteroalkylene, R ５ａ 、R ５ｂ 、R ６ａ and R ６ｂ each of which is independently H, -OR ７ or -NR ８ａ R ８ｂ and R ７ Each of which is independently H, C １ to C ６ alkyl, a hydroxy protecting group, or 【Chemical 166】 and is, R ８ａ and R ８ｂ each of which is independently H, optionally substituted C １ -C ６ -alkyl, -C(=O)(C １ -C ６ -alkyl), -C(=O)phenyl, an amino protecting group, or 【Chemical 167】 or R ８ａ and R ８ｂ together form a divalent amino protecting group, L ３ａ and L ３ｂ each of which is independently C １ to C ２０ alkylene, 2- to 20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 3- to 10-membered heterocyclylene, or optionally substituted C ３ to C １０ cycloalkylene, or one or more methylene repeating units are each independently replaced by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 3- to 10-membered heterocyclylene, optionally substituted C ３ to C １０ cycloalkylene, -C(=O)-, -CH=CH-, and -C≡C-, and is C １ to C ２０ alkylene or 2- to 20-membered heteroalkylene, R ９ａ 、R ９ｂ 、R １０ａ and R １０ｂ each of which, independently, is H, -OR １１ or -NR １２ａ R １２ｂ and R １１ Each of which is, independently, H, C １ ~C ６ alkyl, a hydroxy protecting group, or 【Chemical 168】 and is, R １２ａ and R １２ｂ each of which is independently H, C １ ~C ６ alkyl, -C(=O)(C １ ~C ６ alkyl), -C(=O)phenyl, an amino protecting group, or 【Chemical 169】 or R １２ａ and R １２ｂ together form a divalent amino protecting group, L ４ａ and L ４ｂ each of which is independently C １ to C ２０ alkylene, 2- to 20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 3- to 10-membered heterocyclylene, or optionally substituted C ３ to C １０ cycloalkylene, or one or more methylene repeating units are each independently replaced by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 3- to 10-membered heterocyclylene, optionally substituted C ３ to C １０ cycloalkylene, -C(=O)-, -CH=CH-, and -C≡C-, and is C １ to C ２０ alkylene or 2- to 20-membered heteroalkylene, R １３ａ 、 R １３ｂ 、 R １４ａ and R １４ｂ each of which is, independently, H, -OH, protected hydroxy, -NH ２ , -NH (optionally substituted C １ to C ６ alkyl), or protected amino, each of m1, m2, m3, m4, m5, m6 and m7 is independently 0 or 1, j is an integer from 15 to 1500].

2. The polymer according to claim 1, wherein R is methyl.

3. W is -CH ２ CH ２ The polymer according to claim 1 or 2, wherein NH- is used.

4. W is -CH ２ CH ２ The polymer according to claim 1 or 2, wherein O- is present.

5. W is -CH ２ CH ２ The polymer according to claim 1 or 2, wherein it is -

6. L １ is C ２ to C １０ The polymer according to any one of claims 1 to 5, which is an alkylene linker.

7. L １ The polymer according to any one of claims 1 to 5, wherein L is a 3- to 12-membered heteroalkylene containing 1, 2 or 3 heteroatoms selected from N, O and S.

8. Q is 【Chemical 170】 【Chemical 171】 The polymer according to any one of claims 1 to 7, selected from the group consisting of

9. Each R ３ The polymer according to any one of claims 1 to 8, wherein each R is independently H or a hydroxy protecting group.

10. One R ３ is H or a hydroxy protecting group, and the other R ３ is 【Chemical 172】 The polymer according to any one of claims 1 to 8, which is

11. Each R ４ａ is H, and each R ４ｂ is, independently, H, -C(=O)(C １ ~C ６ alkyl), -C(=O)phenyl, an amino protecting group, or 【Chemical 173】 or -NHR ４ｂ in which hydrogen is absent and R ４ｂ is a divalent amino protecting group, the polymer according to any one of claims 1 to 8.

12. Each R ４ｂ is, independently, H, -C(=O)CH ３ or an amino protecting group, the polymer according to claim 11.

13. One R ４ｂ is H or an amino protecting group, and the other R ４ｂ is 【Chemical 174】 The polymer according to claim 11, which is

14. R ３ each of which is independently 【Chemical 175】 and R ４ｂ each of which is, independently, 【Chemical 176】 The polymer according to claim 11, which is

15. The polymer according to any one of claims 10, 11, 13 and 14, wherein each of m2 and m3 is 1.

16. L ２ａ and L ２ｂ each of which is, independently, C ２ to C ６ alkylene, or 3- to 12-membered heteroalkylene containing 1, 2 or 3 heteroatoms selected from N, O and S, the polymer according to any one of claims 10, 11 and 13 to 15.

17. L ２ａ and L ２ｂ each of which is independently a C replaced by a phenylene optionally substituted or a phenylene in which one methylene unit is optionally substituted by a phenylene ２ to C ６ The polymer according to any one of claims 10, 11 and 13 to 15, which is alkylene or 3- to 12-membered heteroalkylene

18. Each R ８ａ is H, and R ５ａ , R ５ｂ , R ６ａ and R ６ｂ each, independently, is H, -OR ７ or -NHR ８ｂ and each R ８ｂ is, independently, H, -C(=O)(C １ ~C ６ alkyl), -C(=O)phenyl, an amino protecting group, or 【Chemical 177】 or -NHR ８ｂ wherein the hydrogen of ８ｂ is absent and R is a divalent amino protecting group, the polymer according to any one of claims 1 to 17.

19. R ５ａ 、 R ５ｂ 、 R ６ａ and R ６ｂ each of which is, independently, H, -OH, protected hydroxy, -NH ２ , -NHC(=O)CH ３ or protected amino, the polymer according to claim 18.

20. R ５ａ , R ５ｂ , R ６ａ and R ６ｂ The polymer according to claim 19, wherein each of R

21. R ５ａ 、 R ５ｂ 、 R ６ａ and R ６ｂ The polymer according to claim 19, wherein each of

22. R ５ａ 、R ５ｂ 、R ６ａ and R ６ｂ at least one of which is, independently, -OR ７ or -NHR ８ｂ and at least one of R ７ is 【Chemical 178】 and R ８ｂ at least one of which is 【Chemical 179】 The polymer according to claim 18, which is

23. R ５ａ 、R ５ｂ 、R ６ａ and R ６ｂ each of which is, independently, -OR ７ or -NHR ８ｂ and each of R ７ is, independently, 【Chemical 180】 and each R ８ｂ is, independently, 【Chemical 181】 The polymer according to claim 18, which is

24. The polymer according to claim 22 or 23, wherein each of m4 and m5 is 1.

25. L ３ａ and L ３ｂ each of which is, independently, C ２ to C ６ alkylene, or 3- to 12-membered heteroalkylene containing 1, 2 or 3 heteroatoms selected from N, O and S, the polymer according to any one of claims 22 to 24.

26. L ３ａ and L ３ｂ each of which is independently C ２ to C ６ alkylen or 3- to 12-membered heteroalkylen which is optionally replaced by phenylene which is optionally substituted or phenylene in which one methylene unit is optionally substituted, the polymer according to any one of claims 22 to 24.

27. Each R １２ａ is H, and R ９ａ , R ９ｂ , R １０ａ and R １０ｂ each independently is H, —OR １１ or —NHR １２ｂ where each R １２ｂ independently is H, —C(═O)(C １ to C ６ alkyl), —C(═O)phenyl, an amino protecting group, or 【Chemical 182】 or -NHR １２ｂ in which hydrogen is absent and R １２ｂ is a divalent amino protecting group, the polymer according to any one of claims 1 to 26.

28. R ９ａ 、R ９ｂ 、R １０ａ and R １０ｂ each of which is, independently, H, -OH, protected hydroxy, -NH ２ , -NHC(=O)CH ３ or protected amino, the polymer according to claim 27

29. R ９ａ 、 R ９ｂ 、 R １０ａ and R １０ｂ The polymer according to claim 28, wherein each of them is hydroxy or protected hydroxy.

30. R ９ａ 、 R ９ｂ 、 R １０ａ and R １０ｂ The polymer according to claim 28, wherein each of is amino or protected amino.

31. R ９ａ 、R ９ｂ 、R １０ａ and R １０ｂ at least one of which is, independently, -OR １１ or -NHR １２ｂ and at least one of R １１ is 【Chemical 183】 and at least one R １２ｂ is 【Chemical 184】 The polymer according to claim 27, which is

32. R ９ａ 、 R ９ｂ 、 R １０ａ and R １０ｂ each of which is, independently, -OR １１ or -NHR １２ｂ and each of R １１ is 【Chemical Formula 185】 and each R １２ｂ is 【Chemical 186】 The polymer according to claim 27, which is

33. The polymer according to claim 31 or 32, wherein each of m6 and m7 is 1.

34. L ４ａ and L ４ｂ each of which is, independently, C ２ to C ６ an alkylene, or a 3- to 12-membered heteroalkylene containing 1, 2 or 3 heteroatoms selected from N, O and S, the polymer according to any one of claims 31 to 33.

35. R １３ａ 、 R １３ｂ 、 R １４ａ and R １４ｂ each of which is, independently, H, -OH, protected hydroxy, -NH ２ , -NHC(=O)CH ３ , or protected amino, the polymer according to any one of claims 31 to 34.

36. Formula (Ia): 【Chemical 187】 The polymer according to claim 1, having the structure of

37. Formula (Ib), (Ib-1), (Ic) or (Ic-1): 【Chemical 188】 The polymer according to claim 1, having the structure of

38. The structure of the formula (Ib-1) is the formula (Ib-2) or (Ib-3): 【Chemical 189】 The polymer according to claim 37, having the structure of

39. The structure of the formula (Ib-1) is the formula (Ib-4): 【Chemical 190】 The polymer according to claim 37, having the structure of

40. R ５ｂ and R ６ｂ each independently is --NH ２ or --NHAc, the polymer according to claim 37.

41. R ５ｂ and R ６ｂ each of which is —OH, the polymer according to claim 37.

42. The structure of the formula (I) is the formula (Id), (Ie), or (If): 【Chemical Formula 191】 The polymer according to claim 1, having the structure of

43. The polymer according to any one of claims 1 to 42, wherein j is an integer from 400 to 500.

44. The polymer according to any one of claims 1 to 43, wherein the polymer has an average molecular weight of about 15 kDa to about 30 kDa.

45. The polymer according to any one of claims 1 to 44, wherein the polymer has an average molecular weight of about 20 kDa.

46. A polymer for liquid-phase oligonucleotide synthesis having the structure of formula (II) 【Chemical 192】 [wherein, A is a carbon atom, C ２ ~C ２０ alkylene, 2- to 20-membered heteroalkylene, phenylene, 5- to 10-membered heteroarylene, C ５～１０ cycloalkylene, and 5- to 10-membered heterocycloalkylene, and is selected from the group consisting of Each R １５ is independently selected from the group consisting of -O-C １ to C ６ alkyl, -OC(O)(C １ to C ６ alkyl), -OC(O)phenyl, -NHC(O)(C １ to C ６ alkyl), -NHC(O)phenyl, -NHC(O)phenylen-acetoxymethyl, protected hydroxy, or protected amino Each R １６ is, independently, -OH or -NH ２ and q １ and q ２ each of which is, independently, an integer from 10 to 500, each of s and t is independently an integer from 1 to 4, provided that s + t is equal to 2 or greater than that].

47. The polymer according to claim 46, wherein s + t is 4.

48. The polymer according to claim 46 or 47, wherein s is 2 and t is 2.

49. Formula (IIa): 【Chemical Formula 193】 The polymer according to any one of claims 46 to 48, having the structure of.

50. The polymer according to any one of claims 46 to 49, wherein A is a carbon atom or phenylene.

51. The polymer according to claim 46, wherein s + t is 8.

52. The polymer according to claim 46 or 47, wherein s is 5 and t is 3.

53. Formula (IIb): 【Chemical 194】 The polymer according to any one of claims 46, 51 and 52, having the structure of.

54. each q １ The polymer according to any one of claims 46 to 53, wherein each q is independently from about 30 to about 150, from about 40 to about 100, or from about 50 to about 75.

55. each q ２ The polymer according to any one of claims 46 to 54, wherein each q is independently from about 30 to about 150, from about 40 to about 100, or from about 50 to about 75.

56. The polymer according to any one of claims 46 to 55, wherein the average molecular weight of the polymer is about 10 kDa, about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

57. A method for preparing an oligonucleotide by liquid-phase oligonucleotide synthesis, dissolving the polymer according to any one of claims 1 to 56 in a first solvent to form a reaction matrix, and reacting the polymer with one or more nucleoside analogs to form a first bioconjugate containing the structure of formula (III) 【Chemical Formula 195】 [wherein, B １ is a nitrogenous base, G １ is a 5'-hydroxy blocking group, X is O or NR ２０ and R ２０ is H or C １ -C ６ alkyl, and R ａ is -H, -OH, halogen, -O-(C １ ~C ６ alkyl), -O-(C １ ~C ６ haloalkyl), or -OY, where Y is a 2'-hydroxy protecting group, L ５ is a cleavable heteroalkylene linker, where one or more carbon atoms are replaced by O, S, N, C(=O) or C(=S)] A method including.

58. The method according to claim 57, wherein the structure of formula (III) is also represented by formula (IIIa): 【Chemical Formula 196】

59. B １ The method according to claim 57 or 58, wherein B is, independently, adenine optionally protected, deazaadenine optionally protected, cytosine optionally protected, guanine optionally protected, deazaguanine optionally protected, thymine optionally protected or uracil optionally protected.

60. B １ is 【Chemical 197】 and in the formula, R ｘ is hydrogen, unsubstituted or substituted C １ to C ６ alkyl, or an amino protecting group, or the hydrogen of -NHR ｘ is absent, and R ｘ is a divalent amino protecting group, the method according to any one of claims 57 to 59.

61. G １ The method according to any one of claims 57 to 60, wherein G is a trityl-type hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.

62. G １ The method according to claim 38, wherein G is bis(4-methoxyphenyl)phenylmethyl.

63. The method according to any one of claims 57 to 62, wherein the polymer has an average molecular weight of about 10 kDa, about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

64. Removing the 5'-hydroxy blocking group (G １ ) to form a first bioconjugate with an unblocked 5'-end; Isolating the first bioconjugate in which the 5' is not blocked The method according to any one of claims 57 to 63, further comprising

65. The method according to claim 64, wherein the isolation of the first bioconjugate with an unblocked 5' is achieved by precipitation, dialysis or filtration.

66. The method according to claim 65, wherein the isolation of the first bioconjugate with an unblocked 5' is achieved by precipitation.

67. The method according to claim 66, wherein the precipitation is achieved in diethyl ether or isopropanol.

68. (a) reacting the first bioconjugate with an unblocked 5' in a second solvent with one or more nucleoside phosphoramidite analogs to form a second bioconjugate having the structure of formula (IV); 【Chemical 198】 [wherein, G ２ is a 5'-hydroxy blocking group, B ２ is a nitrogenous base, R ｅ is a phosphite protecting group], (b) oxidizing the phosphite moiety of formula (IV); (c) removing said 5'-blocking group G ２ to form a second bioconjugate that is not 5'-blocked and contains the structure of formula (IV'); and 【Chemical 199】 [wherein, Z is O or S], (d) isolating the second bioconjugate with an unblocked 5'; The method according to any one of claims 57 to 67, further comprising

69. The method according to claim 68, wherein the structure of formula (IV) is also represented by (IVa), and formula (IV') is also represented by formula (IV'a). 【Chemical 200】

70. The method according to claim 68 or 69, further comprising, prior to step (b), blocking the unreacted 5'-hydroxy group of the first bioconjugate with an unblocked 5'.

71. B ２ The method according to any one of claims 68 to 70, wherein B is, independently, adenine optionally protected, deazaadenine optionally protected, cytosine optionally protected, guanine optionally protected, deazaguanine optionally protected, thymine optionally protected, or uracil optionally protected.

72. B ２ is 【Chemical 201】 and in the formula, R ｘ is hydrogen, unsubstituted or substituted C １ to C ６ alkyl, or an amino protecting group, or the hydrogen of -NHR ｘ is absent, and R ｘ is a divalent amino protecting group, the method according to claim 71.

73. G ２ The method according to any one of claims 68 to 72, wherein G is a trityl type hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.

74. G ２ The method according to claim 73, wherein G is bis(4-methoxyphenyl)phenylmethyl.

75. The method according to any one of claims 68 to 74, wherein the isolation of the second bioconjugate with an unblocked 5' is achieved by precipitation, filtration or dialysis.

76. The method according to any one of claims 68 to 75, wherein steps (a) to (d) are repeated in multiple cycles until oligonucleotides of one or more desired lengths are synthesized.

77. The method according to claim 76, wherein steps (a) to (d) are repeated at least about 10 cycles.

78. The method according to claim 76 or 77, further comprising removing the oligonucleotide from the polymer.

79. The method according to any one of claims 68 to 78, wherein each of the first solvent and the second solvent comprises one or more aprotic polar solvents, or a combination thereof.

80. The method according to claim 79, wherein the one or more aprotic polar solvents comprise acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or a combination thereof.

81. The method according to claim 80, wherein the one or more aprotic polar solvents is acetonitrile.

82. An oligonucleotide prepared by the method according to any one of claims 57 to 81.

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