Oligonucleotide synthesis method

Abstract

This specification discloses a process or method for producing high-purity and high-yield oligonucleotides on a large, single-batch scale, without a capping step.

Description

[Technical Field]

[0001] This disclosure relates to solid-phase oligonucleotide synthesis, and more particularly to a process or method using a solid-phase reactor system for producing oligonucleotides of various lengths and sequences, without the use of a capping step. [Background technology]

[0002] Having processes and methods for synthesizing high-purity oligonucleotides of various nucleotide sequences and lengths in sufficiently high yields and at minimal cost is of great interest to the therapeutic, diagnostic, reagent, and biological assay industries. Conventional oligonucleotide synthesis generally involves starting with a nucleotide or another similar portion located at the 3' end of the final oligonucleotide, attaching it to a support (e.g., a polymer or glass resin), and then sequentially adding nucleotides to the 5' end by covalent bonding using chemical steps to produce the final oligonucleotide. This synthesis includes a capping step as part of each nucleotide monomer addition, which allows for the removal of shortmers from the reaction, thereby improving the purity of the resulting product. At the end of the synthetic run, the synthesized oligonucleotide is cleaved from the resin support.

[0003] Synthetic methods that utilize a capping step typically require additional time to complete, which can slow down the workflow, reduce the scalability of production, and increase costs. Conventional synthetic methods also typically require the use of a purification step to remove shorter from the reaction product in order to obtain a sufficiently pure yield. Furthermore, the capping step can result in a lower purity of the overall final yield. [Overview of the project] [Problems that the invention aims to solve]

[0004] Therefore, especially for the large-scale production of oligonucleotides, there is a need for improved methods to synthesize high-purity oligonucleotides with various nucleotide sequences in a timely manner and in sufficiently high yields.

[0005] This disclosure is based, at least in part, on the discovery of a method for synthesizing high-yield, long (e.g., more than 10-mer) oligonucleotides using a method that does not involve a capping step. [Means for solving the problem]

[0006] In some embodiments, the Disclosure provides a method for producing oligonucleotides. In some embodiments, the Disclosure provides a method for producing oligonucleotides, comprising: (a) preparing a first nucleotide containing a first trityl group, wherein the first nucleotide is a support nucleotide on a substrate; (b) detritylating the first nucleotide with a detritylating reagent to form a detritylated nucleotide, wherein the detritylating reagent comprises acetic acid or a salt thereof, and the acetic acid may be trifluoroacetic acid (TFA) or a salt thereof; (c) washing the detritylated nucleotide with a washing solution; and (d) coupling a second nucleotide to a detritylated nucleotide under reaction conditions that promote the coupling of a second nucleotide to a detritylated nucleotide, wherein the substrate is a solid support; and the method provides a method for producing oligonucleotides that does not include a capping step.

[0007] In some embodiments, the washing solution comprises (i) a neutralizing solvent, (ii) an alcohol or water, and (iii) an organic solvent. In some embodiments, the washing solution comprises pyridine, methanol, and toluene. In some embodiments, the method further comprises a second washing step after coupling the second nucleotide to the first nucleotide. In some embodiments, the second washing step comprises acetonitrile.

[0008] In some embodiments, the method further includes an oxidation step after coupling a second nucleotide to a first nucleotide, the oxidation step being carried out using an oxidizing reagent. In some embodiments, the oxidizing reagent includes a halogen, a base, and water. In some embodiments, the oxidizing reagent includes iodine, pyridine, and water. In some embodiments, the method further includes an additional washing step after the oxidation step. In some embodiments, the additional washing includes acetonitrile.

[0009] In some embodiments, the method further includes a thiolation step after coupling a second nucleotide to a first nucleotide, the thiolation step being carried out using a thiolation reagent. In some embodiments, the thiolation reagent includes xanthan hydride. In some embodiments, the method further includes an additional washing step after the thiolation step. In some embodiments, the additional washing includes acetonitrile.

[0010] In some embodiments, the method further comprises an oxidation step and a thiolation step. In some embodiments, the method further comprises an additional washing step after the oxidation step, after the thiolation step, and / or after the oxidation step and the thiolation step. In some embodiments, the additional washing comprises acetonitrile.

[0011] In some embodiments, the present disclosure provides a method for producing an oligonucleotide, the method comprising: (a) preparing a first nucleotide containing a first trityl group, wherein the first nucleotide is a carrier-supported nucleotide on a substrate, and the substrate is a solid-phase support; (b) detritylating the first nucleotide using a detritylating reagent containing trifluoroacetic acid (TFA) to form a detritylated nucleotide; (c) washing the detritylated nucleotide with a washing solution containing pyridine and methanol; (d) coupling a second nucleotide to a detritylated nucleotide under reaction conditions that promote the coupling of a second nucleotide to a detritylated nucleotide, wherein the second nucleotide contains a second trityl group; (e) washing the oligonucleotide as appropriate; (f) oxidizing the oligonucleotide to form an oxidized oligonucleotide, or thiolating the oligonucleotide to form a thiolated oligonucleotide; and (g) washing the oxidized or thiolated oligonucleotide, or a method essentially composed of these.

[0012] In some embodiments, a method for producing oligonucleotides yields a higher crude yield of oligonucleotides compared to the same method for producing oligonucleotides that includes a capping step. In some embodiments, a method for producing oligonucleotides yields a higher final yield of oligonucleotides compared to the same method for producing oligonucleotides that includes a capping step. In some embodiments, a method for producing oligonucleotides yields a smaller short mer compared to the same method for producing oligonucleotides that includes a capping step.

[0013] In some embodiments, steps (a) to (d) of the method for producing an oligonucleotide are repeated one or more times, and the second nucleotide containing the second trityl group obtained from step (d) is used as the first nucleotide containing the first trityl group in the next repetition of step (a).

[0014] In some embodiments, the solid support comprises polystyrene or controlled porous glass (CPG). In some embodiments, the support-carrying nucleotide comprises one or more non-nucleotide moieties between the solid support and the first nucleotide.

[0015] In some embodiments, the detritylation reagent contains trifluoroacetic acid. In some embodiments, the detritylation reagent contains trifluoroacetic acid at a concentration of about 1 to about 15% by volume. In some embodiments, the detritylation reagent contains trifluoroacetic acid at a concentration of about 1 to about 10% by volume. In some embodiments, the detritylation reagent contains trifluoroacetic acid at a concentration of about 6% by volume. In some embodiments, the detritylation reagent contains an aromatic solvent or a halogenated solvent. In some embodiments, the detritylation reagent contains toluene, 2,2,2-trifluoroethanol, dichloromethane, or a mixture thereof.

[0016] In some embodiments, reaction conditions that promote coupling include the use of an activator. In some embodiments, the activators are 1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), 5-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 2-bromo-4,5-dicyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, and 1-(cyanomethyl)piperidinium tetraflu The activator comprises olobolate, 1-methyl-1H-benzimidazole-3-ium trifluoromethanesulfonate, 1-phenyl-1H-imidazole-3-ium trifluoromethanesulfonate, 1H-benzimidazole-3-ium trifluoromethanesulfonate, 1H-imidazole-3-ium trifluoromethanesulfonate, 5-nitro-1H-benzimidazole-3-ium trifluoromethanesulfonate, benzimidazolium triflate, and / or imidazolium triflate. In some embodiments, the activator comprises ETT.

[0017] In some embodiments, the oligonucleotide is approximately 5 to approximately 49 nucleotides long, or approximately 10 to approximately 49 nucleotides long, or approximately 15 to approximately 49 nucleotides long, or approximately 20 to approximately 49 nucleotides long, or approximately 25 to approximately 49 nucleotides long, or approximately 30 to approximately 49 nucleotides long, or approximately 35 to approximately 49 nucleotides long, or approximately 38 to approximately 49 nucleotides long, or approximately 19 to approximately 23 nucleotides long, or approximately 19 nucleotides long, or approximately 20 nucleotides long, or approximately 21 nucleotides long, or approximately 22 nucleotides long, or approximately 23 nucleotides long. In some embodiments, the oligonucleotide is 19 to 23 nucleotides long. In some embodiments, the oligonucleotide is 19 nucleotides long. In some embodiments, the oligonucleotide is 20 nucleotides long. In some embodiments, the oligonucleotide is 21 nucleotides long. In some embodiments, the oligonucleotide is 22 nucleotides long. In some embodiments, the oligonucleotide is 23 nucleotides long. In some embodiments, the oligonucleotide contains at least one modified nucleotide. In some embodiments, the oligonucleotide contains at least one modified oligonucleotide linkage. In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide.

[0018] In some embodiments, the oligonucleotide is an oligonucleotide dimer. In some embodiments, the oligonucleotide dimer is about 5 to about 49 nucleotides long, or about 10 to about 49 nucleotides long, or about 15 to about 49 nucleotides long, or about 20 to about 49 nucleotides long, or about 25 to about 49 nucleotides long, or about 30 to about 49 nucleotides long, or about 35 to about 49 nucleotides long, or about 38 to about 49 nucleotides long, or about 38 to about 46 nucleotides long, or about 39 to about 42 nucleotides long, or about 38 nucleotides long, or about 39 nucleotides long, or about 40 nucleotides long, or about 41 nucleotides long, or about 42 nucleotides long. In some embodiments, the oligonucleotide dimer is 38 to 46 nucleotides long. In some embodiments, the oligonucleotide dimer is 39 to 42 nucleotides long. In some embodiments, the oligonucleotide dimer is 38 nucleotides long. In some embodiments, the oligonucleotide dimer is 39 nucleotides long. In some embodiments, the oligonucleotide dimer is 40 nucleotides long. In some embodiments, the oligonucleotide dimer is 41 nucleotides long. In some embodiments, the oligonucleotide dimer is 42 nucleotides long. In some embodiments, the oligonucleotide dimer is 43 nucleotides long. In some embodiments, the oligonucleotide dimer is 44 nucleotides long. In some embodiments, the oligonucleotide dimer is 45 nucleotides long. In some embodiments, the oligonucleotide dimer is 46 nucleotides long. In some embodiments, the oligonucleotide dimer comprises a first oligonucleotide of 19 nucleotides and a second oligonucleotide of 21 nucleotides. In some embodiments, the oligonucleotide dimer contains at least one modified nucleotide. In some embodiments, the oligonucleotide dimer contains at least one modified nucleotide in each strand. In some embodiments, the oligonucleotide dimer contains at least one modified oligonucleotide bond.In some embodiments, the oligonucleotide dimer contains two single-stranded oligonucleotides linked together. In some embodiments, the oligonucleotide dimer contains two double-stranded oligonucleotides linked together. In some embodiments, the oligonucleotide dimer contains at least one spacer. In some embodiments, the spacer is a hexaethylene glycol spacer (e.g., spacer 18, Sp18). In some embodiments, the spacer is an adenosine trimer spacer (AAA).

[0019] In some embodiments, the oligonucleotide is an oligonucleotide trimer. In some embodiments, the oligonucleotide trimer contains three single-stranded oligonucleotides linked together. In some embodiments, the oligonucleotide trimer contains three double-stranded oligonucleotides linked together. In some embodiments, the oligonucleotide trimer is about 10 to about 49 nucleotides long, or about 15 to about 49 nucleotides long, or about 20 to about 49 nucleotides long, or about 25 to about 49 nucleotides long, or about 30 to about 49 nucleotides long, or about 35 to about 49 nucleotides long, or about 38 to about 49 nucleotides long. In some embodiments, the oligonucleotide trimer contains at least one modified nucleotide. In some embodiments, the oligonucleotide trimer contains at least one modified nucleotide in each strand. In some embodiments, the oligonucleotide trimer contains at least one spacer. In some embodiments, the spacer is an Sp18 spacer.

[0020] In some embodiments, the method provides a crude yield that is at least 2% higher compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method provides a crude yield that is at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method provides a final yield that is at least 3% higher compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method provides a final yield that is at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method results in at least a 3% reduction in shortmers compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method results in at least a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% reduction in shortmers compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method results in at least a 2% increase in crude purity compared to the same method for producing an oligonucleotide that includes a capping step. In some embodiments, the method results in at least a 3%, 4%, 5%, 6%, 7%, 8%, or more than 8% increase in crude purity compared to the same method for producing an oligonucleotide that includes a capping step.

[0021] In some embodiments, the present disclosure provides an oligonucleotide synthesized using the method of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising the oligonucleotide of the present disclosure and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1]Figure 1 illustrates a comparison of reversed-phase high-performance liquid chromatography-mass spectrometry (RP HPLC-MS) chromatograms of the crude yield of SEQ ID NO: 1 synthesized by the method disclosed in Example 1. [Figure 2] Figure 2 illustrates a comparison of RP HPLC-MS chromatograms of the crude yield of SEQ ID NO: 2 synthesized by the method disclosed in Example 2. [Figure 3] Figure 3 illustrates a comparison of RP HPLC-MS chromatograms of the crude yield of SEQ ID NO: 3 synthesized by the method disclosed in Example 3. [Figure 4] Figure 4 illustrates a comparison of RP HPLC-MS chromatograms of the crude yield of SEQ ID NO: 4 synthesized by the method disclosed in Example 4. [Figure 5] Figure 5 illustrates a comparison of RP HPLC-MS chromatograms of the crude yield of SEQ ID NO: 2 synthesized by the method disclosed in Example 6. DESCRIPTION OF EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0023] The term “oligonucleotide” as used herein refers to a compound comprising two or more nucleotides bound together. Oligonucleotides are typically less than approximately 100 nucleotides in length (e.g., 2 to 50 nucleotides in length). Examples of oligonucleotides for use in accordance with this disclosure include, but are not limited to, DNA, RNA [such as messenger RNA (mRNA)], DNA and / or RNA hybrids (such as their chemically modified variants), RNA and / or chemically modified RNA capable of inducing RNA interference (RNAi) mechanisms, such as RNAi agents, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and other oligonucleotides disclosed in detail herein or known in the art.

[0024] In this specification, the terms “carrier-supported” or “substrate-supported” refer to nucleotide or oligonucleotide strands supported on a solid carrier or substrate. Nucleotides may be supported directly on a carrier / substrate or as components of multiple bound nucleotides and / or non-nucleotide moieties supported on a carrier / substrate. Nucleotides may be covalently bonded.

[0025] The term “nucleotide” refers to an organic compound comprising a nucleoside and a phosphate group. The term includes organic compounds comprising a nucleic acid base (e.g., adenine, cytosine, guanine, thymine, or uracil) covalently attached to a pentose sugar. The term “nucleic acid base” as used herein refers to the heterocyclic portion of a nucleoside. In some embodiments, the nucleic acid bases of this disclosure are standard nucleic acid bases (A, G, T, C, U). In some embodiments, the nucleic acid base is a purine base—adenine (A) or guanine (G). In some embodiments, the nucleic acid base is a pyrimidine base—thymine (T), cytosine (C), or uracil (U). As used herein, the term “nucleotide” includes ribonucleotides (2'-OH sugar rings) and deoxyribonucleotides (2'-H sugar rings), and also includes modified nucleotides, unless otherwise specified. As described more fully elsewhere in this specification, modified nucleotides are nucleotides in which, for example, nucleic acid bases, pentoses, and / or phosphate groups (or nucleoside-to-nucleoside bonds) are chemically modified from those of ribonucleotides or deoxyribonucleotides. In some embodiments, the nucleotide is a modified nucleotide containing a modified nucleic acid base (i.e., a nucleotide other than a standard nucleic acid base). In some embodiments, the nucleotide is a modified nucleotide containing a modified sugar ring (e.g., a PNA nucleotide). In some embodiments, the nucleotide is a modified nucleotide containing a modified phosphate group (e.g., a phosphorothioate or phosphorodithioate group or other modified nucleoside-to-nucleoside bond). Exemplary modified nucleotides suitable for use in the methods disclosed herein are provided elsewhere in this specification, including in examples, and are also well known in the art.

[0026] In this specification, “non-nucleotide carrier-supported portion” or “non-nucleotide substrate-supported portion” refers to a portion that is a non-nucleotide supported on a solid carrier or substrate. Non-nucleotides may be supported directly on a carrier / substrate or as components of multiple bound nucleotides and / or non-nucleotide portions supported on a carrier / substrate. In some embodiments, the “non-nucleotide carrier-supported portion” is a compound directly attached to the resin / carrier [e.g., UnyLinker solid carrier (NittoPhase, Kinovate Lifesciences), reverse (or inverted) debase solid carrier (NittoPhase, Kinovate Lifesciences), etc.] or a protective phosphoramidite [e.g., Spacer phosphoramidite 18 (18-O-dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research), reverse (or inverted) debase phosphoramidite (3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) (Hongene The spacers can be commercially available as thiol-modified C6S-S linkers (1-O-dimethoxytrityl-hexyl-disulfide, 1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) (Hongene Biotech). In some embodiments, the non-nucleotide support moiety may be a targeted ligand or targeting group to directly assist the oligonucleotide in vivo, a PK / PD modifier to improve the pharmacokinetic / pharmacodynamic (PK / PD) properties of the oligonucleotide, a terminal cap to protect the oligonucleotide from enzymatic degradation, or a binding group such as an NH2-C6 ("AminoLink" trademark) group or an SP18 spacer that enables the coupling of other nucleotide or non-nucleotide groups, a prolinol moiety, or other chemical moieties that confer beneficial or other desirable properties to the oligonucleotide.Non-nucleotide moieties suitable for use with oligonucleotides are described in more detail elsewhere in this specification and are also well known in the art.

[0027] In this specification, the term "coupling efficiency" refers to the reaction yield of coupling a new nucleotide to a strand supported on a solid carrier.

[0028] In this specification, the term "cycle efficiency" refers to the yield of the supported oligonucleotide after a complete reaction cycle, including, for example, detritylation, coupling, and oxidation.

[0029] In this specification, the term "crude yield" refers to the yield of the crude oligonucleotide product before purification. For example, in the synthesis of a 21-mer oligonucleotide, the crude yield refers to the number of moles of the desired 21-mer obtained before purification, divided by the number of moles of oligonucleotide initially supported on the solid support. The number of moles of the desired 21-mer obtained before purification can be calculated, for example, by cutting strands from the resin and then analyzing and measuring the resulting product by reverse-phase high-performance liquid chromatography-mass spectrometry (RP HPLC-MS).

[0030] In this specification, the term "crude purity" refers to the amount of crude product oligonucleotide compared to the total yield from the reaction without purification. Crude purity can be measured by HPLC, for example, as the area under the product peak for the target oligonucleotide relative to the total peak area across the overall chromatogram for the crude product. The amount can be expressed as a percentage.

[0031] In this specification, the term "final yield" refers to the yield of the final purified oligonucleotide product. For example, in the synthesis of a 21-mer oligonucleotide, the final yield refers to the number of moles of the desired 21-mer obtained after purification, divided by the number of moles of oligonucleotide initially supported on the solid support. The number of moles of the desired 21-mer can be calculated after cutting strands from the resin, purifying them using anion exchange chromatography and / or tangent flow filtration, then desalting and freeze-drying the product, and measuring the yield using, for example, UV spectroscopy at 260 nm.

[0032] In this specification, the term "final purity" refers to the amount of the final purified oligonucleotide compared to the total yield from the reaction without purification. Final purity can be measured by HPLC, for example, as the area under the product peak for the target oligonucleotide relative to the total peak area across the overall chromatogram for the product. The amount can be expressed as a percentage. In some cases, final purity may refer to the yield of desalted oligonucleotide. In other cases, final purity may refer to the yield of column-purified and desalted oligonucleotide.

[0033] In this specification, the term "monomer" or "nucleotide monomer" refers to a single nucleotide.

[0034] In this specification, the term “dimer” or “nucleotide dimer” refers to an oligonucleotide containing two nucleotides (i.e., a nucleotide monomer linked to another nucleotide monomer).

[0035] In this specification, the term “trimer” or “nucleotide trimer” refers to an oligonucleotide containing three nucleotides (i.e., three linked nucleotide monomers).

[0036] In this specification, the term "oligonucleotide dimer" refers to two oligonucleotides linked together (for example, the following structure)

[0037] [ka] This refers to an oligonucleotide containing (using an Sp18 spacer having [a specific characteristic]).

[0038] In this specification, the term "oligonucleotide trimer" refers to an oligonucleotide comprising three oligonucleotides linked together (for example, using an Sp18 spacer).

[0039] In this specification, the term "shortmer" refers to an oligonucleotide that is shorter in length than the length intended in the synthetic reaction. Shortmers may lack terminal nucleotides at the 5' and / or 3' ends, and / or one or more internal nucleotides from the intended oligonucleotide sequence. Shortmers may, in some cases, have a length of 10 or fewer nucleotides.

[0040] In this specification, the term "about" as used herein means within 10% of the stated quantity.

[0041] Unless otherwise indicated, all technical and scientific terms herein have the same meanings as those commonly understood by those skilled in the art. All scopes include the endpoint. The term “or” means “and / or” unless otherwise indicated by the context. Methods and materials equivalent to or comparable to those disclosed herein may be used in the practice or testing of this disclosure, but preferred, exemplary methods and materials are disclosed below. The materials, methods, and examples disclosed herein are intended to be illustrative only and not limiting.

[0042] Other features and advantages of this disclosure will become apparent from the following detailed description and from the claims.

[0043] oligonucleotides overview This disclosure provides a method for synthesizing oligonucleotides. This disclosure also provides oligonucleotides prepared by the method of this disclosure. Oligonucleotides are typically polymers of conjugated nucleotides, each having a length of less than 100 nucleotides. In some embodiments, the oligonucleotides of this disclosure contain at least 3 nucleotides. In some embodiments, the oligonucleotides contain 3 to 100 nucleotides. In some embodiments, the oligonucleotides contain 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 6 nucleotides. In some embodiments, the oligonucleotides contain 2 nucleotides (i.e., dinucleotides). In some embodiments, the oligonucleotides contain 3 nucleotides (i.e., trinucleotides). In one embodiment, the oligonucleotide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In one embodiment, the oligonucleotide contains 16 to 26 nucleotides. In one embodiment, the oligonucleotide contains 19 to 23 nucleotides. In one embodiment, the oligonucleotide contains 18 to 20 nucleotides. In one embodiment, the oligonucleotide contains 19 nucleotides. In one embodiment, the oligonucleotide contains 22 to 24 nucleotides. In one embodiment, the oligonucleotide contains 23 nucleotides.

[0044] In some embodiments, the oligonucleotides are modified, for example, the oligonucleotides may include one or more modified nucleotides and / or one or more modified nucleoside bonds. For example, when used in various oligonucleotide constructs, modified nucleotides and modified nucleoside bonds can function to preserve the activity of oligonucleotide compounds in cells and / or to increase the serum stability of these compounds, and may also be used to minimize the possibility of activating human interferon activity at the time of administration.

[0045] Modified nucleotides In some embodiments, the oligonucleotides include modified nucleotides. In this specification, “modified nucleotide” refers to nucleotides other than naturally occurring ribonucleotides (nucleotides containing a 2'-OH sugar ring) or naturally occurring deoxyribonucleotides (nucleotides containing a 2'-H sugar ring). In some embodiments, at least 5% of the nucleotides (e.g., at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) are modified nucleotides. In some embodiments, 100% of the nucleotides are modified nucleotides. In some embodiments, 0% of the nucleotides are modified nucleotides. In this specification, modified nucleotides include, but are not limited to, 2'-modified nucleotides, debasalized nucleotides, 3'-3' linked (i.e., inverted) nucleotides, nucleotides containing modified nucleic acid bases, cross-linked nucleotides, 2',3'-seconucleotide mimetic (i.e., unlocked nucleic acid base analogs), locked nucleotides, 3'-O-methoxy(2'-nucleoside-linked) nucleotides, 2'-F-arabinonucleotides, 5'-methyl-2'-fluoronucleotides, morpholine nucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides. 2'-modified nucleotides (i.e., nucleotides having a group other than an -H or -OH group at the 2' position of a five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoronucleotides (or referred to as 2'-fluoronucleotides), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-aminonucleotides, and 2'-alkyl nucleotides. Not all positions of a given oligonucleotide need to be uniformly modified. Furthermore, two or more modifications can be incorporated at various nucleotides to which modifications are added, or even at a single nucleotide of the oligonucleotide.Modifications on one type of nucleotide may be separate from modifications on other nucleotides.

[0046] Modified nucleic acid bases include synthetic and natural nucleic acid bases, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) derivatives of adenine and guanine, and other alkyl derivatives. Examples include 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azocymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0047] In some embodiments, all or substantially all of the nucleotides in the oligonucleotide are modified nucleotides. In this specification, an oligonucleotide in which substantially all of the present nucleotides are modified nucleotides is an oligonucleotide having about 20% or less of unmodified ribonucleotides or unmodified deoxyribonucleotides.

[0048] Inter-modified nucleoside bonding In some embodiments, one or more nucleotides of oligonucleotides synthesized using the methods disclosed herein are linked by non-standard bonds or skeletons (i.e., bonds or skeletons between modified nucleosides). The bonds or skeletons between modified nucleosides include, but are not limited to, phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylenephosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidates, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl phosphonates, thionoalkylphosphotriesters, morpholino bonds, boranophosphates having a regular 3'-5' bond, 2'-5' bonded analogs of boranophosphates, or boranophosphates with inverted polarity in which adjacent pairs of nucleoside units are bonded from 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the bonds or skeletons between modified nucleosides lack a phosphorus atom. Examples of modified nucleoside bonds lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl intersugar bonds, mixed heteroatoms and alkyl or cycloalkyl intersugar bonds, or one or more short-chain heteroatom or heterocyclic intersugar bonds. Examples of modified nucleoside skeletons in some embodiments include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0049] In some embodiments, oligonucleotides synthesized using the methods disclosed herein contain one or more modified nucleotides and one or more modified nucleoside bonds. In some embodiments, a 2'-modified nucleoside is combined with a modified nucleoside bond.

[0050] Binding groups, targeting groups, and other non-nucleotide groups In some embodiments, oligonucleotides synthesized using the methods disclosed herein contain one or more binding groups, targeting groups, and / or other non-nucleotide groups. Binding groups can be added to the oligonucleotide to facilitate the binding of additional compounds, such as targeting groups or other non-nucleotide groups, such as PK enhancers. Targeting groups (or targeting moieties) are compounds that improve the pharmacokinetic or biodistribution properties of the attached oligonucleotide in order to improve the cell-specific (in some cases, organ-specific) distribution and cell-specific (or organ-specific) absorption of the oligonucleotide. Targeting groups may include one targeting ligand, two targeting ligands (referred to as "bidentate"), three targeting ligands ("terdentate"), four targeting ligands ("quadentate"), or more than four targeting ligands. Examples of targeting groups include compounds that have affinity for cell surface molecules or cell receptors. PK enhancers (also known as “pharmacokinetic (PK) modifiers”) are compounds that, when bound to oligonucleotides, can increase the in vivo systemic circulating time (i.e., increased half-life or plasma residence time) of a compound compared to the free form of an oligonucleotide by restricting its renal excretion without interfering with the delivery of the oligonucleotide to target cells or tissues. In some embodiments, PK enhancers can include molecules that are fatty acids, lipids, albumin binders, antibody binders, polyesters, polyacrylates, polyamino acids, and linear or branched polyethylene glycol (PEG) moieties having about 20 to 900 ethylene oxide (CH2-CH2-O) units.

[0051] For example, in some embodiments, oligonucleotides may be synthesized using phosphoramidites containing binding groups, such as TFA aminolink phosphoramidites, which may be commercially available (ThermoFisher) and which may be used to introduce a reactive linker (e.g., NH2-C6) to the 5' end of the oligonucleotide. In other embodiments, targeting group-containing phosphoramidite compounds may be used. These may include three N-acetylgalactosamines known to have affinity for asialoglycoprotein receptors and may target compounds that are attached to hepatocytes (and hepatocytes in particular), for example, phosphoramidite compounds having the following structure.

[0052] [ka]

[0053] In further embodiments, phosphoramidite compounds containing terminal alkyne groups may be used to facilitate binding to various targeting groups and / or other non-nucleotide groups. These types of binders may be added according to the methods disclosed herein. Examples of preferred trialryakhine binders include:

[0054] [ka] TIFF2026530566000004.tif198123 TIFF2026530566000005.tif254118

[0055] oligonucleotide synthesis Solid-phase oligonucleotide synthesis method This disclosure provides methods for synthesizing oligonucleotides. In some embodiments, the methods for synthesizing oligonucleotides disclosed herein are carried out in a reaction flask or reaction vessel. In some embodiments, the reaction flask or reaction vessel is equipped with a lower filter to allow for the removal of the solution from a solid support held in the vessel by a filter. In some embodiments, the pores of the filter are small enough to prevent target support particles from passing through the filter, and large enough to allow impurities (e.g., unused reagents and other synthesis by-products) to be removed from the flask or vessel. Generally, a reaction column is packed with a solid support, and the solid support in the column remains stationary, while the solvent and reagents flow through the column. A solid support may also be used in a reaction flask or reaction vessel, but the solid support is generally stirred or agitated to create movement. The solvent and reagents in the reaction flask or reaction vessel get the opportunity to interact with the molecules supported on the solid support by stirring or agitation.

[0056] In some embodiments, the reaction flask or reaction vessel may be equipped with a stirrer, a mixer, or other mechanism for stirring or agitating the solid support. By stirring or agitating the solid support, the effective layer depth of the solid support may be increased compared to the layer depth of an equivalent column reactor (typically about 5–10 cm). Thus, stirring or agitating the solid support results in more efficient use of the solid support. In some embodiments, the solid support used in the methods disclosed herein includes polystyrene or controlled porous glass (CPG). In some embodiments, soluble support, such as polynorbornene or other soluble support useful in cyclic synthesis, is used. Other examples of soluble support that may be used are disclosed, for example, in PCT Publication No. 2012 / 165545.

[0057] In some embodiments, the method for synthesizing oligonucleotides disclosed herein involves stirring or agitating a reaction mixture in one or more (e.g., all) steps of the method. In some embodiments, the reaction mixture is agitated at a speed of at least 10 revolutions per minute (rpm). In some embodiments, the reaction mixture is agitated at a speed of at least about 10, 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, or about 1000 revolutions per minute (rpm). In some embodiments, the reaction mixture is agitated at a speed of about 100 rpm to about 600 rpm. In some embodiments, some or all of the reactions disclosed herein occur in a rocker that agitates the reaction mixture.

[0058] Any suitable solid support known in the art may be used to carry out the methods disclosed herein. In some embodiments, the solid support is a polymer resin, for example, Nittophase® or TentaGel®, or controlled porous glass (CPG). TentaGel® resin is a graft copolymer consisting of a low-crosslinked polystyrene matrix grafted with poly(ethylene glycol) (PEG or POE). Nittophase® resin is a crosslinked polystyrene polymer particle with a diameter of approximately 90 microns.

[0059] The reaction flasks or vessels used in the methods disclosed herein may be of any design known in the art. In some embodiments, larger vessels, such as those larger than a 50 L reactor, are used in the methods. Those skilled in the art will recognize that performing oligonucleotide synthesis in such larger reaction vessels allows for larger batch sizes. The methods disclosed herein do not require the use of specialized column reactors (in the case of flow-through methods), thus effectively eliminating the limitations on batch size.

[0060] In some embodiments, oligonucleotide assemblies are generated on a solid support through a stepwise reaction cycle, for example, a series of steps in a stepwise reaction cycle disclosed herein. Exemplary conditions for each reaction step are further disclosed in the examples.

[0061] In some embodiments, the reaction process (e.g., a stepwise reaction cycle) includes a deblocking step, which involves deblocking the trityl group of a support nucleotide (e.g., a single support nucleotide or the terminal nucleotide of a plurality of supported bonded nucleotides), thereby resulting in a detritylated support nucleotide. In some embodiments, the support nucleotide comprises a plurality of nucleotides, i.e., oligonucleotides. In some embodiments, the support nucleotide is directly covalently bonded to the substrate. In some embodiments, the support nucleotide is indirectly covalently bonded to the substrate (e.g., by a non-nucleotide moiety). In some embodiments, the support nucleotide is indirectly bonded to the substrate by a non-nucleotide moiety, e.g., an inverted debase moiety, a C3 cap, or a C6 cap.

[0062] In some embodiments, the deblocking step liberates the 5'-hydroxyl group of the detritylated support nucleotide. In some embodiments, deblocking is performed with a reagent comprising (i) an acid and (ii) an organic solvent. In some embodiments, deblocking is the first step in a stepwise reaction cycle.

[0063] In some embodiments, the reaction process (e.g., a stepwise reaction cycle) includes a washing step, which involves washing the detritylated support nucleotide. In some embodiments, the washing solution comprises (i) a neutralizing solvent, (ii) an alcohol or water, and (iii) an organic solvent. In some embodiments, the washing solution comprises pyridine, methanol, and toluene.

[0064] In some embodiments, the reaction process (e.g., a stepwise reaction cycle) includes a coupling step, which involves coupling a nucleotide building block to a detritylated support nucleotide, thereby forming a new phosphite bond between the nucleotide building block and the support nucleotide. In some embodiments, the nucleotide building block comprises a phosphoramidite or an N-acetylgalactosamine (NAG) ligand.

[0065] In some embodiments, the NAG ligand is the NAG37 ligand shown below: (NAG37)

[0066] [ka] NAG37 can be synthesized according to methods known in the art; see U.S. Publication No. 20180064819, which is incorporated herein by reference in its entirety, including those relating to the synthesis of the NAG37 ligand.

[0067] In some embodiments, the NAG ligand is the NAG52 ligand shown below.

[0068] [ka]

[0069] In some embodiments, the coupling step uses coupling activators, such as 5-(ethylthio)-1H-tetrazole (ETT), 4,5-dicyanoimidazole (DCI), N-methylimidazole (NMI), and / or 1H-tetrazole. Other examples of coupling activators include, but are not limited to, 2-benzylthiotetrazole, 45-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 2-bromo-4,5-dicyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, 1-(cyanomethyl)piperidinium tetrafluoroborate, and 1-methyl-1H-benzoimi Examples include dazole-3-ium trifluoromethanesulfonate, 1-phenyl-1H-imidazole-3-ium trifluoromethanesulfonate, 1H-benzimidazole-3-ium trifluoromethanesulfonate, 1H-imidazole-3-ium trifluoromethanesulfonate, 5-nitro-1H-benzimidazole-3-ium trifluoromethanesulfonate, benzimidazolium triflate, or imidazolium triflate. In some embodiments, the nucleotide building block is ligated to a detritylated carrier-supported nucleotide after a washing step.

[0070] In some embodiments, the reaction process (e.g., a stepwise reaction cycle) includes an oxidation step, comprising oxidizing the phosphite bond between the nucleotide building block and the carrier-supported nucleotide using an oxidizing reagent to form a phosphate triester bond. In some embodiments, the oxidizing reagent is a halogen / base / aqueous solution (e.g., iodine / pyridine / aqueous solution). In some embodiments, the base includes pyridine or a derivative thereof. Other suitable bases include, but are not limited to, pyridine derivatives, e.g., lutidine and colidine. Other suitable oxidizing reagents include tert-butyl hydroperoxide (TBHP) and (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO), which may be used under anhydrous conditions. In some embodiments, the reaction process (e.g., a stepwise reaction cycle) includes a thiolation step, comprising thiolation of the phosphite bond between the nucleotide building block and the carrier-supported nucleotide using a thiolation reagent to form a phosphorothioate bond. Examples of suitable thiolation reagents include xanthan hydrides.

[0071] In some embodiments, the reaction process is repeated one or more times. In some embodiments, at the beginning of a new cycle, the 5'-O-DMT group of the linked product is deblocked (e.g., through the same deblocking step), and subsequently, another nucleotide is added to the molecular chain (e.g., through the same coupling step). In some embodiments, the process is repeated until an oligomer of the desired length is obtained.

[0072] In some embodiments, by-products and excess reagents in the solution are removed after some or all of the reaction steps by filtration and by washing the carrier with a washing solution, such as acetonitrile solvent.

[0073] In some embodiments, the crude reaction product is analyzed and quality-checked, for example, by RP HPLC-MS, before further / final purification.

[0074] In some embodiments, the method of the Disclosure yields a yield of 95% or more per cycle, for example, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more. In some embodiments, the method of the Disclosure yields a yield of about 70% or more for the final synthetic oligonucleotide composition (e.g., after final purification). In some embodiments, the method of the Disclosure yields a total yield of at least 0.5 mmol of oligonucleotide after a single reactor run. In some embodiments, the method yields a total yield of at least 20 mmol, at least 60 mmol, at least 1 mole, at least 1.5 moles, or at least 2 moles after a single reactor run.

[0075] In some embodiments, the method of the present disclosure has a coupling efficiency of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.

[0076] In some embodiments, the method of the present disclosure produces oligonucleotides that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% free of shorters and / or other contaminants (e.g., stray nucleotides or other oligonucleotide fragments).

[0077] Detritylation In some embodiments, the methods of the present disclosure (e.g., stepwise reaction cycles) include a deblocking step, including detritylation. In some embodiments, oligonucleotide synthesis includes protecting the 5'-ribose alcohol of the nucleotide with, for example, a triphenylmethyl (i.e., "trityl") protecting group during synthesis. In some embodiments, the trityl group is 4,4'-dimethoxytrityl or DMT or 5'-DMT. An exemplary DMT protecting group is shown in the following reaction scheme as a protecting group for a solid carrier-bound nucleoside containing the terminal 3' base of the oligonucleotide. In some embodiments, the DMT protecting group prevents polymerization of the nucleoside during the functionalization of the solid carrier resin.

[0078] In some embodiments, the oligonucleotide synthesis method includes removing the trityl group to yield a free alcohol, as shown in the reaction scheme below. In some embodiments, the 2'-ribose alcohol of the nucleotide is protected with a protecting group, for example, a tert-butyldimethylsilyl (TBS) protecting group, as shown in the reaction scheme below. In some embodiments, the phosphoramidite monomer used in oligonucleotide synthesis contains a TBS group attached to the 2'-OH of a 5'-DMT-protected nucleoside, yielding a 2'-TBS-protected nucleoside.

[0079] [ka]

[0080] In some embodiments, the detritylation reaction is carried out using acetic acid and an organic solvent. In some embodiments, the acetic acid includes haloacetic acid [e.g., trifluoroacetic acid (TFA) or dichloroacetic acid (DCA)]. In some embodiments, the acetic acid includes fluoroacetic acid [e.g., trifluoroacetic acid (TFA) or pyridine trifluoroacetate (PTA)]. In some embodiments, the acetic acid includes trifluoroacetic acid (TFA). In some embodiments, the acetic acid includes pyridine trifluoroacetate (PTA). Without being bound by theory, it is considered that using fluoroacetic acid, such as TFA, in the detritylation step of an oligonucleotide synthesis method can reduce or prevent shorter formation compared to a method that does not include, for example, a capping step. For example, Examples 1 to 4 disclose studies of oligonucleotide synthesis in which TFA is used for detritylation in process 1 and the subsequent capping step is not included. In contrast to process 1, process 2 uses DCA for detritylation and does not include a washing step including pyridine and MeOH, but includes a capping step. Compared to Process 2, Process 1 was shown to yield similar product purity and reduced shorter formation despite the differences mentioned above (such as the absence of a capping step).

[0081] In some embodiments, acetic acid includes chlorinated acetic acid. In some embodiments, acetic acid is chloroacetic acid. In some embodiments, acetic acid is dichloroacetic acid (DCA). In some embodiments, acetic acid is trichloroacetic acid. In some embodiments, acetic acid is a combination of chloroacetic acid, dichloroacetic acid, and trichloroacetic acid.

[0082] In some embodiments, the organic solvent used in the detritylation reaction includes an arene. In some embodiments, the arene is selected from alkylbenzenes, alkenylbenzenes, alkynylbenzenes, or any combination thereof. In some embodiments, the arene is toluene, xylene, hemimelitene, pseudodocumene, mesitylene, prenitene, isodurene, durene, pentamethylbenzene, hexamethylbenzene, ethylbenzene, ethyltoluene, propylbenzene, propyltoluene, butylbenzene, pentanylbenzene, pentanyltoluene, hexanylbenzene, hexanyltoluene, diphenylmethane, triphenylmethane, tetraphenylmethane, or 1,2-diphenylethane, or a combination thereof.

[0083] In some embodiments, the organic solvent used in detritylation includes dichloromethane.

[0084] In some embodiments, the organic solvent includes toluene, xylene, dichloromethane, or a combination thereof.

[0085] In some embodiments, the detritylation reaction is carried out using acetic acid in an organic solvent, with an acetic acid-to-organic solvent ratio of about 1 to about 30 volume% acetic acid to about 70 to about 99 volume% organic solvent. In some embodiments, the acetic acid-to-organic solvent ratio in the detritylation reagent is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 volume% acetic acid to about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 volume% organic solvent.

[0086] In some embodiments, the detritylation reaction may be carried out using acetic acid in a detritylation solution at concentrations of about 0.005 M to about 0.2 M, for example, about 0.0075 M to about 0.2 M, about 0.008 M to about 0.2 M, about 0.01 M to about 0.2 M, about 0.015 M to about 0.2 M, about 0.01 M to about 0.2 M, about 0.02 M to about 0.2 M, about 0.04 M to about 0.2 M, about 0.05 M to about 0.2 M, about 0.1 M to about 0.2 M, about 0.005 M to about 0.1 M, about 0.01 M to about 0.1 M, about 0.02 M to about 0.1 M, about 0.04 M to about 0.1 M, and about 0.05 M to about 0.1 M. In some embodiments, the detritylation reaction may be carried out using acetic acid at a concentration of about 0.01 M to about 0.2 M in the detritylation solution.

[0087] In some embodiments, the organic solvent used in the detritylation reaction includes protonating solvents. Protonating solvents are solvents having hydrogen atoms bonded to oxygen (such as a hydroxyl group), hydrogen atoms bonded to nitrogen (such as an amine group), or hydrogen atoms bonded to a fluoride (such as hydrogen fluoride). Non-limiting examples of protonating solvents include formic acid, n-butanol, isopropanol (IPA), nitromethane, ethanol (EtOH), methanol (MeOH), acetic acid (AcOH), and water.

[0088] In some embodiments, the detritylation reaction involves an alcohol. In some embodiments, the alcohol is a sterically hindered alcohol. In some embodiments, the alcohol is a general formula

[0089] [ka] It has, in the formula: R 1 , R 2 , and R 3 Each of these is independently a hydrogen atom, a halogen, an aliphatic chain (e.g., linear or branched alkyl, alkenyl, alkynyl), a heteroaliphatic chain (e.g., linear or branched alkoxy, thioalkoxy, amine), a carbocyclic ring, or a heterocyclic ring; however, R 1 , R2 and R 3 , no more than one of which is hydrogen; or R 1 and R 2 or R 2 and R 3 , together with the carbon atoms therebetween, form a carbocyclic ring or a heterocyclic ring.

[0090] In some embodiments, the alcohol used in the detritylation step comprises tert-butanol (TBA), isopropanol (IPA), neo-pentanol, trifluoroethanol (TFE), phenol (PhOH), benzyl alcohol (BnOH), cyclohexanol, sterol, or a combination thereof. In some embodiments, the alcohol used in the detritylation step comprises trifluoroethanol (TFE).

[0091] Coupling In some embodiments, the method of the present disclosure (e.g., a stepwise reaction cycle) comprises a coupling step comprising coupling a nucleotide building block to a carrier-supported nucleotide. In some embodiments, the 5' end of the carrier-supported nucleotide is deblocked and washed, and then a new nucleotide is linked to the carrier-supported nucleotide. A coupling reaction using a phosphoramidite is exemplified in the scheme below.

[0092]

Chemical Formula

[0093] In some embodiments, as shown in the scheme above, the nucleotide added to the strand is a phosphoramidite. In some embodiments, the nucleotide added to the strand is a phosphate. In some embodiments, the nucleotide added to the strand is a phosphonate. In some embodiments, the nucleotide to be added comprises a plurality of linked nucleotides, for example, two or more linked nucleotides comprising phosphate.

[0094] In some embodiments, the nucleotides to be attached to the strand are added to a solution containing a solvent. In some embodiments, the nucleotides are added to a solution, and the solvent is acetonitrile. In some embodiments, the nucleotides to be attached to the strand are added to a solution, and the nucleotide concentration in the solution is approximately 0.05 to approximately 2.0 M. In some embodiments, the nucleotides to be attached to the strand are added to a solution, and the nucleotide concentrations are approximately 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1. From 5, 1.6, 1.7, 1.8, or 1.9M up to approximately 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0M.

[0095] In some embodiments, the nucleotides added to the strand are added in a low molar equivalent relative to the nucleotides supported on the solid carrier. In some embodiments, the additional nucleotides are added in amounts ranging from 1.0 equivalent to about 5.0 equivalents. In some embodiments, the added nucleotides are in amounts of about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, or 4 The amount added ranges from 0.9 molar equivalents to approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 molar equivalents relative to the nucleotides supported on the strand.

[0096] In some embodiments, the coupling reaction is carried out using an activator. In some embodiments, the activator is an acidic azole. In some embodiments, the activator is a tetrazole derivative or a salt complex thereof. In some embodiments, the activator is selected from 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, and 4,5-dicyanoimidazole. Examples of other coupling activators, but not limited to, include 2-benzylthiotetrazole, 5-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 2-bromo-4,5-dicyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, 1-(cyanomethyl)piperidinium tetrafluoroborate, and 1-methyl-1H-benzoimidazole. Examples include dazole-3-ium trifluoromethanesulfonate, 1-phenyl-1H-imidazole-3-ium trifluoromethanesulfonate, 1H-benzimidazole-3-ium trifluoromethanesulfonate, 1H-imidazole-3-ium trifluoromethanesulfonate, 5-nitro-1H-benzimidazole-3-ium trifluoromethanesulfonate, benzimidazolium triflate, or imidazolium triflate. In some embodiments, the activator is 5-ethylthio-1H-tetrazole (ETT). In some embodiments, the activator is added in an amount of about 1 to about 30 molar equivalents relative to the amount of nucleotides supported on the solid carrier. In some embodiments, the activator is added in amounts ranging from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 molar equivalents relative to the amount of nucleotides supported on the solid carrier, up to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 molar equivalents.

[0097] In some embodiments, the reagents used in the coupling step include a nucleotide (or multiple linked nucleotides) to be added to the strand, an activator, and a solvent. In some embodiments of the coupling step, the activator is concentrated to about 0.1 M to about 5 M. In some embodiments, the activator concentration is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, and also The range is from 4.9M to approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or up to 5.0M. In some embodiments, the reagent used in the coupling step contains an activator and the nucleotide to be added in a ratio of approximately 0.1 moles of activator to approximately 5.0 moles of nucleotide to be added to the strand.In some embodiments, the ratio of the activator to the nucleotides added to the strand is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4. The ratio of 8 or 4.9 moles to the nucleotides added to the strand is approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 moles. In some embodiments, the ratio of activator to nucleotides added to the strand is approximately 1:1.

[0098] In some embodiments, the coupling process is performed in a time of approximately 1 minute to approximately 60 minutes. In some embodiments, the coupling process is performed in a time of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, It will run from 59 minutes, or up to approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes.

[0099] Oxidation / thiolation In some embodiments, the coupling of a new nucleotide to a strand of a support nucleotide (e.g., by using a phosphoramidite) yields a coupling product containing phosphite. In some embodiments, the method of the Disclosure (e.g., a stepwise reaction cycle) includes an oxidation step comprising oxidizing the phosphite bond between the added nucleotide building block and the support nucleotide. In some embodiments, the method of the Disclosure (e.g., a stepwise reaction cycle) includes a thiolation step comprising thioling the phosphite bond between the added nucleotide building block and the support nucleotide.

[0100] In some embodiments, the oxidation / thiolation step includes oxidizing / thioling phosphorous acid to phosphoric acid or phosphorothioate, as exemplified in the following reactions.

[0101] [ka]

[0102] In some embodiments, the phosphorus atom is oxidized to produce phosphoric acid. In some embodiments, oxidation is carried out using a halogen in the presence of water and a base, such as pyridine, lutidine, and colidine. In some embodiments, oxidation is carried out using iodine (I2) in a solvent of water and pyridine. In some embodiments, the water-to-pyridine ratio in the oxidation step is about 1 vol% to about 20 vol% water to about 80 vol% to about 99 vol% pyridine. In some embodiments, the water-to-pyridine ratio is about 10 vol% water to about 90 vol% pyridine.

[0103] In some embodiments, oxidation is carried out under anhydrous conditions using tert-butyl hydroperoxide (TBHP), (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO), or a combination thereof.

[0104] In some embodiments, the phosphorus atom is thiolated to produce a phosphorothioate. In some embodiments, the thiolation reaction is carried out using a thiolation reagent, such as 3-(dimethylaminomethylidene)amino-3H-1,2,4,-dithiazole-3-thion (DDTT). In some embodiments, the thiolation reagent is xanthan hydride.

[0105] In some embodiments, the iodine concentration in the oxidation solution is about 10 mM to about 200 mM. In some embodiments, the iodine concentration in the oxidation solution ranges from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 mM to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mM.

[0106] In some embodiments, the oxidation process is carried out in about 1 to 30 minutes. In some embodiments, the oxidation step is carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 minutes, or for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes.

[0107] Capping The capping step typically involves capping unreacted solid support-borne 5'-OH groups or other free alcohols produced by unintended side reactions. In some embodiments, the capping reagents include hydroxy protecting groups, such as esters and ethers. Non-limiting examples of suitable ethers and esters include methoxymethyl ether or MOM ether, tetrahydropyranyl ether or THP ether, tert-butyl ether, allyl ether, benzyl ether, tert-butyldimethylsilyl ether or TBDMS ether, tert-butyldiphenylsilyl ether or TBDPS ether, acetate esters, pivalates, benzoates, and the like. In one embodiment, unreacted 5'-OH groups are capped with acetic anhydride.

[0108] Oligonucleotide synthesis methods known in the art typically employ a capping step to reduce or prevent the formation of shorters from the reaction and to improve the purity of the final yield. In some embodiments, the present disclosure employs oligonucleotide synthesis methods that do not involve a capping step. In some embodiments, avoiding the capping step in the oligonucleotide synthesis method results in a higher crude yield compared to an equivalent method that includes a capping step. In some embodiments, the crude yield of the capping-free method is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% higher than the crude yield of an equivalent method that includes a capping step.

[0109] In some embodiments, the absence of a capping step in the oligonucleotide synthesis method results in a higher overall final yield compared to an equivalent method that includes a capping step. In some embodiments, the final yield of the capped method is approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% higher than the final yield of the equivalent method that includes a capping step.

[0110] In some embodiments, the absence of a capping step in the oligonucleotide synthesis method results in a reduction in shorting compared to an equivalent method that includes a capping step.

[0111] Cleaning In some embodiments, the washing step is performed after some or all of the oligonucleotide synthesis steps disclosed herein (e.g., detritylation, ligation, oxidation). In some embodiments, the washing step is performed by draining the reactants from a container. In some embodiments, the washing solution comprises (i) a neutralizing solvent, (ii) an alcohol or water, and (iii) an organic solvent. In some embodiments, the washing solution comprises pyridine, methanol, and toluene. In some embodiments, the washing solution (e.g., a washing solution containing an organic solvent) is added to the reaction flask and stirred to remove unwanted components from the product. In some embodiments, the washing solution (e.g., a washing solution containing an organic solvent) is added to the reaction vessel, and the vessel is stirred or agitated for at least 30 seconds before draining the organic solvent. In some embodiments, the washing step is repeated two or more times to remove unwanted substances.

[0112] Non-limiting examples of suitable solvents for the washing step include pyridine, methanol, tetrahydrofuran (THF), ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), nitromethane, propylene carbonate, or combinations thereof. In some embodiments, the washing solvent includes acetonitrile (MeCN), pyridine, methanol, or combinations thereof. Without being bound by theory, the use of a pyridine and methanol combination in the washing step of a method that does not include a capping step is thought to help reduce or prevent shorter formation. [Examples]

[0113] Further explanation is provided in the following examples.

[0114] The following abbreviations are used in the examples to describe nucleotides or oligonucleotides: a=2'-O-methyladenosine-3'-phosphate as=2'-O-methyladenosine-3'-phosphorothioate c=2'-O-methylcytidine-3'-phosphate g = 2'-O-methylguanosine-3'-phospho gs = 2'-O-methylguanosine-3'-phosphorothioate i=2'-O-methylinosine-3'-phosphate u = 2'-O-methyluridine-3'-phosphate us=2'-O-methyluridine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Gf = 2'-fluoroguanosine-3'-phospho Uf = 2'-fluorouridine-3'-phosphorate Ufs = 2'-fluorouridine-3'-phosphorothioate invAb = Inverted debasal deoxyribonucleotide: If placed inside:

[0115] [ka] If placed at the 3' end:

[0116] [ka] invAbs = Inverted debase deoxyribonucleotide-5'-phosphorothioate: If placed inside:

[0117] [ka] s = phosphorothioate bond Sp18s = Spacer 18 having the following structure, hexaethylene glycol:

[0118] [ka] [Example 1]

[0119] Comparison of oligonucleotide synthesis methods in the synthesis of SEQ ID NO:1 In this example, oligonucleotides containing SEQ ID NO:1(5'-NAG37s,invAbs,c,c,u,g,u,u,u,u,Gf,c,Uf,u,Uf,u,g,u,a,a,c,u,us,invAb-3') were synthesized according to various process parameters and reaction conditions, and are summarized in Tables 1 and 2 below.

[0120] [Table 1]

[0121] [Table 2]

[0122] Process 1 Synthesis: 1 mmol of invAb-packed Nittophase HL (250 μmol / g) was packed into a Fineline 35 synthesis column. Oligonucleotide SEQ ID NO: 1 was synthesized on a 1 mmol scale in an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 1. After synthesis, the support was dried, and the oligonucleotide was then cleaved from the solid support using 50 mL of methylamine (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm polyethersulfone (PES) membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed in an AKTA Crossflow equipped with a 2 kDa molecular weight cutoff (MWC) regenerated cellulose membrane for diafiltration and desalting. The solution was first concentrated to 300 mL, dialyzed with 7 diameters against 0.2 M NaCl, then dialyzed with 10 diameters against deionized water, and subsequently concentrated to a final volume of 150 mL before being eluted from the system. The crude material and final product were analyzed by RP HPLC-MS.

[0123] Process 2 Synthesis: 1 mmol of invAb-packed Nittophase HL (250 μmol / g) was packed into a Fineline 35 synthesis column. The oligonucleotide with SEQ ID NO: 1 was synthesized on a 1 mmol scale using an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 2. After synthesis, the support was dried, and the oligonucleotide was then cleaved from the solid support using 50 mL of methylamine solution (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was packed into a 5 × 19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 μm). Crude material was purified using a B10–60% gradient over a 15CV column volume (mobile phase A: 20 mM sodium phosphate, 10% ACN pH 11.0 and mobile phase B: 20 ​​mM sodium phosphate, 1 M NaBr, 10% ACN pH 11.0). The pure fraction was pooled and then placed on an AKTA Crossflow with a 2 kDa MWC regenerated cellulose membrane for desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 10 Dia volume against deionized water, then concentrated to a final volume of 150 mL, and eluted from the system. Crude material and final product were analyzed by RP HPLC-MS.

[0124] Results: The crude yield, crude purity, final yield after purification, final purity, and -NAG impurities for Process 1 and Process 2 are disclosed in Table 3 below. Figure 1 shows RP HPLC-MS chromatograms comparing Process 1 (underlined in Figure 1) and Process 2 (top line in Figure 1).

[0125] [Table 3] [Example 2]

[0126] Comparison of oligonucleotide synthesis methods in the synthesis of SEQ ID NO:2 In this example, the oligonucleotide containing SEQ ID NO:2(5'-as,Afs,g,u,u,a,c,a,a,a,a,Gf,c,Af,a,Af,a,c,as,gs,g-3') was synthesized according to the process parameters and reaction conditions of Process 1 and Process 2 listed in Tables 1 and 2 of Example 1 above.

[0127] Process 1 Synthesis: 1 mmol of 2'OMe-G packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. Oligonucleotides with SEQ ID NO: 2 were synthesized on a 1 mmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 1. After synthesis, the support was dried, and the oligonucleotides were then cleaved from the solid support using 50 mL of methylamine (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed in an AKTA Crossflow equipped with a 2 kDa MWC regenerated cellulose membrane for diafiltration and desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 7 diavolts against 0.2 M NaCl, dialyzed at 10 diavolts against deionized water, and then concentrated to a final volume of 150 mL before being eluted from the system. Crude materials and final products were analyzed by RP HPLC-MS.

[0128] Process 2 Synthesis: 1 mmol of 2'OMe-G packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. The oligonucleotide with SEQ ID NO: 2 was synthesized on a 1 mmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 2. After synthesis, the support was dried, and the oligonucleotide was then cleaved from the solid support using 50 mL of methylamine solution (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was packed into a 5 × 19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 μm). Crude material was purified using a B10–60% gradient over a 15CV column volume (mobile phase A: 20 mM sodium phosphate, 10% ACN pH 11.0 and mobile phase B: 20 ​​mM sodium phosphate, 1 M NaBr, 10% ACN pH 11.0). The pure fraction was pooled and then placed on an AKTA Crossflow with a 2 kDa MWC regenerated cellulose membrane for desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 10 Dia volume against deionized water, then concentrated to a final volume of 150 mL, and eluted from the system. Crude material and final product were analyzed by RP HPLC-MS.

[0129] Results: The crude yield, crude purity, final yield after purification, final purity, and -NAG impurities for Process 1 and Process 2 are disclosed in Table 4 below. Figure 2 shows RP HPLC-MS chromatograms comparing Process 1 (underlined in Figure 2) and Process 2 (top line in Figure 2).

[0130] [Table 4] [Example 3]

[0131] Comparison of oligonucleotide synthesis methods in the synthesis of SEQ ID NO:3 In this example, the oligonucleotide containing SEQ ID NO:3(5'-NAG37s,invAbs,a,c,c,c,u,a,c,u,Cf,Uf,Gf,u,u,g,u,u,c,g,a,a,as,invAb-3') was synthesized according to the process parameters and reaction conditions of Process 1 and Process 2 listed in Tables 1 and 2 of Example 1 above.

[0132] Process 1 Synthesis: 1 mmol of 2'OMe-G packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. The oligonucleotide with SEQ ID NO: 3 was synthesized on a 1 mmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 1. After synthesis, the support was dried, and the oligonucleotide was then cleaved from the solid support using 50 mL of methylamine (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed in an AKTA Crossflow equipped with a 2 kDa MWC regenerated cellulose membrane for diafiltration and desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 7 diavolts against 0.2 M NaCl, dialyzed at 10 diavolts against deionized water, and then concentrated to a final volume of 150 mL before being eluted from the system. Crude materials and final products were analyzed by RP HPLC-MS.

[0133] Process 2 Synthesis: 1 mmol of 2'OMe-G packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. The oligonucleotide with SEQ ID NO: 3 was synthesized on a 1 mmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 2. After synthesis, the support was dried, and the oligonucleotide was then cleaved from the solid support using 50 mL of methylamine solution (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was packed into a 5 × 19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 μm). Crude material was purified using a B10–60% gradient over a 15CV column volume (mobile phase A: 20 mM sodium phosphate, 10% ACN pH 11.0 and mobile phase B: 20 ​​mM sodium phosphate, 1 M NaBr, 10% ACN pH 11.0). The pure fraction was pooled and then placed on an AKTA Crossflow with a 2 kDa MWC regenerated cellulose membrane for desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 10 Dia volume against deionized water, then concentrated to a final volume of 150 mL, and eluted from the system. Crude material and final product were analyzed by RP HPLC-MS.

[0134] Results: The crude yield, crude purity, final yield after purification, final purity, and -NAG impurities for Process 1 and Process 2 are disclosed in Table 5 below. Figure 3 shows RP HPLC-MS chromatograms comparing Process 1 (underlined in Figure 2) and Process 2 (top line in Figure 2).

[0135] [Table 5] [Example 4]

[0136] Comparison of oligonucleotide synthesis methods in the synthesis of SEQ ID NO:4 In this example, the oligonucleotide containing SEQ ID NO:4(5'-us,Ufs,us,Cf,g,Af,a,c,a,a,c,Af,g,Af,g,Uf,a,Gf,Gf,gs,u-3') was synthesized according to the process parameters and reaction conditions of Process 1 and Process 2 listed in Tables 1 and 2 of Example 1 above.

[0137] Process 1 Synthesis: 1 mmol of 2'OMe-U packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. Oligonucleotides with SEQ ID NO: 4 were synthesized on a 1 mmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 1. After synthesis, the support was dried, and the oligonucleotides were then cleaved from the solid support using 50 mL of methylamine (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed in an AKTA Crossflow equipped with a 2 kDa MWC regenerated cellulose membrane for diafiltration and desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 7 diavolts against 0.2 M NaCl, dialyzed at 10 diavolts against deionized water, and then concentrated to a final volume of 150 mL before being eluted from the system. Crude materials and final products were analyzed by RP HPLC-MS.

[0138] Process 2 Synthesis: 1 mmol of 2'OMe-G packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. Oligonucleotides with SEQ ID NO: 4 were synthesized on a 1 mmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 2. After synthesis, the support was dried, and the oligonucleotides were then cleaved from the solid support using 50 mL of methylamine solution (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was packed into a 5 × 19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 μm). Crude material was purified using a B10–60% gradient over a 15CV column volume (mobile phase A: 20 mM sodium phosphate, 10% ACN pH 11.0 and mobile phase B: 20 ​​mM sodium phosphate, 1 M NaBr, 10% ACN pH 11.0). The pure fraction was pooled and then placed on an AKTA Crossflow with a 2 kDa MWC regenerated cellulose membrane for desalting. The solution was first concentrated to a volume of 300 mL, dialyzed at 10 Dia volume against deionized water, then concentrated to a final volume of 150 mL, and eluted from the system. Crude material and final product were analyzed by RP HPLC-MS.

[0139] Results: The crude yield, crude purity, final yield after purification, final purity, and -NAG impurities for Process 1 and Process 2 are disclosed in Table 6 below. Figure 4 shows RP HPLC-MS chromatograms comparing Process 1 (underlined in Figure 4) and Process 2 (top line in Figure 4).

[0140] [Table 6] [Example 5]

[0141] Process 3: Method for synthesizing long-chain (dimeric) oligonucleotides In this embodiment, an oligonucleotide having the following characteristics was synthesized: NAG37s was attached at the 5' end to a 19-nucleotide segment of a chemically modified nucleotide (either a 2'-fluoro-modified nucleotide or a 2'-methoxy-modified nucleotide) having an inverted debasement cap portion at its 3' end, and then this was attached as an Sp18s spacer to a second 19-nucleotide segment of a chemically modified nucleotide (either a 2'-fluoro-modified nucleotide or a 2'-methoxy-modified nucleotide) having an inverted debasement cap portion at its 3' end.

[0142] NAG37s,c,g,u,u,u,u,Gf,c,Uf,u,Uf,u,g,u,a,a,c,u,us,invAb,Sp18s,gs,g,g,a,c,a,Gf,Uf,Af,u,u,c,u,c,a,g,u,i,as,invAb(SEQ ID NO:5). The abbreviation is deciphered from the above paragraph.

[0143] It is located at (

[0107] ).

[0144] The oligonucleotides were synthesized according to the process parameters and reaction conditions of Process 3, and are summarized in Table 7 below.

[0145] [Table 7]

[0146] Process 3 for long-chain synthesis: 220 μmol of invAb-packed Nittophase HL (100 μmol / g) was packed into a 24 mL synthesis column. Oligonucleotides were synthesized on a 220 μmol scale using an AKTA oligopilot100 synthesizer according to the parameters and reaction conditions in Table 7. After synthesis, the support was dried, and then half of the support (110 μmol) was cleaved from the oligonucleotides using 11 mL of ammonium hydroxide solution (28% in water) at room temperature for approximately 7 days. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 250 mL with deionized water. The crude solution was packed into a 5 × 13.7 cm column packed with Tosoh TSKGel SuperQ-5PW (20 μm). Crude material was purified using a B10–60% gradient over a 15CV column (mobile phase A: 20 mM sodium phosphate, 10% ACN pH 11.0 and mobile phase B: 20 ​​mM sodium phosphate, 1 M NaBr, 10% ACN pH 11.0). The pure fraction was pooled. Crude material and final product were analyzed by RP HPLC-MS and AEX HPLC.

[0147] Results: For Process 3, the crude yield, crude purity, final yield after purification, final purity, and -NAG impurities are disclosed in Table 8 below.

[0148] [Table 8] [Example 6]

[0149] Comparison of Extended OCD Synthesis Methods In this embodiment, oligonucleotides from Example 2 (SEQ ID NO:2) were synthesized according to the process parameters and reaction conditions of Process 4 and Process 5, listed in Tables 9 and 10 below, using an Oligopilot 400 synthesizer (including longer reaction times and slower linear flow rates required for large-scale production), but designed to simulate a six-fold increase in scale using smaller-scale packing. Process 4 is equivalent to Process 1 from Example 2, including an extended reaction time. Process 5 is equivalent to Process 2 from Example 2, including an extended reaction time.

[0150] [Table 9]

[0151] [Table 10]

[0152] 1 mmol of 2'OMe-G packed Nittophase HL (350 μmol / g) was packed into a Fineline 35 synthesis column. Oligonucleotides were synthesized on an AKTA oligopilot 100 synthesizer on a 1 mmol scale using the parameters and reaction conditions for both Process 4 and Process 5 as shown in Tables 9 and 10. The flow rates for these syntheses were capped to simulate the contact time required to synthesize 428.5 mmol on an Oligopilot 400, and further longer contact times were also obtained. After synthesis, the support was dried, and the oligonucleotides were then cleaved from the support using 50 mL of methylamine (40% in water) at room temperature for 100 minutes. The crude solution was filtered using a 0.45 μm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude material and final product were analyzed by RP HPLC-MS.

[0153] Results: The crude products for Process 4 and Process 5, along with those for Process 1 and Process 2 from Example 2, are disclosed in Table 11 below. Figure 5 shows RP HPLC-MS chromatograms comparing Process 2 (top line in Figure 5), Process 4 (bottom line in Figure 5), and Process 5 (midline in Figure 5).

[0154] [Table 11] [Example 7]

[0155] Synthesis of NAG52 (compound 9) Synthesis of Compounds 1 to 3 In this embodiment, Compound 1, Compound 2, and Compound 3 were synthesized according to previously published procedures (see, for example, U.S. Patent Application Publication No. 2002 / 0107224).

[0156] Cbz-NH-Glu-Glu-OH:

[0157] [ka] It was synthesized according to the procedure described in International Patent Application Publication: International Publication No. 2017 / 156012, the details of which are incorporated herein by reference as relating to the synthesis of Cbz-NH-Glu-Glu-OH.

[0158] Compound 1 is synthesized via the following route:

[0159] [ka] It was synthesized from.

[0160] Compound 3 is synthesized via the following route:

[0161] [ka] It was synthesized from.

[0162] Synthesis of Compound 5 Compound 3 (4.61 g, 12.3 mmol) and Boc-N-amide-PEG2-NHS ester (CAS2183440-73-3, 4.61 g, 12.3 mmol) were dissolved in anhydrous DCM (100 mL), followed by the addition of triethylamine (3.4 mL, 24.6 mmol). The reaction mixture was stirred at room temperature (rt) for 2 hours. The solution was concentrated to 30 mL under reduced pressure and diluted with chloroform (300 mL). The resulting solution was washed first with brine / citric acid (1:1, 30 mL) and then with brine / saturated bicarbonate solution (1:1, 30 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and purified by silica column (100% DCM to 20% MeOH in DCM).

[0163] The fractions containing the desired product, compound 4, were combined, the solvent was removed under reduced pressure, and the resulting foaming residue was redissolved in 1,4-dioxane in 4M HCl (100 mL). The reaction mixture was stirred at room temperature for 1 hour, and the solvent was removed under reduced pressure. The resulting residue was suspended in toluene and dried under reduced pressure to obtain the desired product, compound 5, as an HCl salt (5.30 g, 9.30 mmol, 76% yield). Calculated MW533.26. Detected by ESI MS. + m / z = 534.23[M+H + ].

[0164] [ka]

[0165] Synthesis of Compound 7 Compound 5 (5.30 g, 9.94 mmol) was dissolved in anhydrous DMF, followed by the addition of DIPEA (4.4 mmol, 45.97 mmol). Z-bisGlu (1.12 g, 2.73 mmol) and TBTU (3.03 g, 7.99 mmol) were added with vigorous stirring. The solution turned a light chestnut color and deepened over time. The reaction mixture was left to stand at room temperature with stirring for 2 hours, during which time no unreacted starting materials could be detected by LC-MS. The solvent was removed by simultaneous evaporation with toluene, and the residue was redissolved in chloroform (400 mL). The resulting solution was washed first with brine / water (1:1, 60 mL), and then with brine / bicarbonate solution (1:1, 60 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and purified by silica column (100% DCM to 20% MeOH in DCM).

[0166] The fractions containing the desired product, compound 6, were combined, the solvent was removed under reduced pressure, and the resulting foaming residue was redissolved in methanol (200 mL). Pd / C (0.70 g) was added to the solution, and the suspension was hydrogenated overnight at 1 atm. The reaction mixture was stirred overnight in hydrogen at room temperature. The solution was filtered through a Celite pad and concentrated under reduced pressure to yield the desired product, compound 7 (3.80 g, 2.09 mmol, 77% yield), which was used in subsequent steps. Calculated MW1821.84. Detected ESI MS. + m / z = 912.13[M+2H + ].

[0167] [ka]

[0168] Synthesis of compound 9 Compound 7 (3.80 g, 2.09 mmol) was dissolved in anhydrous DMF (30 mL) and slowly added to a solution in anhydrous DMF (30 mL) containing 4-hydroxycyclohexanecarboxylic acid (0.35 g, 2.43 mmol), TBTU (0.82 g, 2.16 mmol), and DIPEA (1.12 mL, 6.44 mmol). The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed by simultaneous evaporation with toluene, and the residue was redissolved in chloroform (300 mL). The solution was washed with brine / citric acid 5% (1:1, 30 mL), dried over Na2SO4, concentrated under reduced pressure, and purified by silica column (100% DCM to 30% MeOH in DCM) to obtain the desired product, Compound 8 (2.40 g, 1.23 mmol, 59% yield). Calculated MW 1947.90. Detected by ESI MS. + m / z = 975.46[M+2H + ].

[0169] [ka]

[0170] Compound 8 (2.40 g, 1.23 mmol) was completely dried by co-evaporating DCM with toluene and then dried under vacuum for 30 minutes. A stirring bar and a pre-treated molecular sieve were placed in a round-bottom flask and purged with nitrogen. The flask was filled with DCM (100 mL) and the molecular sieve was gently stirred for 10 minutes. Diisopropylammonium tetrazolide (1.40 g, 8.19 mmol) was added to the solution and the reaction mixture was stirred for a further 30 minutes. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (0.55 mL, 1.73 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour, during which time no unreacted starting materials could be detected by LC-MS. The solution was filtered through a Celite pad to remove the molecular sieve and diluted with saturated bicarbonate solution (100 mL) while stirring. After 15 minutes, the organic layer was separated, and the aqueous layer was extracted with chloroform (2 × 200 mL). The organic fractions were combined, dried over Na₂SO₄, concentrated under reduced pressure, and purified by silica column chromatography [100% DCM (+ 0.1% triethylamine) to 10% MeOH in DCM (+ 0.1% triethylamine)].

[0171] The fraction containing the desired product and compound 9 (NAG52) was combined and concentrated under reduced pressure. The product was evaporated twice simultaneously with toluene to remove any residual triethylamine, yielding the desired product (2.4 g, 1.16 mmol, 94% yield) as an off-white solid.

[0172] [ka]

[0173] Tris(αCNAGAc3Peg2)bisGluNHCO-CH-PA: 1H NMR (DMSO-d6): 1.14 d (12H), 1.44 m (7H), 1.62-1.90 m (11H), 1.80 s (9H), 1.94 s (9H), 2.00 s (9H), 2.07 s (9H), 2.03-2.16 m (4H), 2.20-2.31 m (6H), 2.76 t (2H), 2.88-2.98 m (3H), 3.12- 3.23 m (10H), 3.34-3.42 m (6H), 3.46 s (12 H), 3.57 t (8H), 3.62- 3.76 m (2H), 3.98-4.20 m (15H), 4.20-4.30 m (3H), 4.96 dd (3H), 5.28, d( 3H), 7.56- 8.00 m (8H), 8.12 d (3H). 31 P NMR (DMSO-d6): 145.84, 146.01. [Example 8]

[0174] Comparison of Oligonucleotide Dimer Synthesis Methods In this example, SEQ ID NO:6 (NAG52s,g,g,a,c,c,u,Gf,u,Uf,Uf,u,g,c,u,u,u,u,g,us,invAb,Sp18,gs,g,g,a,c,a,Gf,u,Af,u,Uf,c,u,c,a,g,u,i,as,invAbs,C6-NH2) was synthesized using processes 6 to 8 described below (on a 500 μmol scale).

[0175] [Table 12]

[0176] [Table 13]

[0177] [Table 14]

[0178] Results: A comparison of crude yield, crude purity, final yield after purification, and final purity for processes 6 to 8 is disclosed in Table 15 below.

[0179] [Table 15]

[0180] The TFA Nitto condition yielded the highest crude purity (78%). The DCA Nitto condition yielded the highest final purity (88.6%), and the TFA Nitto condition yielded the second highest final purity (87.6%). The TFA Nitto condition yielded the highest crude yield (11.08 g / mmol) and the highest final yield (7.9 g / mmol).

Claims

1. A method for producing oligonucleotides, a. To prepare a first nucleotide containing a first trityl group, wherein the first nucleotide is a support-supported nucleotide on a substrate, and the substrate is a solid-phase support. b. Forming a detrithylated nucleotide by detritylation of the first nucleotide using a detritylation reagent, wherein the detritylation reagent contains acetic acid or a salt thereof, and the acetic acid may be trifluoroacetic acid (TFA) or a salt thereof. c. Washing the detritylated nucleotide with a washing solution, and d. Producing the oligonucleotide by coupling the second nucleotide to the detritylated nucleotide under reaction conditions that promote the coupling of the second nucleotide to the detritylated nucleotide. Includes, A method that does not include a capping step.

2. The method according to claim 1, wherein the washing solution comprises (i) a neutralizing solvent, (ii) an alcohol or water, and (iii) an organic solvent; the washing solution may also comprise pyridine, methanol, and toluene.

3. The method according to claim 1 or 2, further comprising a second washing step after coupling the second nucleotide with the first nucleotide; the second washing step may include acetonitrile.

4. The method according to any one of claims 1 to 3, further comprising an oxidation step, after coupling the second nucleotide to the first nucleotide, wherein the oxidation step is carried out using an oxidizing reagent; the oxidizing reagent may include a halogen, a base, and water.

5. The method according to claim 4, wherein the oxidizing reagent comprises iodine, pyridine, and water.

6. The method according to any one of claims 1 to 5, further comprising a thiolation step after coupling the second nucleotide to the first nucleotide, wherein the thiolation step is carried out using a thiolation reagent.

7. The method according to claim 6, wherein the thiolation reagent comprises xanthan hydride.

8. The method according to any one of claims 4 to 7, further comprising an additional washing step after the oxidation step or the thiolation step; the additional washing may include acetonitrile.

9. The method described above is a. To prepare a first nucleotide containing a first trityl group, wherein the first nucleotide is a carrier-supported nucleotide on a substrate, and the substrate is a solid-phase support. b. The first nucleotide is detritylated using a detritylation reagent containing trifluoroacetic acid (TFA) to form a detritylated nucleotide. c. Washing the detritylated nucleotide with a washing solution containing pyridine and methanol, and d. Producing the oligonucleotide by coupling the second nucleotide to the detritylated nucleotide under reaction conditions that promote the coupling of the second nucleotide to the detritylated nucleotide, wherein the second nucleotide contains a second trityl group, and e. Wash the oligonucleotide as appropriate. f. Oxidizing the oligonucleotide to form an oxidized oligonucleotide, or thiolating the oligonucleotide to form a thiolated oligonucleotide, and g. Washing the oxidized oligonucleotide or the thiolated oligonucleotide. The method according to any one of claims 1 to 8, comprising the above.

10. The method according to any one of claims 1 to 9, wherein the method yields a higher crude yield of the oligonucleotide compared to the same method for producing the oligonucleotide, which includes a capping step.

11. The method according to any one of claims 1 to 10, wherein the method results in a higher final yield of the oligonucleotide compared to the same method for producing the oligonucleotide, which includes a capping step.

12. The method according to any one of claims 1 to 11, wherein the method yields less shorting compared to the same method for producing the oligonucleotide, which includes a capping step.

13. The method according to any one of claims 1 to 12, wherein steps (a) to (d) are repeated one or more times, and the second nucleotide containing the second trityl group obtained from step (d) is used as the first nucleotide containing the first trityl group in the next repetition of step (a).

14. The method according to any one of claims 1 to 13, wherein the solid phase support comprises polystyrene or controlled porous glass (CPG).

15. The method according to any one of claims 1 to 14, wherein the carrier-supported nucleotide includes one or more non-nucleotide moieties between the solid support and the first nucleotide.

16. The method according to any one of claims 1 to 15, wherein the detritylation reagent contains trifluoroacetic acid at a concentration of about 1 to about 15 volume%, may contain it at a concentration of about 1 to about 10 volume%, or may contain it at a concentration of about 6 volume%.

17. The method according to any one of claims 1 to 16, wherein the detritylation reagent further comprises an aromatic solvent or a halogenated solvent, and the detritylation reagent may further comprise toluene, 2,2,2-trifluoroethanol, dichloromethane, or a mixture thereof.

18. The method according to any one of claims 1 to 17, wherein the reaction conditions that promote coupling include the use of an activator.

19. The aforementioned activators include 1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), 5-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 2-bromo-4,5-dicyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, 1-(cyanomethyl)piperidinium tetrafluoroborate, and 1-methyl The method according to claim 18, wherein the member is 1H-benzimidazole-3-ium trifluoromethanesulfonate, 1-phenyl-1H-imidazole-3-ium trifluoromethanesulfonate, 1H-benzimidazole-3-ium trifluoromethanesulfonate, 1H-imidazole-3-ium trifluoromethanesulfonate, 5-nitro-1H-benzimidazole-3-ium trifluoromethanesulfonate, benzimidazolium triflate, and / or imidazolium triflate.

20. The method according to claim 19, wherein the activator is ETT.

21. The method according to any one of claims 1 to 20, wherein the oligonucleotide is approximately 5 to approximately 49 nucleotides long, approximately 10 to approximately 49 nucleotides long, approximately 15 to approximately 49 nucleotides long, approximately 20 to approximately 49 nucleotides long, approximately 25 to approximately 49 nucleotides long, approximately 30 to approximately 49 nucleotides long, approximately 35 to approximately 49 nucleotides long, or approximately 38 to approximately 49 nucleotides long.

22. The method according to claim 21, wherein the oligonucleotide is 19 nucleotides to 23 nucleotides in length.

23. The method according to claim 22, wherein the oligonucleotide has a length of 21 nucleotides.

24. The method according to claim 22, wherein the oligonucleotide has a length of 23 nucleotides.

25. The method according to claim 22, wherein the oligonucleotide is 19 nucleotides long.

26. The method according to claim 21, wherein the oligonucleotide is 38 nucleotides to 46 nucleotides in length.

27. The method according to claim 26, wherein the oligonucleotide is 38 nucleotides long.

28. The method according to claim 26, wherein the oligonucleotide is 40 nucleotides long.

29. The method according to claim 26, wherein the oligonucleotide is 42 nucleotides long.

30. The method according to any one of claims 1 to 29, wherein the oligonucleotide comprises at least one modified nucleotide.

31. The method according to any one of claims 1 to 30, wherein the oligonucleotide comprises at least one modified oligonucleotide bond.

32. The method according to any one of claims 1 to 31, wherein the oligonucleotide is a single-stranded oligonucleotide.

33. The method according to any one of claims 1 to 31, wherein the oligonucleotide is a double-stranded oligonucleotide.

34. The method according to any one of claims 1 to 33, wherein the oligonucleotide is an oligonucleotide dimer.

35. The method according to claim 34, wherein the oligonucleotide dimer comprises two single-stranded oligonucleotides linked together.

36. The method according to claim 34, wherein the oligonucleotide dimer comprises two double-stranded oligonucleotides linked together.

37. The method according to any one of claims 1 to 33, wherein the oligonucleotide is an oligonucleotide trimer.

38. The method according to claim 37, wherein the oligonucleotide trimer comprises three single-stranded oligonucleotides linked together.

39. The method according to claim 37, wherein the oligonucleotide trimer comprises three double-stranded oligonucleotides linked together.

40. The method according to any one of claims 34 to 39, wherein the oligonucleotide dimer or the oligonucleotide trimer includes a spacer.

41. The method according to claim 40, wherein the spacer is Sp18.

42. The method according to any one of claims 1 to 41, wherein the method yields a gross yield at least 2% higher than the same method for producing the oligonucleotide, comprising a capping step, and optionally at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher.

43. The method according to any one of claims 1 to 42, wherein the method yields a final yield at least 3% higher than the same method for producing the oligonucleotide, comprising a capping step, and optionally at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher.

44. The method according to any one of claims 1 to 43, wherein the method results in a reduction of at least 3% of the shorter, and optionally a reduction of at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% of the shorter, compared to the same method for producing the oligonucleotide, which includes a capping step.

45. The method according to any one of claims 1 to 44, wherein the method results in an increase of at least 2% in crude purity compared to the same method for producing the oligonucleotide, comprising a capping step, and optionally an increase of at least 3%, 4%, 5%, 6%, 7%, 8%, or more than 8% in crude purity.

46. Oligonucleotides synthesized using the method described in any one of claims 1 to 45.

47. A pharmaceutical composition comprising the oligonucleotide and a pharmaceutically acceptable carrier as described in claim 46.

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