Oligonucleotide synthesis methods

Abstract

Disclosed herein are processes or methods for producing oligonucleotides, including in large single batch scales and with high purity and yields without a capping step.

Description

OLIGONUCLEOTIDE SYNTHESIS METHODS

[0001] The disclosure pertains to solid phase oligonucleotide synthesis and, in particular, to processes or methods utilizing a solid phase reactor system for making oligonucleotides of various lengths and sequences without the use of a capping step.

[0002] It is of great interest for the therapeutic, diagnostic, reagent, and biological assay industries to have processes and methods for synthesizing highly pure oligonucleotides of various nucleotide sequences and lengths, at sufficiently high yields, and at minimal cost. Current oligonucleotide syntheses generally involve starting with a nucleotide or other similar moiety that is to be positioned at the 3’ end of the final oligonucleotide, which is attached to a support - e.g., a polymer or glass resin - and then employing chemical steps to sequentially add nucleotides, by covalent attachment, to the 5’ end to produce the final oligonucleotide. This synthesis includes a capping step as part of each nucleotide monomer addition that allows for exclusion of shortmers from the reaction, thereby improving the purity of the resulting product. At the end of the synthesis run, the synthesized oligonucleotide is cleaved from the resin support.

[0003] Syntheses methods that employ a capping step typically take additional time to complete, which may slow down workflow, reduce production scalability, and add to cost. Current syntheses methods also typically require the use of a purification step in order to remove shortmers from the reaction product in order to achieve sufficiently pure yields. Furthermore, a capping step may result in reduced purity of the overall final yield.

[0004] Thus, there is a need for improved methods to synthesize highly pure oligonucleotides, with various nucleotide sequences, in a timely manner and at sufficiently high yields, particularly for a large-scale production of oligonucleotides.SUMMARY

[0005] The present disclosure is based, at least in part, on the discovery of a method for synthesizing long (e.g., greater than 10-mer) oligonucleotides, including in high yields, using a method without a capping step.

[0006] In some embodiments, the present disclosure provides a method of producing an oligonucleotide. In some embodiments, the present disclosure provides a method of producing an oligonucleotide comprising: (a) providing a first nucleotide comprising a first trityl group, wherein the first nucleotide is a support-bound nucleotide on a substrate; (b) detritylating the first nucleotide with a detritylating reagent to form a detritylated nucleotide, wherein the detritylating reagent comprises an acetic acid or a salt thereof, optionally wherein the aceticacid is trifluoroacetic acid (TFA) or a salt thereof; (c) washing the detritylated nucleotide with a wash; and (d) coupling a second nucleotide to the detritylated nucleotide under reaction conditions that promote coupling of the second nucleotide to the detritylated nucleotide to produce the oligonucleotide; wherein the substrate is a solid phase support; and wherein the method does not comprise a capping step.

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

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

[0009] In some embodiments, the method further comprises a thiolation step after coupling the second nucleotide to the first nucleotide, wherein the thiolation step is carried out using a thiolation reagent. In some embodiments, the thiolation reagent comprises xanthane hydride. In some embodiments, the method further comprises an additional wash step after the thiolation step. In some embodiments, additional wash comprises 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 wash step after the oxidation step, after the thiolation step, and / or after the oxidation step and the thiolation step. In some embodiments, the additional wash comprises acetonitrile.

[0011] In some embodiments, the present disclosure provides a method of producing an oligonucleotide, the method consisting of, or consisting essentially of: (a) providing a first nucleotide comprising a first trityl group, wherein the first nucleotide is a support-bound nucleotide on a substrate, wherein the substrate is a solid phase support; (b) detritylating the first nucleotide with a detritylating reagent comprising trifluoroacetic acid (TFA) to form a detritylated nucleotide; (c) washing the detritylated nucleotide with a wash comprising pyridine and methanol; (d) coupling a second nucleotide to the detritylated nucleotide under reaction conditions that promote coupling of the second nucleotide to the detritylatednucleotide to produce the oligonucleotide, wherein the second nucleotide comprises a second trityl group; (e) optionally, washing the oligonucleotide; (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 thiolated oligonucleotide.

[0012] In some embodiments, the method of producing an oligonucleotide results in a higher crude yield of the oligonucleotide compared to the same method of producing the oligonucleotide with a capping step. In some embodiments, the method of producing an oligonucleotide results in a higher final yield of the oligonucleotide compared to the same method of producing the oligonucleotide with a capping step. In some embodiments, the method of producing an oligonucleotide results in fewer shortmers compared to the same method of producing the oligonucleotide with a capping step.

[0013] In some embodiments, steps (a) through (d) of the method of producing an oligonucleotide are repeated one or more times, wherein the second nucleotide comprising the second trityl group from step (d) is used as the first nucleotide comprising the first trityl group in the subsequent repeat of step (a).

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

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

[0016] In some embodiments, the reaction conditions that promote coupling comprise the use of an activator. In some embodiments, the activator comprises IH-tetrazole, 2- benzylthiotetrazole, 4, 5 -di cyanoimidazole (DCI), 5-ethylthio-lH-tetrazole (ETT), 5-(4- nitrophenyl)- 1 H-tetrazole, 5 -(bi s-3 , 5 -trifluorom ethyl -phenyl)- 1 H-tetrazole, 5 -methylthio- 1 H- tetrazole, 2-bromo-4,5-di cyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, 1- (cyanomethyl)piperidinium tetrafluoroborate, 1 -methyl- IH-benzimidazol -3 -ium trifluoromethanesulfonate, l-phenyl-lH-imidazol-3-ium trifluoromethanesulfonate, 1H-benzimidazol-3-ium trifluoromethanesulfonate, lH-imidazol-3-ium trifluoromethanesulfonate, 5-nitro-lH-benzimidazol-3-ium trifluoromethanesulfonate, benzimidazolium triflate, and / or imidazolium triflate. In some embodiments, the activator comprises ETT.

[0017] In some embodiments, the oligonucleotide 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 19 to about 23 nucleotides long, or about 19 nucleotides long, or about 20 nucleotides long, or about 21 nucleotides long, or about 22 nucleotides long, or about 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 comprises at least one modified nucleotide. In some embodiments, the oligonucleotide comprises 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, theoligonucleotide 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 19 nucleotides long, and a second oligonucleotide 21 nucleotides long. In some embodiments, the oligonucleotide dimer comprises at least one modified nucleotide. In some embodiments, the oligonucleotide dimer comprises at least one modified nucleotide on each strand. In some embodiments, the oligonucleotide dimer comprises at least one modified oligonucleotide linkage. In some embodiments, the oligonucleotide dimer comprises two single-stranded oligonucleotides coupled together. In some embodiments, the oligonucleotide dimer comprises two double-stranded oligonucleotides coupled together. In some embodiments, the oligonucleotide dimer comprises at least one spacer. In some embodiments, the spacer is a hexaethyleneglycol spacer (e.g., Spacer 18, Spl8). 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 comprises three single-stranded oligonucleotides coupled together. In some embodiments, the oligonucleotide trimer comprises three doublestranded oligonucleotides coupled 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 comprises at least one modified nucleotide. In some embodiments, the oligonucleotide trimer comprises at least one modified nucleotide on each strand. In some embodiments, the oligonucleotide trimer comprises at least one spacer. In some embodiments, the spacer is an Spl8 spacer.

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

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

[0022] FIG. 1 depicts a comparison of reverse phase high performance liquid chromatography and mass spectrometry (RP HPLC-MS) chromatogram of the crude yield of SEQ ID NO: 1 synthesized by processes disclosed in Example 1.

[0023] FIG. 2 depicts a comparison of RP HPLC-MS chromatogram of the crude yield of SEQ ID NO: 2 synthesized by processes disclosed in Example 2.

[0024] FIG. 3 depicts a comparison of RP HPLC-MS chromatogram of the crude yield of SEQ ID NO: 3 synthesized by processes disclosed in Example 3.

[0025] FIG. 4 depicts a comparison of RP HPLC-MS chromatogram of the crude yield of SEQ ID NO: 4 synthesized by processes disclosed in Example 4.

[0026] FIG. 5 depicts a comparison of RP HPLC-MS chromatogram of the crude yield of SEQ ID NO: 2 synthesized by processes disclosed in Example 6.DETAILED DESCRIPTION

[0027] The term "oligonucleotide", as used herein, refers to a compound that includes two or more nucleotides linked together. An oligonucleotide is typically less than about 100 nucleotides in length (e.g., 2-50 nucleotides in length). Examples of oligonucleotides for use in accordance with the present disclosure include, but are not limited to, DNA, RNA (including messenger RNA (mRNA)), hybrids of DNA and / or RNA (including chemically modified variants thereof), RNAi agents comprised of RNA and / or chemically modified RNA that are able to induce the RNA interference (RNAi) mechanism such as short or small interfering RNAs(siRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), antisense and sense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, and other oligonucleotides disclosed in detail herein or generally known in the art.

[0028] As used herein, the terms "support-bound" or "substrate-bound" refer to nucleotides or oligonucleotide strands that are bound to a solid support or substrate. The nucleotide can be bound directly to the support / substrate, or can be bound as a member of a plurality of linked nucleotides and / or non-nucleotide moieties which are bound to the support / substrate. The nucleotide can be bound covalently.

[0029] The term "nucleotide" refers to an organic compound which includes a nucleoside and a phosphate group. The term encompasses an organic compound comprising a nucleobase (e.g., adenine, cytosine, guanine, thymine, or uracil) covalently attached to a pentose sugar. The term "nucleobase," as used herein, refers to a heterocyclic moiety of a nucleoside. In some embodiments, a nucleobase of the present disclosure is a canonical nucleobase (A, G, T, C, U). In certain embodiments, the nucleobase is a purine base — adenine (A) or guanine (G). In certain embodiments, the nucleobase is a pyrimidine base — thymine (T), cytosine (C), or uracil (U). As used herein the term "nucleotide," unless expressly provided otherwise, includes ribonucleotides (2'-OH sugar ring) and deoxyribonucleotides (2'-H sugar ring), and also includes modified nucleotides. As described more fully elsewhere in the specification, a modified nucleotide is a nucleotide where, for example, the nucleobase, the pentose sugar, and / or the phosphate group (or internucleoside linkage) is modified chemically from that of a ribonucleotide or deoxyribonucleotide. In some embodiments, the nucleotide is a modified nucleotide comprising a modified nucleobase (i.e., a nucleotide other than a canonical nucleobase). In some embodiments, the nucleotide is a modified nucleotide comprising a modified sugar ring (e.g., a PNA nucleotide). In some embodiments, the nucleotide is a modified nucleotide comprising a modified phosphate group (e.g., a phosphorothioate or phosphorodithioate group or other modified internucleoside linkage). Exemplary modified nucleotides suitable for use with the methods disclosed herein are provided for elsewhere in the specification including in the Examples, and are further generally known in the art.

[0030] As used herein, a "non-nucleotide support-bound moiety" or a "non-nucleotide substrate-bound moiety" refers to a moiety that is a non-nucleotide that is bound to a solid support or substrate. The non-nucleotide can be bound directly to the support / substrate, or can be bound as a member of a plurality of linked nucleotides and / or non-nucleotide moieties which are bound to the support / substrate. In some embodiments, a "non-nucleotide support-bound moiety" can be acquired commercially as a compound directly attached to the resin / support (for example, UnyLinker Solid Support (NittoPhase, Kinovate Life Sciences), Reverse (or inverted) Abasic Solid Support (NittoPhase, Kinovate Life Sciences), etc.), or as protected phosphorami dites (for example, a spacer such as Spacer Phosphorami dite 18 (18-0- Dimethoxytritylhexaethyleneglycol,l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research), reverse (or inverted) abasic phosphoramidite (3-O-Dimethoxytrityl-2- deoxyribose-5-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) (Hongene Biotech), thiol- modified C6 S-S Linker (l-O-Dimethoxytrityl-hexyl-disulfide,l'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite) (Hongene Biotech), etc.). In some embodiments the nonnucleotide support-bound moiety can be a targeting ligand or targeting group to help direct the oligonucleotide in vivo, a pharmacokinetic / pharmacodynamic (PK / PD) modulator that enhance the PK / PD properties of the oligonucleotide, an end cap to protect the oligonucleotide from enzyme degradation, a linking group such as an NH2-C6 ("AminoLink™") group or an SP18 spacer that allows for the coupling of other nucleotide or non-nucleotide groups, a prolinol moiety, or another other chemical moiety that confers beneficial or otherwise desirable properties to the oligonucleotide. Suitable non-nucleotide moi eties for use with oligonucleotides are described in greater detail elsewhere in the specification, and are further generally known in the art.

[0031] The term "coupling efficiency" as used herein refers to the reaction yield of the coupling of a new nucleotide to a strand bound to a solid support.

[0032] The term "cycle efficiency" as used herein refers to the yield of support-bound oligonucleotide following a full reaction cycle including, e.g., detrityl ati on, coupling, and oxidation.

[0033] The term "crude yield" as used herein refers to the yield of the crude product oligonucleotide without purification. For example, in the synthesis of a 21-mer oligonucleotide, the crude yield refers to the number of moles of desired 21-mer obtained before purification divided by the number of moles of oligonucleotide which were initially bound to the solid support. The number of moles of a desired 21-mer obtained before purification can be calculated, for example, by cleaving the strand from the resin, then analyzing and measuring the resulting product by Reverse Phase High Performance Liquid Chromatography and Mass Spectrometry (RP HPLC-MS).

[0034] The term "crude purity" as used herein refers to the amount of crude product oligonucleotide compared to the total yield from a reaction without purification. Crude puritymay be measured by HPLC, e.g., as the area under a product peak for the target oligonucleotide relative to a total peak area over an entire chromatogram for the crude product. The amount can be expressed as a percentage.

[0035] The term "final yield" as used herein refers to the yield of the final purified product oligonucleotide. For example, in the synthesis of a 21-mer oligonucleotide, the final yield refers to the number of moles of desired 21-mer obtained after purification divided by the number of moles of oligonucleotide which were initially bound to the solid support. The number of moles of a desired 21-mer may be calculated after cleaving the strand from the resin, purifying using anion exchange chromatography and / or tangential flow filtration, then desalting and lyophilizing the product, and measuring yield using UV spectroscopy, e.g., at 260 nm.

[0036] The term "final purity" as used herein refers to the refers to the amount of final purified product oligonucleotide compared to the total yield from a reaction without purification.Final purity may be measured by HPLC, e.g., as the area under a product peak for the target oligonucleotide relative to a total peak area over an entire chromatogram for the product. The amount can be expressed as a percentage. The final purity may in some cases refer to the yield of desalted oligonucleotide. In other instances, it may refer to the yield of an oligonucleotide that has been column purified and desalted.

[0037] As used herein, the term "monomer" or "nucleotide monomer" refers to a single nucleotide.

[0038] As used herein, the term "dimer" or "nucleotide dimer" refers to an oligonucleotide comprising two nucleotides (i.e., a nucleotide monomer coupled to another nucleotide monomer).

[0039] As used herein, the term “trimer” or "nucleotide trimer" refers to an oligonucleotide comprising three nucleotides (i.e., three nucleotide monomers coupled together).

[0040] As used herein, the term "oligonucleotide dimer" refers to an oligonucleotide comprising two oligonucleotides coupled together (e.g., with a Spl8 spacer having the following structure:

[0041] As used herein, the term "oligonucleotide trimer" refers to an oligonucleotide comprising three oligonucleotides coupled together (e.g., with Spl8 spacers).

[0042] As used herein, the term "shortmer" refers to an oligonucleotide shorter in length than the intended length for a synthesis reaction. The shortmer may be missing terminal nucleotidesat the 5’ and / or 3’ end, and / or may be missing one or more internal nucleotide from the intended oligonucleotide sequence. A shortmer may, in some instances, be 10 nucleotide in length or less.

[0043] As used herein, the term "about" as used herein means within 10% of the stated amount.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All ranges include the endpoints. The term "or" shall mean "and / or" unless context dictates otherwise. Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the present disclosure, suitable exemplary methods and materials are disclosed below. The materials, methods, and examples disclosed herein are intended to be illustrative only and are not intended to be limiting.

[0045] Other features and advantages of the disclosure will be apparent from the following detailed description, and from the claims.OligonucleotidesOverview

[0046] The present disclosure provides methods for synthesizing oligonucleotides. The present disclosure also provides oligonucleotides prepared by the methods of the present disclosure. Oligonucleotides are polymers of linked nucleotides, typically less than about 100 nucleotides in length. In some embodiments, oligonucleotides of the present disclosure include at least 3 nucleotides. In some embodiments, the oligonucleotide includes 3 to 100 nucleotides. In some embodiments, the oligonucleotide includes 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 certain embodiments, the oligonucleotide includes 2 nucleotides (i.e., a dinucleotide). In certain embodiments, the oligonucleotide includes 3 nucleotides (i.e., a trinucleotide). In certain embodiments the oligonucleotide includes 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 certain embodiments the oligonucleotide includes 16 to 26 nucleotides. In certain embodiments the oligonucleotide includes 19 to 23 nucleotides. In certain embodiments the oligonucleotide includes 18 to 20 nucleotides. In certain embodiments the oligonucleotide includes 19 nucleotides. In certain embodiments the oligonucleotide includes 22 to 24 nucleotides. In certain embodiments the oligonucleotide includes 23 nucleotides.

[0047] In some embodiments, oligonucleotides are modified, e.g., an oligonucleotide may comprise one or more modified nucleotides and / or one or more modified internucleoside linkages. For example, modified nucleotides and modified intemucleoside linkages, whenused in various oligonucleotide constructs, may serve to preserve activity of the oligonucleotide compound in cells and / or to increase the serum stability of these compounds, and may also be used to minimize the possibility of activating interferon activity in humans upon administration.Modi fied Nucleotides

[0048] In some embodiments, the oligonucleotides include nucleotides that are modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a naturally occurring ribonucleotide (nucleotide with 2'-OH sugar ring) or a naturally occurring deoxyribonucleotide (nucleotide with 2'-H sugar ring). In some embodiments, at least 5% (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%) of the nucleotides are modified nucleotides. In some embodiments, 100% of the nucleotides are modified nucleotides. In some embodiments, 0% of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, 2'-modified nucleotides, abasic nucleotides, 3' to 3' linkages (i.e., inverted) nucleotides, modified nucleobase-comprising nucleotides, bridged nucleotides, 2', 3 '-seco nucleotide mimics (i.e., unlocked nucleobase analogues), locked nucleotides, 3'-O-methoxy (2' intemucleoside linked) nucleotides, 2'-F-Arabino nucleotides, 5'-Methyl-2'-fluoro nucleotides, morpholine nucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides. 2'-modified nucleotides (i.e., a nucleotide with a group other than an -H or -OH group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides (alternatively referred to as 2’ -fluoro nucleotides), 2 '-methoxy ethyl (2'-O-2-methoxylethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given oligonucleotide to be uniformly modified. Further, more than one modification can be incorporated in different nucleotides being added, or even in a single nucleotide of the oligonucleotide. Modification at one nucleotide may be independent of modification at another nucleotide.

[0049] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimi dines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, 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) and other alkyl derivatives of adenine and guanine, 2 -thiouracil, 2 -thiothymine, 2 -thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 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.

[0050] In some embodiments, all or substantially all of the nucleotides of an oligonucleotide are modified nucleotides. As used herein, an oligonucleotide wherein substantially all of the nucleotides present are modified nucleotides is an oligonucleotide having no more than about 20% unmodified ribonucleotides or unmodified deoxyribonucleotides.Modi fied Internucleoside Linkages

[0051] In some embodiments, one or more nucleotides of an oligonucleotide synthesized using the methods disclosed herein are linked by non-standard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). Modified intemucleoside linkages or backbones include, but are not limited to, phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3 '-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3 '-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl- phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2'-5' to 5 '-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0052] In some embodiments, the oligonucleotides synthesized using the methods disclosed herein contain one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2'-modified nucleoside is combined with modified intemucleoside linkage.Linking Groups, Targeting Groups and Other Non-Nucleotide Groups

[0053] In some embodiments, the oligonucleotides synthesized using the methods disclosed herein contain one or more linking groups, targeting groups, and / or other non-nucleotide groups. Linking groups can be added to and positions on the oligonucleotide to facilitate the linking of additional compounds, such as targeting groups or other non-nucleotide groups such as PK enhancers. Targeting groups (or targeting moieties) are compounds that enhance the pharmacokinetic or biodistribution properties of the oligonucleotide to which they are attached, to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the oligonucleotide. Targeting groups may include one targeting ligand, or two targeting ligands (referred to as "bidentate"), three targeting ligands ("tridentate"), four targeting ligands ("tetradentate"), or more than four targeting ligands. Examples of targeting groups include compounds having affinity to cell surface molecules or cell receptors. PK enhancers (also referred to as "pharmacokinetic (PK) modifiers") are compounds that, when linked to an oligonucleotide are able to increase systemic circulation time of the compound in vivo (i.e., increased half-life or plasma residence time) versus the free form of the oligonucleotide by limiting renal excretion, without impeding delivery of the oligonucleotide to the targeted cells or tissues. In some embodiments, PK enhancers may include molecules that are fatty acids, lipids, albuminbinders, antibody -binders, polyesters, polyacrylates, poly-amino acids, and linear or branched polyethylene glycol (PEG) moieties having about 20-900 ethylene oxide (CH2-CH2-O) units.

[0054] For example, in some embodiments, the oligonucleotides can be synthesized using phosphoramidites that include linking groups, such as TFA aminolink phosphoramidites that can be commercially purchased (ThermoFisher) and used to introduce reactive group linkers (e.g., NH2-C6) to the 5’ end of an oligonucleotide. In other embodiments, targeting-group containing phosphoramidite compounds can be used. These can include three N-acetyl- galactosamines which are known to have affinity for the asialoglycoprotein receptor and are able to target the compounds to which they are attached to liver cells (and hepatocytes in particular), such as the phosphoramidite compounds having the following structures:

[0055] In further embodiments, phosphoramidite compounds that include terminal alkyne groups can be used to facilitate the linkage to various targeting groups and / or other nonnucleotide groups. These types of linking agents may be added according to the methods disclosed herein. Examples of suitable trialkyne linking groups include the following:Oligonucleotide SynthesisSolid Phase Oligonucleotide Synthesis Methods

[0056] The present disclosure provides methods for synthesizing oligonucleotides. In some embodiments, the methods of synthesizing oligonucleotides disclosed herein are carried out in a reaction flask or a reaction vessel. In some embodiments, the reaction flask or reaction vessel includes a filter at the bottom to allow drainage of solutions from a solid phase support retained in the vessel by the filter. In some embodiments, pores in the filter are small enough to prevent the target support particles from passing through the filter, and are also large enough to allow impurities (e.g., unused reagents and other synthesis by-products) to drain out of the flask or vessel. In general, reaction columns are packed with a solid support, and the solid support in the column remains stationary while solvents and reagents flow through the column. In a reaction flask or reaction vessel, a solid support may also be used, but the solid support is generally stirred or agitated to create motion. The solvents and reagents in a reaction flask or vessel have the opportunity to interact with molecules bound to the solid support because of the stirring or agitation.

[0057] In some embodiments, the reaction flask or vessel may be equipped with a stir bar, agitator or other mechanism for stirring or agitating the solid support. By stirring or agitating the solid support, the effective bed-depth of the solid support may be increased relative to the bed-depth of a comparable column reactor (typically about 5-10 cm). Stirring or agitating the solid support can thus result in a more efficient use of the solid support. In some embodiments, a solid support used in a method disclosed herein comprises polystyrene or controlled pore glass (CPG). In some embodiments, a soluble support such as polynorbornene or other soluble support useful in cyclic syntheses is used. Other examples of soluble supports that may be used are disclosed in, for example, PCT Publication No. WO / 2012 / 165545.

[0058] In some embodiments, a method for synthesizing oligonucleotides disclosed herein includes stirring or agitation of the reaction mixture in one or more (e.g., all) of the steps inthe method. In some embodiments, the reaction mixture is stirred at a rate of at least 10 revolutions per minute (rpm). In some embodiments, the reaction mixture is stirred at a rate 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 stirred at rate of about 100 rpm to about 600 rpm. In some embodiments, some or all of the reactions disclosed herein take place on a rocker which agitates the reaction mixture.

[0059] 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 either a polymer resin, e.g., Nittophase® or Tentagel®, or a controlled porous glass (CPG). TentaGel® resins are grafted copolymers consisting of a low crosslinked polystyrene matrix on which poly (ethylene glycol) (PEG or POE) is grafted. Nittophase® resins are cross-linked polystyrene polymer particles having a diameter of about 90 microns.

[0060] The reaction flask or vessel used in the methods disclosed herein may be of any design known in the art. In some embodiments, large vessels, such as greater than 50L reactors, are employed in the methods. As one skilled in the art will appreciate, carrying out oligonucleotide synthesis in such large-scale reaction vessels allows for greater batch sizes. Because the methods disclosed herein do not require the use of a specialized column reactor (as in a flow-through method), batch size limitations are effectively eliminated.

[0061] In some embodiments, oligonucleotide assembly occurs on a solid support via a series of steps in a step-wise reaction cycle, such as a step-wise reaction cycle disclosed herein. Exemplary conditions for each reaction step are further disclosed in the Examples.

[0062] In some embodiments, the reaction process (e.g., step-wise reaction cycle) includes a deblocking step which comprises deblocking a trityl group of a support-bound nucleotide (e.g., a single support-bound nucleotide or a terminal nucleotide in a support-bound plurality of linked nucleotides), thereby providing a detritylated support-bound nucleotide. In some embodiments, the support-bound nucleotide comprises a plurality of nucleotides, i.e., an oligonucleotide. In some embodiments, the support-bound nucleotide is covalently linked directly to the substrate. In some embodiments, the support-bound nucleotide is covalently linked indirectly (e.g., through a non-nucleotide moiety) to the substrate. In some embodiments, the support-bound nucleotide is indirectly linked to the substrate through a non-nucleotide moiety, such as an inverted abasic moiety, a C3 cap, or a Ce cap.

[0063] In some embodiments, the deblocking step liberates a 5'-hydroxyl group on the detritylated support-bound nucleotide. In some embodiments, the deblocking is with a reagent comprising (i) an acid, and (ii) an organic solvent. In some embodiments, the deblocking is the first step in a step-wise reaction cycle.

[0064] In some embodiments, the reaction process (e.g., step-wise reaction cycle) includes a washing step which comprises washing the detritylated support-bound nucleotide. In some embodiments, the wash comprises: (i) a neutralizing solvent, (ii) alcohol or water, and (iii) an organic solvent. In some embodiments, the wash comprises pyridine, methanol, and toluene.

[0065] In some embodiments, the reaction process (e.g., step-wise reaction cycle) includes a coupling step which comprises coupling a nucleotide building block to the detritylated support-bound nucleotide, thereby forming a new phosphite linkage between the nucleotide building block and the support-bound nucleotide. In some embodiments, the nucleotide building block comprises a phosphoramidite or an A-acetylgalactosamine (NAG) ligand.

[0066] In some embodiments, the NAG ligand is an NAG37 ligand, depicted below:NAG37 can be synthesized according to methods known in the art; see US Published Application 20180064819, which is incorporated here by reference in its entirety, including as related to the synthesis of the NAG37 ligand.

[0067] . In some embodiments, the NAG ligand is a NAG52 ligand: depicted below:

[0068] In some embodiments the coupling step uses a coupling activator, such as 5- (Ethylthio)-lH-tetrazole (ETT), 4,5-dicyanoimidazole (DCI), A'-m ethyl imidazole (NMI), and / or I rt- tetrazole. Examples of other coupling activators include, but are not limited to, 2- benzylthiotetrazole, 45-(4-nitrophenyl)-lH-tetrazole, 5-(bis-3,5-trifluoromethyl-phenyl)-lH- tetrazole, 5-methylthio-lH-tetrazole, 2-bromo-4,5-dicyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, l-(cyanomethyl)piperidinium tetrafluoroborate, 1 -methyl- 1H- benzimidazol-3-ium trifluoromethanesulfonate, l-phenyl-lH-imidazol-3-ium trifluoromethanesulfonate, lH-benzimidazol-3-ium trifluoromethanesulfonate, IH-imidazol- 3-ium trifluoromethanesulfonate, 5-nitro-lH-benzimidazol-3-ium trifluoromethanesulfonate, benzimidazolium tritiate, or imidazolium tritiate. In some embodiments, the nucleotide building block is coupled to the detritylated support-bound nucleotide after a washing step.

[0069] In some embodiments, the reaction process (e.g., step-wise reaction cycle) includes an oxidation step which comprises oxidizing the phosphite linkage between the nucleotide building block and the support-bound nucleotide with an oxidation reagent, to form a phosphate triester linkage. In some embodiments, the oxidation reagent is a halogen / base / water solution e.g., iodine / pyridine / water solution). In some embodiments, the base comprises pyridine or a derivative thereof. Examples of other suitable bases include, but are not limited to, pyridine derivatives such as lutidine and collidine. Examples of other suitable oxidation reagents include tert-butyl hydroperoxide (TBHP) and (1 S)-(+)-(10- camphorsulfonyl)-oxaziridine (CSO), which may be used in anhydrous conditions. In some embodiments, the reaction process (e.g., step-wise reaction cycle) includes a thiolation stepwhich comprises thiolating the phosphite linkage between the nucleotide building block and the support-bound nucleotide with a thiolation reagent to form a phosphorothioate linkage. Examples of suitable thiolation reagents include xanthane hydride.

[0070] In some embodiments, the reaction process is repeated one or more times. In some embodiments, at the start of a new cycle, a 5’-O-DMT-group of the coupled product is deblocked (e.g., through the same deblocking step), followed by addition of another nucleotide to the chain (e.g., through the same coupling step). In some embodiments, the process is repeated until an oligomer of the desired length is obtained.

[0071] In some embodiments, by-products and excess reagents in solution are removed by filtration and washing the support with a wash solution, for example, acetonitrile solvent after some or all of the reaction steps.

[0072] In some embodiments, crude reaction product is analyzed and quality-confirmed, e.g., by RP HPLC-MS, before additional / final purification.

[0073] In some embodiments, methods of the present disclosure produce yields of 95% or more, e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more, per cycle. In some embodiments, methods of the present disclosure produce a yield of about 70% or more for the final synthetic oligonucleotide composition (e.g., after final purification). In some embodiments, methods of the present disclosure produce a total yield of at least 0.5 mmol oligonucleotide after a single reactor run. In some embodiments, methods produce a total yield of at least 20 mmol, at least 60 mmol, at least 1 mol, at least 1.5 mol, or at least 2 mol after a single reactor run.

[0074] In some embodiments, methods of the present disclosure have at least a 95% coupling efficiency, at least a 96% coupling efficiency, at least a 97% coupling efficiency, at least a 98% coupling efficiency, at least a 99% coupling efficiency, or at least a 99.5% coupling efficiency.

[0075] In some embodiments, methods of the present disclosure produce an oligonucleotide that is at least 85%, at least 90% free, at least 95% free, at least 96% free, at least 97% free, or at least 98% free of shortmers and / or other contaminants (e.g., stray nucleotides or other oligonucleotide fragments).Detritylation

[0076] In some embodiments, methods of the present disclosure (e.g., a step-wise reaction cycle) include a deblocking step which comprises detritylation. In some embodiments, oligonucleotide synthesis includes protecting the 5' ribose alcohol of nucleotides during thesynthesis, e.g., by a triphenylmethyl (i.e., "trityl") protecting group. In some embodiments, the trityl group is 4,4'-dimethoxytrityl or DMT or 5'-DMT. An exemplary DMT protecting group is shown in the reaction scheme below as the protecting group on the solid-support- linked nucleoside that contains the terminal 3' base of the oligonucleotide. In some embodiments, a DMT protecting group prevents polymerization of the nucleoside during functionalization of the solid support resin.

[0077] In some embodiments, oligonucleotide synthesis methods include removing a trityl group to provide a free alcohol, as shown in the reaction scheme below. In some embodiments, the 2' ribose alcohol of a nucleotide is protected, e.g., by a tertbutyldimethylsilyl (TBS) protecting group, as shown in the reaction scheme below. In some embodiments, a phosphoramidite monomer used in the oligonucleotide synthesis includes a TBS group added to the 2'-OH of the 5' -DMT -protected nucleoside, to yield a 2'-TBS protected nucleoside.

[0078] In some embodiments, detritylation reactions are carried out using an acetic acid and an organic solvent. In some embodiments, the acetic acid comprises a halo-acetic acid (e.g., trifluoroacetic acid (TFA) or di chloroacetic acid (DCA)). In some embodiments, the acetic acid comprises a fluoro-acetic acid (e.g., trifluoroacetic acid (TFA) or pyridine trifluoroacetate (PTA)). In some embodiments, the acetic acid comprises trifluoroacetic acid (TFA). In some embodiments, the acetic acid comprises pyridine trifluoroacetate (PTA). Without being bound by theory, it is believed that using fluoroacetic acids such as TFA in the detritylation step of an oligonucleotide synthesis method may reduce or prevent production of shortmers, e.g., as compared to methods that do not include a capping step. For instance, Examples 1-4 disclose oligonucleotide synthesis studies in which TFA was used for detritylation in Process 1, without a subsequent capping step. In contrast to Process 1, Process 2 uses DCA for detritylation, does not include a wash step comprising pyridine and MeOH, and includes a capping step. When compared to Process 2, Process 1 was shown to provide a similar product purity and reduced shortmer production, despite the noted differences from Process 2 (including not including a capping step).

[0079] In some embodiments, the acetic acid comprises a chlorinated acetic acid. In some embodiments, the acetic acid is chloroacetic acid. In some embodiments, the acetic acid is di chloroacetic acid (DCA). In some embodiments, the acetic acid is trichloroacetic acid. In some embodiments, the acetic acid is a combination of chloroacetic acid, dichloroacetic acid, and trichloroacetic acid.

[0080] In some embodiments, the organic solvent used in the detritylation reaction comprises an arene. In some embodiments, the arene is selected from: alkylbenzene, an alkenylbenzene, an alkynyl benzene, or any combination thereof. In some embodiments, the arene is toluene, xylene, hemimellitene, pseudodocumene, mesitylene, prehnitene, isodurene, durene pentamethylbenzene, hexamethylbenzene, ethylbenzene, ethyltoluene, propylbenzene, propyltoluene, butylbenzene, pentanylbenzene, pentanyl toluene, hexanyl benzene, hexanyl toluene, diphenylmethane, triphenylmethane, tetraphenylmethane, or 1,2-diphenylethane, or combinations thereof.

[0081] In some embodiments, the organic solvent used in the detritylation comprises di chi orom ethane .

[0082] In some embodiments, the organic solvent comprises toluene, xylene, dichloromethane, or combinations thereof.

[0083] In some embodiments, the detritylation reaction is carried out using an acetic acid in an organic solvent, wherein the ratio of acetic acid to organic solvent is about 1 to about 30% by volume of acetic acid to about 70 to about 99% by volume organic solvent. In some embodiments, the ratio of acetic acid to organic solvent 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% by volume of 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% by volume of organic solvent.

[0084] In some embodiments, detritylation reactions may be carried out using an acetic acid at a concentration in detritylation solution of about 0.005 M to about 0.2 M, e.g., 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, about 0.05 M to about 0.1 M. In some embodiments, detritylationreactions may be carried out using an acetic acid at a concentration of about 0.01 M to about 0.2 M in a detritylation solution.

[0085] In some embodiments, the organic solvent used in the detritylation reaction comprises a protic solvent. A protic solvent is a solvent that has a hydrogen atom bound to an oxygen (as in a hydroxyl group), a nitrogen (as in an amine group), or fluoride (as in hydrogen fluoride). Non-limiting examples of a protic solvent are formic acid, w-butanol, isopropanol (IP A), nitromethane, ethanol (EtOH), methanol (MeOH), acetic acid (AcOH), and water.

[0086] In some embodiments, the detritylation reaction comprises an alcohol. In some embodiments, the alcohol is a sterically-hindered alcohol. In some embodiments, the alcohol has a general formulawherein: R1, R2, and R3are each independently hydrogen, 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; provided that no more than one of R1, R2, and R3is hydrogen; and alternatively, R1and R2or R2and R3, together with their intervening carbon atom, form a carbocyclic ring, or a heterocyclic ring.

[0087] In some embodiments, the alcohol used in the detritylation step comprises tert-butanol (TBA), isopropanol (IP A), weo-pentanol, trifluoroethanol (TFE), phenol (PhOH), benzyl alcohol (BnOH), cyclohexanol, a sterol, or a combination thereof. In some embodiments, the alcohol used in the detritylation step comprises trifluoroethanol (TFE).Coupling

[0088] In some embodiments, methods of the present disclosure (e.g., a step-wise reaction cycle) include a coupling step which comprises coupling a nucleotide building block to a support-bound nucleotide. In some embodiments, the 5' end of the support-bound nucleotide has been deblocked and washed, and a new nucleotide is then coupled to the support-bound nucleotide. A coupling reaction using a phosphoramidite is exemplified in the following scheme:

[0089] In some embodiments, the nucleotide to be added to the strand is a phosphoramidite, as shown in the scheme above. In some embodiments, the nucleotide to be added to the strand is a phosphate. In some embodiments, the nucleotide to be added to the strand is a phosphonate. In some embodiments, the nucleotide to be added comprises a plurality of linked nucleotides, e.g., two or more linked nucleotides comprising a phosphate.

[0090] In some embodiments, the nucleotide to be added to the strand is added in a solution comprising a solvent. In some embodiments, the nucleotide is added in a solution, and the solvent is acetonitrile. In some embodiments, the nucleotide to be added to the strand is added in a solution, wherein the concentration of the nucleotide in the solution is about 0.05 to about 2.0 M. In some embodiments, the nucleotide to be added to the strand is added in a solution wherein the concentration of the nucleotide is about 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, 1.5,1.6, 1.7, 1.8, or 1.9 M to about 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 2.0 M.

[0091] In some embodiments, the nucleotide to be added to the strand is added in a low molar equivalence relative to the nucleotide bound to the solid support. In some embodiments, the additional nucleotide is added in an amount of 1.0 equivalents to about 5.0 equivalents. In some embodiments, the nucleotide to be added is added in an amount 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 4.9 mole equivalents relative to the nucleotide bound to the strand to about 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 mole equivalents relative to the nucleotide bound to the strand.

[0092] In some embodiments, coupling reactions are 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 IH-tetrazole, 5-ethylthio-lH-tetrazole, 2-benzylthiotetrazole, and 4, 5 -di cyanoimidazole. Examples of other coupling activators include, but are not limited to, 2-benzylthiotetrazole, 5-(4-nitrophenyl)-lH-tetrazole, 5-(bis-3,5-trifluoromethyl-phenyl)-lH-tetrazole, 5- methylthio-lH-tetrazole, 2-bromo-4,5-di cyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, l-(cyanomethyl)piperidinium tetrafluoroborate, 1 -methyl- IH-benzimidazol- 3-ium trifluoromethanesulfonate, l-phenyl-lH-imidazol-3-ium trifluoromethanesulfonate, lH-benzimidazol-3-ium trifluoromethanesulfonate, lH-imidazol-3-ium trifluoromethanesulfonate, 5-nitro-lH-benzimidazol-3-ium trifluoromethanesulfonate, benzimidazolium triflate, or imidazolium triflate. In some embodiments, the activator is 5- ethylthio-lH-tetrazole (ETT). In some embodiments, the activator is added in an amount of from about 1 to about 30 mole equivalents relative to the amount of nucleotide bound to the solid support. In some embodiments, the activator is added in an amount of 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 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 mole equivalents relative to the amount of nucleotide bound to the solid support.

[0093] In some embodiments, the reagent used in the coupling step comprises a nucleotide (or a plurality of linked nucleotides) to be added to the strand, an activator, and a solvent. In some embodiments of the coupling step, the activator is in a concentration of about 0.1 M to about 5 M. In some embodiments, the concentration of the activator is about 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, or 4.9 M to about 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 M. In some embodiments, the reagent used in the coupling step comprises the activator and the nucleotide to be added in a ratio of about 0.1 moles activator to about 5.0 moles nucleotide to be added to the strand. In some embodiments, the ratio of the activator and the nucleotide to be added to the strand is about 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, or 4.9 moles activator to about 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 nucleotide to be added to the strand. In some embodiments, the ratio of activator to nucleotide to be added to the strand is about 1 : 1.

[0094] In some embodiments, the coupling step is carried out for a time of about 1 minute to about 60 minutes. In some embodiments, the coupling step is carried out 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, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, or 59 minutes 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, 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.Oxidation / Thiolation

[0095] In some embodiments, the coupling of a new nucleotide to a strand of support-bound nucleotides (e.g., by using a phosphoramidite) results in a coupling product that comprises a phosphite. In some embodiments, methods of the present disclosure (e.g., a step-wise reaction cycle) include an oxidation step which comprises oxidizing the phosphite linkage between an added nucleotide building block and a support-bound nucleotide. In some embodiments, methods of the present disclosure (e.g., a step-wise reaction cycle) include a thiolation step which comprises thiolating the phosphite linkage between an added nucleotide building block and a support-bound nucleotide.

[0096] In some embodiments, the oxidation / thiolation step comprises oxidizing / thiolating the phosphite to a phosphate or phosphorothioate, as exemplified in the following reaction:

[0097] In some embodiments, the phosphorous atom is oxidized to create a phosphate. In some embodiments, the oxidation is performed using a halogen in the presence of water and a base such as pyridine, lutidine, and collidine. In some embodiments, the oxidation is performed using iodine (I2) in a combined water and pyridine solvent. In some embodiments,the ratio of water to pyridine in the oxidation step is about 1% to about 20% by volume water to about 80% to about 99% by volume pyridine. In some embodiments the ratio of water to pyridine is about 10% by volume water to about 90% by volume pyridine.

[0098] In some embodiments, the oxidation is performed in anhydrous conditions using tertbutyl hydroperoxide (TBHP), (lS)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO), or a combination thereof.

[0099] In some embodiments, the phosphorous atom is thiolated to create a phosphorothioate. In some embodiments, a thiolation reaction is performed using a thiolation reagent such as 3-(Dimethylaminomethylidene)amino-3H-l,2,4,-dithiazole-3-thione (DDTT). In some embodiments, the thiolation reagent is xanthane hydride.

[0100] In some embodiments, the concentration of the iodine in the oxidation solution is about 10 mM to about 200 mM. In some embodiments, the concentration of the iodine in the oxidation solution is 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.

[0101] In some embodiments, the oxidation step is carried out for about 1 to about 30 minutes. In some embodiments, the oxidation step is carried out 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 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 minutes. Capping

[0102] A capping step typically involves capping unreacted solid-support bound 5 ’-OH groups or other free alcohols produced by unintended side reactions. In some embodiments, a capping reagent comprises hydroxy protecting groups, such as esters and ethers. Non-limiting examples of suitable ethers and esters are methoxymethyl or MOM ether, tetrahydropyranyl or THP ether, tert-butyl ether, allyl ether, benzyl ether, tert-butyldimethylsilyl or TBDMS ether, tert-butyldiphenylsilyl or TBDPS ether, acetic acid ester, pivalic acid ester, benzoate ester, etc. In one embodiment, the unreacted 5 ’-OH groups are capped with acetic anhydride.

[0103] Oligonucleotide synthesis methods known in the art usually employ a capping step to reduce or prevent the production of shortmers 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 comprise a capping step. In some embodiments, avoiding a capping step in an oligonucleotide synthesis method results in higher crude yield compared toa comparable method that includes a capping step. In some embodiments, crude yield for a method that does not include a capping step 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 a comparable method that includes a capping step.

[0104] In some embodiments, not including a capping step in an oligonucleotide synthesis method results in higher overall final yield compared to comparable methods that include a capping step. In some embodiments, final yield for a method that does not include a capping step 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 final yield of a comparable method that includes a capping step.

[0105] In some embodiments, not including a capping step in an oligonucleotide synthesis method results in reduced shortmers compared to comparable methods that include a capping step.Washing

[0106] In some embodiments, a washing step is performed after some or all of the oligonucleotide synthesis steps disclosed herein (e.g., detrityl ati on, couple, oxidation). In some embodiments, a washing step is performed to flush the reactants from the vessel. In some embodiments, the wash comprises: (i) a neutralizing solvent, (ii) alcohol or water, and (iii) an organic solvent. In some embodiments, the wash comprises pyridine, methanol, and toluene. In some embodiments, a wash (e.g., a wash comprising an organic solvent) is added to the reaction flask and agitated to remove undesired components from the product. In some embodiments, a wash (e.g., a wash comprising 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 wash step is repeated two or more times to remove undesired materials.

[0107] 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 a combination thereof. In some embodiments, the wash solvent comprises acetonitrile (MeCN), pyridine, methanol, or a combination thereof. Without being bound by theory, it is believed that using a combination of pyridine and methanol in the washing step of a method that does not have a capping step helps reduce or prevent production of shortmers.EXAMPLES

[0108] Further description is provided in the following examples.

[0109] The following abbreviations are used in the examples when describing 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 '-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate i = 2 '-O-methylinosine-3 '-phosphate u = 2'-O-methyluridine-3 '-phosphate us = 2'-O-methyluridine-3'-phosphorothioateAf = 2 '-fluoroadenosine-3 '-phosphateAfs = 2'-fluoroadenosine-3'-phosporothioateCf = 2'-fluorocytidine-3 '-phosphateGf = 2 '-fluoroguanosine-3 '-phosphateUf = 2'-fluorouridine-3 '-phosphateUfs = 2'-fluorouridine-3'-phosphorothioate invAb= inverted abasic deoxyribonucleotide:When positioned internally: linkage towards 5' endlinkage towards 3' endWhen positioned at the 3' terminal end: linkage towards 5' endinvAbs= inverted abasic deoxyribonucleotide-5 '-phosphorothioateWhen positioned internally: linkage towards 5' endlinkage towards 3' end s = phosphorothioate linkageSpl8s = spacer 18, hexaethylene glycol, having the following structure:Example 1 - Comparison of Oligonucleotide Synthesis Methods in the Synthesis of SEQ ID NO: 1

[0110] In this example, an oligonucleotide comprising 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') was synthesized according to different process parameters and reaction conditions, which are summarized in Tables 1 and 2 below.Table 1: Process 1 Parameters and Reaction ConditionsTable 2: Process 2 Parameters and Reaction Conditions

[0111] Process 1 Synthesis: 1 mmol inv Ab-loaded Nittophase HL (250pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 1 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 1. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm poly ethersulfone (PES) membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed on an AKTA Crossflow with a 2KDa molecular weight cutoff (MWC) regenerated cellulose membrane for diafiltration and desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against 0.2M NaCl for 7 diavolumes, dialyzed against deionized water for 10 diavolumes, then 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.

[0112] Process 2 Synthesis: 1 mmol inv Ab-loaded Nittophase HL (250 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 1 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 2. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine solution (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was loaded onto a 5x19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 pm). The crude material was purified using a gradient of 10-60% B over 15 CV column volumes (mobile phase A: 20 mM sodium phosphate, 10% ACN pH11.0 and mobile phase B: 20 mM sodium phosphate, IM NaBr, 10% ACN pH 11.0). Pure fractions were pooled, then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against deionized water for 10 diavolumes, then 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.

[0113] 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. An RP HPLC- MS chromatogram is shown in FIG. 1, comparing Process 1 (bottom line of FIG. 1) and Process 2 (top line of FIG. 1).Table 3: Example 1 ResultsExample 2 - Comparison of Oligonucleotide Synthesis Methods in the Synthesis of SEQ ID NO: 2

[0114] In this example, an oligonucleotide comprising 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 1 and Process 2 process parameters and reaction conditions listed in Tables 1 and 2 of Example 1, above.

[0115] Process 1 Synthesis: 1 mmol 2'OMe-G-loaded Nittophase HL (350 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 2 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 1. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for diafiltration and desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against 0.2 M NaCl for 7 diavolumes, dialyzed against deionized water for 10 diavolumes, then 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.

[0116] Process 2 Synthesis: 1 mmol 2'OMe-G-loaded Nittophase HL (350 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 2 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 2. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine solution (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was loaded onto a 5x19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 pm). The crude material was purified using a gradient of 10-60% B over 15 CV column volumes (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). Pure fractions were pooled, then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against deionized water for 10 diavolumes, then 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.

[0117] 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. An RP HPLC- MS chromatogram is shown in FIG. 2, comparing Process 1 (bottom line of FIG. 2) and Process 2 (top line of FIG. 2).Table 4: Example 2 ResultsExample 3 - Comparison of Oligonucleotide Synthesis Methods in the Synthesis of SEQ ID NO: 3

[0118] In this example, an oligonucleotide comprising 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 Process 1 and Process 2 process parameters and reaction conditions listed in Tables 1 and 2 of Example 1, above.

[0119] Process 1 Synthesis: 1 mmol 2'OMe-G-loaded Nittophase HL (350 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 3 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to theparameters and reaction conditions in Table 1. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for diafiltration and desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against 0.2 M NaCl for 7 diavolumes, dialyzed against deionized water for 10 diavolumes, then 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.

[0120] Process 2 Synthesis: 1 mmol 2'OMe-G-loaded Nittophase HL (350pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 3 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 2. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine solution (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to 1 L with deionized water. The crude solution was loaded onto a 5x19.8 cm column packed with Tosoh TSKGel SuperQ-5PW (20 pm). The crude material was purified using a gradient of 10-60% B over 15 CV column volumes (mobile phase A: 20 mM sodium phosphate, 10% ACN pH 11.0 and mobile phase B: 20 mM sodium phosphate, IM NaBr, 10% ACN pH 11.0). Pure fractions were pooled, then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against deionized water for 10 diavolumes, then 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.

[0121] 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. An RP HPLC- MS chromatogram is shown in FIG. 3, comparing Process 1 (bottom line of FIG. 2) and Process 2 (top line of FIG. 2).Table 5: Example 3 ResultsExample 4 - Comparison of Oligonucleotide Synthesis Methods in the Synthesis of SEQ ID NO: 4

[0122] In this example, an oligonucleotide comprising 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 1 and Process 2 process parameters and reaction conditions listed in Tables 1 and 2 of Example 1, above.

[0123] Process 1 Synthesis: 1 mmol 2’OMe-U-loaded Nittophase HL (350 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 4 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 1. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to IL with deionized water. The crude solution was then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for diafiltration and desalting. The solution was initially concentrated to a volume of 300 mL, dialyzed against 0.2 M NaCl for 7 diavolumes, dialyzed against deionized water for 10 diavolumes, then 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.

[0124] Process 2 Synthesis: 1 mmol 2'OMe-G-loaded Nittophase HL (350 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide of SEQ ID NO: 4 was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 2. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the solid support with 50 mL methylamine solution (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to IL with deionized water. The crude solution was loaded onto a 5x19.8cm column packed with Tosoh TSKGel SuperQ-5PW (20 pm). The crude material was purified using a gradient of 10-60% B over 15 CV column volumes (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). Pure fractions were pooled, then placed on an AKTA Crossflow with a 2 KDa MWC regenerated cellulose membrane for desalting. The solution was initiallyconcentrated to a volume of 300 mL, dialyzed against deionized water for 10 diavolumes, then concentrated to a final volume of 150mL before being eluted from the system. The crude material and final product were analyzed by RP HPLC-MS.

[0125] 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. An RP HPLC- MS chromatogram is shown in FIG. 4, comparing Process 1 (bottom line of FIG. 4) and Process 2 (top line of FIG. 4).Table 6: Example 4 ResultsExample 5 - Process 3 Oligonucleotide Synthesis Method in the Synthesis of a Long- Strand (Dimer) Oligonucleotide

[0126] In this example, an oligonucleotide was synthesized with the follow features: NAG37s at the 5'-terminal end linked to a 19-nucleotide long segment of chemically modified nucleotides (either 2'-fluoro or 2'-methoxy modified nucleotides) with an inverted abasic end cap moiety at the 3' end which is then connected an Spl8s spacer to a second 19- nucleotide long segment of chemically modified nucleotides (either 2'-fluoro or 2'-methoxy modified nucleotides) with an inverted abasic end cap moiety at the 3' terminal end.

[0127] NAG37s,c,g,u,u,u,u,Gf,c,Uf,u,Uf,u,g,u,a,a,c,u,us,invAb,Spl8s,gs,g,g,a,c,a,Gf,Uf,Af,u ,u,c,u,c,a,g,u,i,as,invAb (SEQ ID NO: 5). A key for abbreviations is above in paragraph

[0107] ,

[0128] The oligonucleotide was synthesized according to the Process 3 process parameters and reaction conditions, which are summarized in Table 7 below.Table 7: Process 3 Parameters and Reaction Conditions for Long-Strand Synthesis

[0129] Process 3 for Long- Strand Synthesis: 220 pmol inv Ab-loaded Nittophase HL (100 pmol / g) was loaded into a 24 mL synthesis column. The oligonucleotide was synthesized at 220 pmol scale on an AKTA oligopilot 100 synthesizer according to the parameters and reaction conditions in Table 7. Following synthesis, the support was dried, then half of the support (110 pmol) was cleaved from the oligonucleotide with 11 mL ammonium hydroxide solution (28% in water) for approximately 7 days at room temperature. The crude solution was filtered using a 0.45 pm PES membrane and washed with 30% ethanol. The crude solution was diluted to 250 mL with deionized water. The crude solution was loaded onto a 5x13.7 cm column packed with Tosoh TSKGel SuperQ-5PW (20 pm). The crude material was purified using a gradient of 10-60% B over 15 CV column volumes (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). Pure fractions were pooled. The crude material and final product were analyzed by RP HPLC-MS and AEX HPLC.

[0130] Results: The crude yield, crude purity, final yield after purification, final purity, and - NAG impurities for Process 3 are disclosed in Table 8 below.Table 8: Example 5 ResultsExample 6 - Comparison of Extended Oligonucleotide Synthesis Methods

[0131] In this example, the oligonucleotide from Example 2 (SEQ ID NO: 2) was synthesized according to Process 4 and Process 5 process parameters and reaction conditions listed in Tables 9 and 10 below, which were designed to simulate a six-fold increase in scale using an Oligopilot 400 synthesizer (including longer reaction times and slower linear flow rates required for large-scale production) but using a smaller scale load. Process 4 is comparable to Process 1 from Example 2, with extended reaction times. Process 5 is comparable to Process 2 from Example 2, with extended reaction times.Table 9: Process 4 Parameters and Reaction ConditionsTable 10: Process 5 Parameters and Reaction Conditions

[0132] 1 mmol 2'OMe-G-loaded Nittophase HL (350 pmol / g) was loaded into a Fineline 35 synthesis column. The oligonucleotide was synthesized at 1 mmol scale on an AKTA oligopilot 100 synthesizer using both Process 4 and Process 5 parameters and reaction conditions in Tables 9 and 10. The flow rates for these syntheses were capped to simulate the contact times necessary to perform a 428.5 mmol synthesis on an Oligopilot 400, resulting in much longer contact times. Following synthesis, the support was dried, then the oligonucleotide was cleaved from the support with 50 mL methylamine (40% in water) for 100 minutes at room temperature. The crude solution was filtered using a 0.45 pm 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.

[0133] Results: The crude products for Process 4 and Process 5, along with Process 1 and Process 2 from Example 2, are disclosed in Table 11 below. An RP HPLC-MS chromatogram is shown in FIG. 5, comparing Process 2 (top line of FIG. 5), Process 4 (bottom line of FIG. 5), and Process 5 (middle line of FIG. 5).Table 11: Example 6 Results (% Crude Purity)Example 7 - Synthesis of NAG52 (Compound 9)Synthesis of Compounds 1-3

[0134] In this example, Compounds 1, 2 and 3 below were each synthesized in accordance with previously published procedures (see, e.g., U.S. Patent Application Publication No.was synthesized in accordance with the procedure described in International Patent Application Publication No: WO 2017 / 156012, the contents of which are incorporated herein by reference as related to the synthesis of Cbz- NH-Glu-Glu-OH.

[0136] Compound 1 was synthesized from the following synthetic route:123Synthesis of Compound 5

[0138] Compound 3 (4.61 g, 12.3 mmol) and Boc-N-amido-PEG2-NHS ester (CAS 2183440-73-3, 4.61 g, 12.3 mmol) were dissolved in anhydrous DCM (100 mL) followed by addition of triethylamine (3.4 mL, 24.6 mmol). The reaction mixture was stirred at room temperature (rt) for 2h, the solution was concentrated down to 30 mL under reduced pressure and diluted with chloroform (300 mL). The resulting solution was first washed withbrine / 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 on a silica column (100% DCM to 20% MeOH in DCM).

[0139] Fractions containing the desired product, Compound 4, were combined, the solvent was removed under reduced pressure and the resulting foaming residue was redissolved in 4M HC1 in 1,4-di oxane (100 mL). The reaction mixture was stirred at rt for Ih, and the solvent was removed under reduced pressure. The resulting residue was suspended in toluene and dried under reduced pressure giving the desired product, Compound 5, as an HC1 salt (5.30 g, 9.30 mmol, 76% yield). Calculated MW 533.26 Found ESIMS+m / z = 534.23 [M+H+],Synthesis of Compound 7

[0140] Compound 5 (5.30 g, 9.94 mmol) was dissolved in anhydrous DMF followed by 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) was added upon vigorous stirring. The solution turned light maroon, darkening over time. The reaction mixture was left stirring at rt for 2h when no unreacted starting material could be detected by LC-MS. The solvent was removed by co-evaporation with toluene, and the residue was redissolved in chloroform (400 mL). The resulting solution was first washed 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 on a silica column (100% DCM to 20% MeOH in DCM).

[0141] Fractions containing the desired product, Compound 6, were combined, the solvent was removed under reduced pressure and the resulting foaming residue was redissolved inmethanol (200 mL). Pd / C (0.70 g) was added to the solution, the suspension was hydrogenated under 1 atm overnight. The reaction mixture was stirred under hydrogen at rt overnight. The solution was filtered through a celite pad and concentrated under reduced pressure to yield the desired product, Compound 7, that was used as is in the next step (3.80 g, 2.09 mmol, 77% yield). Calculated MW 1821.84 Found ESIMS+m / z = 912.13 [M+2H+],Synthesis of Compound 9

[0142] Compound 7 (3.80 g, 2.09 mmol) was dissolved in anhydrous DMF (30 mL) and was slowly added to a solution containing 4-hydroxycyclohexane carboxylic acid (0.35 g, 2.43 mmol), TBTU (0.82 g, 2.16 mmol) and DIPEA (1.12 mL, 6.44 mmol) in anhydrous DMF (30 mL). The reaction mixture was stirred at rt for 2h. The solvent was removed by coevaporation with toluene, and the residue was redissolved in chloroform (300 mL). Thesolution was washed with brine / 5% citric acid (1 : 1, 30 mL), dried over ISfeSCU and concentrated under reduced pressure and purified on a silica column (100% DCM to 30% MeOH in DCM) to yield the desired product, Compound 8 (2.40 g, 1.23 mmol, 59% yield).Calculated MW 1947.90 Found ESIMS+m / z = 975.46 [M+2H+],

[0143] Compound 8 (2.40 g, 1.23 mmol) was thoroughly dried by co-evaporating DCM with toluene and dried in vacuo for 30 min. A round-bottom flask was charged with a stir bar and pre-treated molecular sieves, and was purged with nitrogen. The flask was filled with DCM (100 mL), and the molecular sieves were gently stirred for 10 min. Diisopropylammonium tetrazolide (1.40 g, 8.19 mmol) was added to the solution, and the reaction mixture was stirred for another 30 min. 2-Cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (0.55 mL, 1.73 mmol) was added, and the reaction mixture was stirred for Ih at rt when no unreacted starting material could be detected by LC-MS. The solution was filtered through a celite pad to remove molecular sieves and diluted with saturated bicarbonate solution (100 mL) upon stirring. After 15 min, the organic layer was separated, and the aqueous layer wasextracted with chloroform (2 x 200 mL). The organic fractions were combined, dried over Na2SO4, concentrated under reduced pressure and purified on a silica column (100% DCM (+0.1% triethylamine) to 10%MeOH in DCM (+0.1% triethylamine)).

[0144] The fractions containing the desired product, Compound 9 (NAG52), were combined, concentrated under reduced pressure, and the product was co-evaporated twice with toluene to remove any residual triethylamine to give the desired product as an off-white solid (2.4 g, 1.16 mmol, 94% yield).

[0145] Tris (aCNAGAc3Peg2)BisGluNHCO-CH-PA: 'H 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).31P NMR (DMS0-d6): 145.84, 146.01.Example 8 - Comparison of Oligonucleotide Dimer Synthesis Methods

[0146] 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,Spl8,gs,g,g,a,c,a,Gf,u,Af,u,Uf,c,u,c, a,g,u,i,as,invAbs,C6-NH2) was synthesized (500 pmol scale) using Processes 6-8 described below.Table 12: Process 6 Parameters and Reaction ConditionsTEA on Nittophase support (Nittophase 100 97pmol / g loading)Table 13: Process 7 Parameters and Reaction ConditionsDCA on Nittophase support (Nittophase 100 97pmol / g loading)Table 14: Process 8 Parameters and Reaction Conditions DCA on CPG Support (lOOOA CPG 58pmol / g loading)

[0147] Results: Comparisons of the crude yield, crude purity, final yield after purification, and final purity for Processes 6-8 are disclosed in Table 15 below.Table 15: Comparison of Results of Processes 6-8

[0148] TFANitto conditions produced the highest crude purity (78%). DCANitto conditions produced the highest final purity (88.6%) with TFANitto producing second highest (87.6%).TFA Nitto conditions produced the highest crude (11.08 g / mmol) and final yield (7.9g / mmol).

Claims

CLAIMSWhat is claimed is:

1. A method of producing an oligonucleotide comprising a. providing a first nucleotide comprising a first trityl group, wherein the first nucleotide is a support-bound nucleotide on a substrate, and wherein the substrate is a solid phase support; b. detritylating the first nucleotide with a detritylating reagent to form a detritylated nucleotide, wherein the detritylating reagent comprises an acetic acid or a salt thereof, optionally wherein the acetic acid is trifluoroacetic acid (TFA) or a salt thereof; c. washing the detritylated nucleotide with a wash; and d. coupling a second nucleotide to the detritylated nucleotide under reaction conditions that promote coupling of the second nucleotide to the detritylated nucleotide to produce the oligonucleotide; wherein the method does not comprise a capping step.

2. The method of claim 1 wherein the wash comprises (i) a neutralizing solvent, (ii) alcohol or water, and (iii) an organic solvent; optionally wherein the wash comprises pyridine, methanol, and toluene.

3. The method of any one of claims 1-2, further comprising a second wash step after coupling the second nucleotide to the first nucleotide; optionally wherein the second wash step comprises acetonitrile.

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

5. The method of claim 4, wherein the oxidation reagent comprises iodine, pyridine, and water.

6. The method of any one of claims 1-5, 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 of claim 6, wherein the thiolation reagent comprises xanthane hydride.

8. The method of any one of claims 4-7, further comprising an additional wash step after the oxidation step or the thiolation step; optionally wherein the additional wash comprises acetonitrile.

9. The method of any one of claims 1-8, the method consisting of a. providing a first nucleotide comprising a first trityl group, wherein the first nucleotide is a support-bound nucleotide on a substrate, and wherein the substrate is a solid phase support; b. detritylating the first nucleotide with a detritylating reagent comprising trifluoroacetic acid (TFA) to form a detritylated nucleotide; c. washing the detritylated nucleotide with a wash comprising pyridine and methanol; and d. coupling a second nucleotide to the detritylated nucleotide under reaction conditions that promote coupling of the second nucleotide to the detritylated nucleotide to produce the oligonucleotide, wherein the second nucleotide comprises a second trityl group; and e. optionally, washing the oligonucleotide; 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 thiolated oligonucleotide.

10. The method of any one of claims 1-9, wherein the method results in a higher crude yield of the oligonucleotide compared to the same method of producing the oligonucleotide with a capping step.

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

12. The method of any one of claims 1-11, wherein the method results in fewer shortmers compared to the same method of producing the oligonucleotide with a capping step.

13. The method of any one of claims 1-12, wherein steps (a) through (d) are repeated one or more times, wherein the second nucleotide comprising the second trityl group from step (d) is used as the first nucleotide comprising the first trityl group in the subsequent repeat of step (a).

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

15. The method of any one of claims 1-14, wherein the support-bound nucleotide comprises one or more non-nucleotide moieties between the solid phase support and the first nucleotide.

16. The method of any one of claims 1-15, wherein the detritylating reagent comprises trifluoroacetic acid at a concentration of about 1-15% by volume, optionally about 1- 10% by volume, optionally about 6% by volume.

17. The method of any one of claims 1-16, wherein the detritylating reagent further comprises an aromatic or halogenated solvent; optionally wherein the detritylating reagent further comprises toluene, 2,2,2-trifluoroethanol, dichloromethane, or mixtures thereof.

18. The method of any one of claims 1-17, wherein the reaction conditions that promote coupling comprise the use of an activator.19 The method of claim 18, wherein the activator is IH-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-lH-tetrazole (ETT), 5-(4-nitrophenyl)-lH-tetrazole, 5-(bis-3,5-trifluoromethyl-phenyl)-lH-tetrazole, 5-methylthio-lH-tetrazole, 2 -bromo-4,5-di cyanoimidazole, pyridinium chloride, pyridinium trifluoroacetate, 1- (cyanomethyl)piperidinium tetrafluoroborate, 1 -methyl- IH-benzimidazol -3 -ium trifluoromethanesulfonate, l-phenyl-lH-imidazol-3-ium trifluoromethanesulfonate, lH-benzimidazol-3-ium trifluoromethanesulfonate, lH-imidazol-3-ium trifluoromethanesulfonate, 5-nitro-lH-benzimidazol-3-ium trifluoromethanesulfonate, benzimidazolium triflate, and / or imidazolium triflate20. The method of claim 19, wherein the activator is ETT.

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

22. The method of claim 21, wherein the oligonucleotide is from 19 nucleotides long to 23 nucleotides long.

23. The method of claim 22, wherein the oligonucleotide is 21 nucleotides long.

24. The method of claim 22, wherein the oligonucleotide is 23 nucleotides long.

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

26. The method of claim 21, wherein the oligonucleotide is from 38 nucleotides long to46 nucleotides long.

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

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

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

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

31. The method of any of claims 1-30, wherein the oligonucleotide comprises at least one modified oligonucleotide linkage.

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

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

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

35. The method of claim 34, wherein the oligonucleotide dimer comprises two singlestranded oligonucleotides coupled together.

36. The method of claim 34, wherein the oligonucleotide dimer comprises two doublestranded oligonucleotides coupled together.

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

38. The method of claim 37, wherein the oligonucleotide trimer comprises three singlestranded oligonucleotides coupled together.

39. The method of claim 37, wherein the oligonucleotide trimer comprises three doublestranded oligonucleotides coupled together.

40. The method of any one of claims 34-39, wherein the oligonucleotide dimer or trimer comprises a spacer.

41. The method of claim 40, wherein the spacer is Spl8.

42. The method of any of claims 1-41, wherein the method results in at least 2% higher crude yield compared to the same method of producing the oligonucleotide with a capping step, optionally at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher.

43. The method of any of claims 1-42, wherein the method results in at least 3% higher final yield compared to the same method of producing the oligonucleotide with a capping step, optionally at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher.

44. The method of any of claims 1-43, wherein the method results in a reduction of at least 3% of shortmers compared to the same method of producing the oligonucleotide with a capping step; optionally at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10% higher.

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

46. An oligonucleotide synthesized using the method of any one of claims 1-45.

47. A pharmaceutical composition comprising the oligonucleotide of claim 46, and a pharmaceutically acceptable carrier.

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