Liquid-phase oligonucleotide synthesis using 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) as a protecting group

Abstract

A process of liquid-phase oligonucleotide synthesis (LPOS) is provided which uses the photolabile protecting group 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC). The process may be used for synthesizing an NPPOC-protected oligonucleotide or salt thereof or a precursor thereto. Also provided is the use of NPPOC in liquid phase oligonucleotide synthesis, as well as an NPPOC-protected oligonucleotide or salt thereof which is in solution.

Description

[0001] 201393-PCT01-NP P78290WO

[0002] LIQUID-PHASE OLIGONUCLEOTIDE SYNTHESIS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 696,889, filed September 20, 2024, the disclosure of which is incorporated by reference herein.

[0005] SEQUENCE LISTING

[0006] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled P78290WO Sequence Listing, created on 18 September, 2025, which is 7 kB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0007] FIELD

[0008] Provided is a process of liquid-phase oligonucleotide synthesis (LPOS). The process uses the photolabile protecting group 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) within compounds (e.g., oligonucleotide intermediates and / or reactants) which are deprotected in solution. Also provided is a process for synthesizing an NPPOC -protected oligonucleotide or salt thereof, e.g. for use as an intermediate in the aforementioned process. Use of NPPOC as a protecting group in the synthesis of an oligonucleotide or a salt thereof in solution is also provided, as is a solution comprising an NPPOC-protected oligonucleotide or salt thereof.

[0009] SUMMARY

[0010] Oligonucleotides, such as e.g. antisense oligonucleotides, represent an extremely important class of molecules for research and pharmaceutical applications. While oligonucleotides may be synthesized in small amounts for research use, pharmaceutical applications can require large scale production. Owing to their oligomeric structure and the stepwise process which is typically employed for their synthesis, the yield and purity of an oligonucleotide product can be highly sensitive to small improvements in the individual synthetic steps. That sensitivity increases as the length of the oligonucleotide increases, and it becomes especially important during commercial scale production of pharmaceutical oligonucleotides.

[0011] Oligonucleotides are conventionally synthesized by automated solid phase methods that employ the phosphoramidite approach originally developed in the 1980s (see, e.g., Matteucci et al., J. Am. Chem. Soc. (1981) 103:3185-3191; and McBride et al., Tetrahedron Lett. 201393-PCT01-NP P78290WO

[0012] (1983) 24:245-248). Each cycle of reactions is typically responsible for adding one nucleotide to the growing oligonucleotide, and a key step in each cycle is the removal of a protecting group such as 4,4'-dimethoxytrityl (DMT) from the terminal 5 ’-hydroxyl group. This deprotection step is typically carried out using a strong acid such as trifluoroacetic acid or dichloroacetic acid - these reagents pose problems in terms of cost, supply, regulatory compliance and safety. The acidic environment of the deprotection step can also promote degradation of the growing oligonucleotide, e.g., via depurination, and it can lead to sulfur deposition in the reaction vessel. Furthermore, the DMT carbocation and derivatives thereof which form in the deprotection step are problematic for subsequent steps and must be removed from the reaction mixture, typically by a series of steps, including reverse phase chromatography and concentration and desalting (see, e.g., Krotz et al., Organic Process Research & Development (2003) 7 :47-52), diafiltration or tangential flow filtration (see, e.g., US patent publication No. 2021 / 0347800). This introduces additional steps into the reaction cycle which can increase complexity and cost, andean decrease yield. Even after purification, trace amounts of impurities can persist, thereby also affecting purity.

[0013] One way to simplify purification steps during the reaction cycle is to tether the growing oligonucleotide to a solid support such as a polymer bead. Synthesis of the oligonucleotide on solid phase allows impurities to be washed away at the end of a step of the reaction cycle using less solvent and / or without risking loss of the product. However, solid phase oligonucleotide synthesis has its own limitations, including extra material costs and significant difficulties in scaling the process up to commercial scales.

[0014] There is therefore a need for efficient and cost-effective methods for the production of oligonucleotides that reduce the need for purification, have fewer side reactions, minimize or prevent the production of unwanted products or products that interfere with downstream chemistry, limit the use of solvents, and are readily scalable. The present disclosure seeks to address this need by providing a process for synthesizing oligonucleotides which retains advantages of LPOS methods (e.g., as compared to solid phase methods) while avoiding limitations associated with the use of conventional LPOS protecting groups. To this end, the inventors have discovered that 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) can act as a suitable protecting group in a LPOS process, and that the use of NPPOC avoids the above- mentioned difficulties of DMT while also avoiding problems associated with solid phase synthesis. NPPOC has previously been used as a protecting group in the solid phase synthesis of DNA microarrays (see, e.g., Beier et al., Nucleic Acids Res. (2000) 28(4):el 1; and US 201393-PCT01-NP P78290WO patent publication No. 2010 / 0292458), using photolithography to take advantage of the photolabile nature of NPPOC. Its use in LPOS methods as described herein and the advantages associated therewith have not, however, previously been reported.

[0015] In brief, the process of the present disclosure uses NPPOC as a protecting group in a process for synthesizing an oligonucleotide or a salt thereof in solution. The process advantageously does not require the use of a solid support and is, therefore, readily scaleable. Moreover, the byproducts which are liberated following NPPOC deprotection do not destabilise the growing oligonucleotide and do not interfere with subsequent reaction steps. The process thus does not require additional purification steps to remove byproducts resulting from deprotection between cycles such as, e.g., diafiltration. The processes of the invention can thus provide improvements over convention processes in terms of, e.g., increased yield, decreased cost, improved sustainability, and / or improved scalability.

[0016] Summary of the Disclosure

[0017] The present disclosure includes the following aspects and embodiments which are presented as numbered clauses 1 to 22.

[0018] 1. A process for synthezising an oligonucleotide or a salt thereof in liquid phase, wherein 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) is used as a protecting group.

[0019] 2. The process of clause 1, wherein NPPOC is used as a 5 ’-hydroxyl protecting group.

[0020] 3. The process of clause 1 or clause 2 comprising a deprotection step, wherein an NPPOC-protected oligonucleotide or an NPPOC-protected precursor thereto (e.g., an NPPOC-protected growing oligonucleotide) is irradiated in solution to deprotect the oligonucleotide or precursor thereto (either in part or in full).

[0021] 4. The process of clause 3, wherein the irradiation results in at least about 90% deprotection, e.g. at least about 95%, at least about 99%, or about 100% deprotection.

[0022] 5. The process of clause 3 or clause 4, wherein the irradiation is performed with light having a wavelength (e.g., a km;«) of about 350 nm to about 400 nm.

[0023] 6. The process of any one of clauses 3 to 5, wherein the irradiation is performed in the presence of a base, e.g. wherein the base is selected from one or more of zPioNH, 1- methylimidazole, and morpholine, optionally wherein the base is 1 -methylimidazole. 201393-PCT01-NP P78290WO

[0024] 7. The process of any one of clauses 3-6 wherein the reaction mixture is sparged with nitrogen before irradiation.

[0025] 8. The process of any one of clauses 3-7, further comprising a coupling step wherein the deprotected oligonucleotide precursor is reacted with a nucleoside -containing moiety under conditions resulting in chain extension (i.e., forming the oligonucleotide or an extended precursor thereto).

[0026] 9. The process of clause 8, wherein the nucleoside-containing moiety is NPPOC- protected such that the oligonucleotide or extended precursor thereto which is produced is also NPPOC-protected.

[0027] 10. The process of clause 8 or clause 9, wherein the chain extension step is carried out on the deprotected oligonucleotide precursor from the preceding deprotection step without any intervening purification (e.g., diafiltration).

[0028] 11. The process of any one of clauses 8-10, comprising a plurality of coupling (chain extension) and deprotection steps.

[0029] 12. The process of any one of clauses 1-11, wherein one or more steps are performed under continuous flow conditions.

[0030] 13. The process of any one of clauses 1-12, wherein MeCN is used as a solvent.

[0031] 14. The process of any one of clauses 1-13, wherein the oligonucleotide or salt thereof is a 7-mer or longer oligonucleotide, e.g. an 8-mer to 30-mer, such as a 16 -mer oligonucleotide.

[0032] 15. The process of any one of clauses 1-14 wherein the oligonucleotide or salt thereof comprises one or more phosphoro thioate groups.

[0033] 16. The process of any one of clauses 1-15, wherein the oligonucleotide or salt thereof comprises DNA, RNA, or a derivative thereof.

[0034] 17. The process of any one of clauses 1-16, wherein (i) the oligonucleotide product obtained by the process does not comprise more than 5% by dry weight of phosphomannose isomerase inhibitors, for example trityl cation, monomethoxy trityl cation, dimethoxy trityl cation, and / or derivatives thereof; and / or (ii) the total oligonucleotides produced by the process do not comprise more than 5 mol% of oligonucleotides in which one or more nucleotides are depurinated. 201393-PCT01-NP P78290WO

[0035] 18. The process of any one of clauses 1-17, wherein the oligonucleotide or salt thereof represents at least 50% of the total oligonucleotides produced by the process.

[0036] 19. Use of NPPOC as a protecting group in liquid phase oligonucleotide synthesis.

[0037] 20. An NPPOC-protected oligonucleotide or salt thereof, wherein the oligonucleotide or salt thereof is in solution.

[0038] 21. The NPPOC-protected oligonucleotide or salt thereof of clause 20, wherein the oligonucleotide or salt thereof is as defined in any one of clauses 14-19.

[0039] 22. A solution comprising an NPPOC-protected oligonucleotide or salt thereof.

[0040] BRIEF DESCRIPTION OF THE FIGURES

[0041] FIG. 1 shows, for illustrative purposes, a conventional LPOS process for synthesising oligonucleotides which includes a cycle of coupling, sulfurisation, and deprotection steps . DMT is shown as the protecting group, and a step of diafiltration is included after the detritylation step. The “Carrier” in Fig. 1 denotes a molecular support for the (oligo)nucleotide in solution.

[0042] FIG. 2 shows a LPOS process of the disclosure in which NPPOC is used as a protecting group. The process includes a cycle of coupling, oxidation / sulfurisation, and deprotection steps. No additional purification step is included after deprotection. The “Carrier” in Fig. 2 denotes a molecular support for the (oligo )nucleotide in solution, e.g. a star macromolecule which can be loaded with a plurality of nucleosides in step (B).

[0043] FIG. 3a shows a schematic view of an exemplary synthesizer which may be used for the LPOS processes disclosed herein. The reaction vessel includes an output / input loop for a flow photochemistry coil, in which the NPPOC deprotection step can take place.

[0044] FIG. 3b shows a schematic view of an exemplary synthesizer which may be used for the LPOS processes disclosed herein. The reaction vessel includes a photochemical flow reactor, in which the NPPOC deprotection step can take place.

[0045] FIG. 4 shows a LPOS process of the disclosure in which NPPOC is used as a protecting group. In contrast to FIG. 2, step (A) depicts DMT protection rather than NPPOC protection. The process includes a cycle of coupling, oxidation / sulfurisation, and deprotection steps. No additional purification step is included after deprotection. It will be appreciated that a 201393-PCT01-NP P78290WO purification step maybe included immediately before the first round of the chain extension cycle, i.e. after DMT-deprotection. The “Carrier” in Fig. 4 denotes a molecular support for the (oligo )nucleotide in solution, e.g. a star macromolecule which can be loaded with a plurality of nucleosides in step (B).

[0046] DETAILED DESCRIPTION

[0047] Although specific embodiments of the present disclosure will now be described with reference to the description and examples, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments which can represent applications of the principles of the present disclosure. Various changes and modifications will be obvious to those of skill in the art given the benefit of the present disclosure and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

[0048] Definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understoodby one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0050] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of chemical synthesis, tissue culture, immunology, molecular biology, microbiology, cell biology, recombinant DNA, etc., which are within the skill of the art. See, e.g., Michael R. Green and Joseph Sambrook, Molecular Cloning (4thed., Cold Spring Harbor Laboratory Press 2012); the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson etal. (1995) PCR 2: A Practical Approach; Harlow and Laneeds. (1999) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press). 201393-PCT01-NP P78290WO

[0051] All numerical designations, e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges, are approximations which are varied ( + ) or ( - ) by increments of, e.g., 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”, which is used to denote a conventional level of variability. For example, a numerical designation which is “about” a given value may vary by ± 10% of said value; alternatively, the variation may be ± 5%, ± 2%, or ± 1% of the value. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0052] As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0053] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, without excluding other elements. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure. Use of the term “comprising” herein is intended to encompass, and to disclose, the corresponding statements in which the term “comprising” is replaced by “consisting essentially of’ or “consisting of’.

[0054] The term “deprotection” as used herein refers to the removal of a protecting group, such as e.g. NPPOC from a 5’ primary hydroxyl group.

[0055] The term “depurination” as used herein refers to the chemical reaction of purine containing deoxyadenosine or deoxyguanosine, and adenosine or guanosine, in which the P-N-glycosidic 201393-PCT01-NP P78290WO bond is hydrolytically cleaved releasing adenine or guanine, respectively, and leading to the formation of an apurinic site.

[0056] The term “nucleic acid” as used herein includes nucleic acids selected from the group consisting of DNA, RNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA). The nucleic acid may comprise one or more modifications in its nucleobases, sugars, and / or backbone (e.g., phosphate backbone), such as nucleobase methylation, modification of the 2’ position of a ribose (e.g., fluorination, O -methylation, or O-methoxyethylation), sulfurization of the phosphate backbone, etc. The nucleic acid may be a functional nucleic acid, e.g., whereby the functional nucleic acid is selected from the group consisting of mRNA, micro - RNA, gRNA, saRNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acid (e.g., antisense oligonucleotide (ASO)), ribozymes, aptamers and spiegelmers. A “peptide nucleic acid” is a polymer which is similar to DNA or RNA in which the backbone is composed of repeating amino acid (typically A-(2-aminoethyl)-glycine) units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge and a carbonyl group. A “locked nucleic acid” is a nucleic acid in which the 2’ hydroxyl is connected, e.g., by a methylene bridge, to the 4’ carbon of the same ribose sugar.

[0057] The term "abasic" as used herein in connection with nucleotides, refers to moieties lacking a base or having other chemical groups in place of a base at the T position, for example a 3',3' - linked or 5',5'-linked deoxyabasic ribose derivative.

[0058] The term “alternating” as used herein in connection with nucleic acids means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on.

[0059] As used herein, the term “alkyl” means a saturated linear or branched free radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms. The term “alkylene” has the corresponding meaning in connection with the divalent free radical. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, etc. Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. The terms “(Ci-C3)alkyl”, “(Ci-C6)alkyl”, etc., have equivalent meanings, i.e., a saturated linear or branched free radical consisting essentially of 1 to 3 (or 1 to 6) carbon atoms and a corresponding number of hydrogen atoms. The definition of “alkyl” 201393-PCT01-NP P78290WO also applies in the context of other groups which comprise alkyl groups, such as “-0(Cr C3)alkyl”. The term “haloalky 1” means an alkyl group which is substituted by one or more halogens. Exemplary haloalkyl groups include trifluoromethyl, trifluoroethyl, difluoroethyl, pentafluoroethyl, chloromethyl, etc. One or more carbon atoms in the backbone of the alkyl group may be substituted by (or bonded to) a heteroatom by a multiple bond (e.g., a double bond); for example, a carbon atom of the alkyl group may be bonded to oxygen via a double bond (i.e., substituted by oxo to provide a carbonyl function). The presence of such a substituent does not prevent the carbon backbone of the free radical being considered as an alkyl group. In embodiments, the alkyl group is linear. Alkyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0060] As used herein, the term “alkoxy” means a group -O-alkyl, i.e. which is bonded via its oxygen atom to the rest of the molecule. The alkyl portion of the alkoxy is as defined herein, i.e. it may be branched or linear and it may be substituted by one or more optional substituents. Exemplary alkoxy groups include methoxy (-OMe) and ethoxy (-OEt).

[0061] As used herein, the term “alkenyl” means an unsaturated linear or branched free radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms, which free radical comprises at least one carbon-carbon double bond. The term “alkenylene” has the corresponding meaning in connection with the divalent free radical. Exemplary alkenyl groups include ethenyl, prop-l-enyl, prop-2-enyl, isopropenyl, but-l-enyl, 2-methyl-prop-l- enyl, and 2-methyl-prop-2-enyl. The terms “(C2-C6)alkenyl”, etc., have equivalent meanings, i.e., an unsaturated linear or branched free radical consisting essentially of 2 to 6 carbon atoms and a corresponding number of hydrogen atoms. Alkenyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0062] As used herein, the term “alkynyl” means an unsaturated linear or branched free radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms, which free radical comprises at least one carbon-carbon triple bond. The term “alkynylene” has the corresponding meaning in connection with the divalent free radical. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, etc.. The terms “(C2-C6)alkynyl”, etc., have equivalent meanings, i.e., an unsaturated linear or branched free radical consisting essentially of 2 to 6 carbon atoms and a corresponding number of hydrogen atoms. Alkynyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein. 201393-PCT01-NP P78290WO

[0063] As used herein, the term “acyl” means a group -C(O)R*, i.e. which is bonded to the rest of the molecule via the carbon atom of the carbonyl group. The group R* can be a non-cyclic (e.g., a branched or linear aliphatic group) or cyclic group which may be saturated or unsaturated (e.g. aromatic) and may contain one or more heteroatoms. Examples of acyl groups include ethanoyl (R*=Me), propanoyl (R*=Me), and benzoyl (R*=phenyl). Acyl groups may be substituted with one or more optional substituents as defined herein, typically on the R* portion.

[0064] As used herein, the term “cycloalkyl” means a saturated free radical having at least 3 to 9 carbon atoms (i.e., ring atoms) that form a ring. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. It will be appreciated that the cycloalkyl group may be monocyclic or multicyclic (e.g., fused, bridged, or spirocyclic). In the case of multicyclic cycloalkyl groups, there are further rings, e.g. 1 or more further rings, all of which contain from 3 to 7 carbon atoms (i.e., ring atoms). Exemplary cycloalkyl groups having such further rings include bicyclo [l .l .l]pentanyl. One or more ring atoms of the cycloalkyl group may be substitutedby (i.e., bonded to) a heteroatom by a double bond (e.g., cycloalkyl substituted by oxo). The presence of such a substituent does not prevent the carbon backbone of the free radical being considered as a cycloalkyl group. Cycloalkyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0065] As used herein, the term “aryl” means an aromatic free radical having at least 6 carbon atoms (i.e., ring atoms) that form a ring. It will be appreciated that the aryl group may be monocyclic or multicyclic (e.g., fused). In the case of multicyclic aryl groups, there are further rings, e.g. 1 or more further rings, all of which contain at least 3 carbon atoms (i.e., ring atoms). Examples of aryl groups include phenyl and naphthalenyl. The aryl group may contain from 6 to 10 carbon atoms in the ring portion of the group, which may be monocyclic or multicyclic (e.g., fused). In embodiments, aryl is phenyl. Aryl groups of the present disclosure may be substituted with one or more optional substituents as defined herein. The terms “alkaryl” and “aralkyl” will be understood to mean an aryl group bonded to an alkyl group (as defined here) which is connected to the rest of the molecule either via the aryl portion or the alkyl portion, respectively.

[0066] The term “heterocycle” as used herein to refer to a free radical having at least 3 to 10 atoms (i.e., ring atoms) that form a ring, wherein at least 1 to 9 of said ring atoms are carbon and the 201393-PCT01-NP P78290WO remaining at least 1 to 9 ring atom(s) (i.e., hetero ring atom(s)) are selected independently from the group consisting of nitrogen, sulphur, and oxygen. A heterocyclic group may be fully saturated, in which case it may be referred to as a “heterocycloalkyl” group. A heterocyclic group may alternatively be fully unsaturated, in which case it may be referred to as a “heteroaryl” group. Partially saturated heterocyclic group also fall within the meaning of the term “heterocycle” as used herein. A heterocycle may be monocyclic or multicyclic (e.g., fused, bridged, or spirocyclic). In the case of multicyclic heterocycles, there are further rings, e.g. 1 or more further rings, all of which contain from 3 to 7 carbon atoms (i.e., ring atoms). The further rings may also contain heteroatoms, e.g. selected from nitrogen, oxygen and sulphur. One or more ring atoms of the heterocyclic group maybe substituted by (i.e., bonded to) an optional substituent as defined herein. One or more ring atoms of the heterocyclic group may also be substituted by (i.e., bonded to) a heteroatom by a double bond (e.g., substituted by oxo).

[0067] As used herein, the term “heterocycloalkyl” means a saturated free radical having at least 3 to 10 atoms (i.e., ring atoms) that form a ring, wherein at least 1 to 9 of said ring atoms are carbon and the remaining at least 1 to 9 ring atom(s) (i.e., hetero ring atom(s)) are selected independently from the group consisting of nitrogen, sulphur, and oxygen. Heterocycloalkyl rings may have oxo substituents, typically adjacent to a hetero atom (e.g., 2-oxopyrrolidinyl), but the oxygen atom does not form part of the ring and is excluded from the number of ring atoms. The presence of such a substituent does not prevent the ring (or rings) of the free radical being considered as a heterocycloalkyl group. Exemplary heterocycloalkyl groups include tetrahydro furanyl, piperidinyl, morpholinyl and piperazinyl. In the case of multicyclic heterocycloalkyl groups, there are further rings, e.g. 1 or more further rings, all of which contain from 3 to 7 ring atoms selected from carbon, nitrogen, sulphur, and oxygen. The further rings may be saturated, or partially or fully unsaturated (e.g., having aromatic character). Multicyclic heterocycloalkyl groups include fused, bridged and spirocyclic ring systems. Where a multicyclic heterocycloalkyl group contains an unsaturated fused ring, the group is typically not bonded to the rest of the molecule via that fused ring. Heterocycloalkyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0068] As used herein, the term “heteroaryl” means an aromatic (i.e., having aromatic character) free radical typically containing from 6 to 10 ring atoms, wherein 1 to 9 of said ring atoms are carbon and the remaining 1 to 9 ring atom(s) (i.e., hetero ring atom(s)) are selected 201393-PCT01-NP P78290WO independently from the group consisting of nitrogen, sulphur, and oxygen. It will be appreciated that the heteroaryl group may be monocyclic or multicyclic (e.g., fused). In the case of multicyclic heteroaryl groups, there are further rings, e.g. 1 or more further rings, all of which contain at least 3 atoms (i.e., ring atoms), which further rings may optionally be aromatic. Examples of heteroaryl groups include monocyclic groups such as pyridyl, and 2- oxopyridonyl, as well as multicyclic groups such as benzimidazolyl. Where a heteroaryl group is described as being “X- to Y-membered”, this means that the heteroaryl group contains a total number of ring atoms from X to Y. Thus, for example, a “5 - to 10-membered heteroaryl group” contains a total of 5, 6, 7, 8, 9 or 10 ring atoms, for example benzimidazolyl (9 ring atoms). A heteroaryl group may also be referred to as an “aromatic heterocycle”.

[0069] The term "phosphate" is typically used herein to denote a radical (or diradical) group which comprises a central phosphorus atombondedto four oxygen atoms (one via a double bond). The oxygen atoms which are bonded to phosphorus via single bonds may represent points of attachment to the rest of the molecule, or may carry hydrogen atoms, or may be considered to carry a negative charge (e.g., in the case of salts). The term “thiophosphate” is typically used herein to denote a phosphate analogue in which at least one oxygen atom is replaced by sulphur; such groups are also called “phosphorothioates” herein.

[0070] An “optional substituent” is a group which is covalently attached to a moiety (generally via a carbon atom of the moiety, and typically in place of a hydrogen atom on said carbon atom). The optional substituent may be chosen to be a group which does not significantly alter the steric and / or electronic properties of the molecule. In embodiments, each optional substituent is independently selected from the group consisting of: halogen (e.g. , -F, -Cl, -Br or -I); -OH; -SH; -NH2; -NHMe; -NMe2; -(Ci-C3)alkyl (e.g., -Me or -Et); and 3- or 4-membered cycloalkyl or heterocyclo alkyl group (e.g., cyclopropyl or epoxide), which may optionally be substituted with one or more halo. A group defined as “optionally substituted” may be either unsubstituted, or substituted with one or more substituents, e.g. 1, 2, 3, 4, 5, 6, or more substituents. In embodiments, a substituted group has 1 to 4 substituents, e.g. 1, 2, or 3 substituents. In embodiments, a substituted group has 1 or 2 substituents. In embodiments, a substituted group has 3 substituents.

[0071] As used herein, the terms “halo” and “halogen” mean fluorine, chlorine, bromine, or iodine. These terms are used interchangeably and may refer to a halogen free radical group or to a 201393-PCT01-NP P78290WO halogen atom as such. Those of skill in the art will readily be able to ascertain the identification of which in view of the context in which this term is used in the present disclosure. In embodiments, the halogen is fluorine.

[0072] The term “star” is used herein to denote a macromolecule containing a single branch point from which linear chains (i.e., arms) emanate. A star macromolecule with n linear chains attached to the branch point is termed an n-star macromolecule, e.g. 4-star macromolecule contains four arms. The arms may be, for example, PEG or a PEG derivative as described herein. A “regular” star macromolecule contains arms which are essentially identical with respect to constitution and degree of polymerization, whereas a “variegated” star macromolecule contains arms which are not essentially identical, e.g. which are composed of different monomeric units. Star macromolecules in accordance with the present disclosure may be regular star macromolecules or variegated star macromolecules.

[0073] The term “UV purity” is used herein to express the purity of a product of the processes described herein, as determined by liquid chromatography UV (LC-UV) methods. The UV purity may be calculated by integrating the area under the main chromatographic peak detected at 260 nm by LC-UV, e.g. when the LC is performed by ion-pair HPLC.

[0074] The term “full length product (FLP) purity” is used herein to express the purity of a product of the processes described herein. The full length product purity may be calculated by identifying the species present (desired product and impurities) under the main chromatographic HPLC peak, followed by quantification through integration of the extracted ion chromatograms (EIC) of the identified species, and expressing it as a calibration - corrected percentage of the total oligonucleotide ion signal within the main chromatographic (HPLC) peak.

[0075] In some embodiments, the processes of the present disclosure are described, inter alia, by way of structural formulae. It will be appreciated that these formulae typically show only one form (e.g., resonance form, tautomeric form, etc.) of the compound, whereas certain compounds may exist in more than one such form. This will be readily apparent to the skilled reader. The present disclosure includes all possible tautomers of the compounds characterised by the structural formulae herein, including as single tautomers, or as any mixture of tautomers in any ratio. It will also be appreciated that certain of the present compounds may exist in one or more isomeric (e.g., stereoisomeric) forms. The present disclosure includes all possible stereoisomers, enantiomers, diastereomers, etc. of the compounds described 201393-PCT01-NP P78290WO hereinbefore and below, as well as cis- and trans- forms and conformers of the same. The purification and the separation of isomers may be accomplished by methods described hereinafter, as well as by techniques known in the art. For example, optical isomers of the compounds can be obtained by resolution of the racemic mixture of diastereoisomeric salts thereof (e.g., using an optically active acid or base, or by the formation of covalent diastereomers). A different process for separation of optical isomers involves the use of chiral chromatography (e.g., HPLC columns using a chiral phase), with or without conventional derivatization. Enzymatic separation, with or without deriv arization, may also be useful, and optically active compounds of the present disclosure can likewise be obtained by chiral syntheses utilizing optically active starting materials. The present disclosure includes all possible stereoisomers of the compounds described herein as single stereoisomers, or as any mixture of said stereoisomers, e.g. (R)- or (S)- isomers, in any ratio.

[0076] The compounds of the disclosure may exist in the form of free acids or bases, or may exist as addition salts with suitable acids orbases. Methods for forming salts are described below and are also known in the art (see, e.g., Berge et al., J Pharm Sci. (1977) 66:1-19). As used herein, the term “pharmaceutically acceptable” when used in connection with salts means a salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction(s) with any other component of a pharmaceutical composition in which it may be contained.

[0077] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0078] The following abbreviations and empirical formulae are used herein:

[0079] ADP impurity in which guanine replaced by N2-acetyl-2,6-diaminopurine

[0080] ANA altritol nucleic acid

[0081] ASO antisense oligonucleotide

[0082] BIT benzimidazolium triflate

[0083] BNA bridged nucleic acid

[0084] BOP (benzotriazol- l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate 201393-PCT01-NP P78290WO

[0085] BOP-CI bromo trip yrrolidinophosphonium hexafluorophosphate; bis(2-oxo-3- oxazolidinyl)phosphinic chloride

[0086] BTT 5-benzylthio-lH-tetrazole

[0087] Cbz benzyloxycarbonyl

[0088] CDI carbonyldiimidazole

[0089] CFL compact fluorescent lamp

[0090] CNET N3-(2-cyanoethyl)thymine

[0091] CRN conformationally restricted nucleic acid

[0092] DABCO l,4-diazabicyclo[2.2.2]octane

[0093] DBU l,8-diazabicyclo[5.4.0]undec-7-ene

[0094] DCC dicyclohexylcarbodiimide

[0095] DCE dichloroethane

[0096] DCM dichloromethane

[0097] DO 4,5-dicyanoimidazole

[0098] DDT 1 -dodecanethiol

[0099] DEPBT 3-(diethylphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one

[0100] DIC diisopropylcarbodiimide

[0101] DIPEA diisopropylethylamine

[0102] DMF dimethylformamide

[0103] DMSO dimethyl sulfoxide

[0104] DMT 4,4'-dimethoxytrityl

[0105] DNA deoxyribonucleoic acid

[0106] DPS 2,2-dipyridyl disulfide

[0107] DV diavolume(s)

[0108] EDC ethyl-(N’,N’-dimethylamino)propylcarbodiimide hydrochloride

[0109] ETT 5-ethylthio-lH-tetrazole

[0110] GNA glycol nucleic acid rRNA guide RNA

[0111] HATU O-(7 -azabenzotriazol- 1 -yl)-N,N,N’,N’ -tetramethyluronium hexafluorophosphate

[0112] HBTU O-(benzo triazol- 1 -yl)-N,N,N’ ,N’ -tetramethyluronium hexafluorophosphate

[0113] HCTU O-(6-chlorobenzotriazol- 1 -yl)-N,N,N’ ,N’ -tetramethyluronium hexafluorophosphate 201393-PCT01-NP P78290WO

[0114] HNA hexitol nucleic acid

[0115] HPLC high-performance liquid chromatography

[0116] IDP impurity in which guanine replaced by N2-isobutyryl-2,6-diaminopurine

[0117] LCMS liquid chromatography / mass spectrometry

[0118] LED light emitting diode

[0119] LNA locked nucleic acid

[0120] LPOS liquid phase oligonucleotide synthesis

[0121] MMT monomethoxytrityl

[0122] MNA mannitol nucleic acid

[0123] MOE 2 ’-methoxyethoxy substituted ribose mRNA messenger RNA

[0124] NBS N-bromosuccinimide

[0125] NCS N-chlorosuccinimide

[0126] NIS N-iodosuccinimide

[0127] NMA 2'-O-[2-(methylamino)-2-oxoethyl] substituted ribose

[0128] NMI 1 -methylimidazole

[0129] NPPOC 2-(2-nitrophenyl)propyloxycarbonyl

[0130] PBI polybenzimidazole

[0131] PCR polymerase chain reaction

[0132] PEG polyethylene glycol

[0133] PNA peptide nucleic acid

[0134] POS 3-phenyl l,2,4-dithiazoline-5-one

[0135] PyAOP (7 -azabenzo triazol- 1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0136] PyBOP (benzotriazol- l-yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0137] RNA ribonucleic acid

[0138] RRFs relative response factors saRNA small activating RNA shRNA short hairpin RNA siNA / siRNA small interfering (ribo)nucleic acid

[0139] SMI saccharin 1 -methylimidazole

[0140] T3P propylphosphonic anhydride

[0141] TATU O-(7 -azabenzotriazol- 1 -yl)-N,N,N’,N’ -tetramethyluronium tetrafluoroborate 201393-PCT01-NP P78290WO

[0142] TBTU 2-(l H-benzotriazole- 1-yl)- 1, 1 ,3 ,3-tetramethylaminium tetrafluoroborate

[0143] TEA triethylamine

[0144] TFA trifluoroacetic acid

[0145] THF tetrahydrofuran

[0146] THP tetrahydropyran

[0147] TNTU O-(5-norbornene-2,3-dicarboximido)-N,N,N’,N’-tetramethyluronium tetrafluoroborate

[0148] TOTU O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetra methyluronium tetrafluoroborate

[0149] TPTU O-(l,2-dihydro-2-oxo-l-pyridyl-N,N,N’,N’-tetramethyluronium tetrafluoroborate

[0150] TSTU O-(N-succinimidyl)- 1,1,3 ,3-tetramethyl-uronium tetrafluoroborate

[0151] UNA unlocked nucleic acid

[0152] UPEC ultra-performance liquid chromatography

[0153] UV ultraviolet

[0154] Processes for synthesising oligonucleotides

[0155] The present disclosure provides EPOS processes that use 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) as a protecting group. The processes of the disclosure can avoid, in part or completely, the use of conventional LPOS protecting groups such as DMT. The problems associated with the such protecting groups, e.g. the need for additional purification steps and / or difficulties associated with scale up (as described above), may therefore be reduced or avoided.

[0156] Accordingly, in a first aspect the present disclosure provides a process for synthesizing an oligonucleotide or a salt thereof in liquid phase, wherein 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) is used as a protecting group. This process may be described as a liquid -phase oligonucleotide synthesis (LPOS) process. The process will typically not involve the use of any reagents on solid support (e.g., on particles such as polymer beads or magnetic beads, or attached to surfaces such as metal or silica). In embodiments, the process does not involve the use of any solid support, e.g. any solid support attached to any of the growing oligonucleotide chains.

[0157] In embodiments of the present disclosure, NPPOC is used as a 5 ’ -hydroxyl protecting group. 201393-PCT01-NP P78290WO

[0158] Core steps of the chain extension cycle

[0159] As illustrated herein in more detail, NPPOC may be used to protect hydroxyl groups of the growing oligonucleotide and / or of the nucleosides to be added thereto. Thus, NPPOC may be used during chain extension in a LPOS process, i.e. where a nucleoside (such as an activated nucleoside) is added to the growing oligonucleotide chain. At its heart, the LPOS process is typically a cycle which comprises two core steps, namely: (1) coupling of a protected nucleoside to the growing oligonucleotide chain, e.g. to the free 5 ’-hydroxyl group of the growing oligonucleotide chain; and (2) deprotection of the newly added, terminal nucleotide to expose a further hydroxyl group which can participate in another round of the cycle (see, e.g., steps D and F of Fig. 2). An optional step of base and / or backbone modification can be included between steps (1) and (2) such as, e.g., oxidation or sulfurization of one or more phosphorus atoms in the backbone (see, e.g., step E of Fig. 2). A particular advantage of using NPPOC is that, after deprotection, it does not interfere with subsequent steps in the reaction cycle. As such, there is no need for additional purification steps to remove products of the deprotection reaction. This contrasts with conventional LPOS protecting agents such as DMT. It also permits deprotection in the same reaction medium (solution) with the use of less chemical reagents.

[0160] Thus, in embodiments, the process of the disclosure comprises:

[0161] (i) a coupling step in which a nucleobase-containing moiety (e.g., a 3 ’-hydroxylprotected nucleoside, or the growing oligonucleotide chain) is reacted with an activated nucleoside (e.g., a 3’-phosphoramidite activated nucleoside) which comprises an NPPOC-protected hydroxyl group (e.g., an NPPOC -protected 5’- hydroxyl group) to form a product having one nucleobase more than said nucleobase-containing moiety;

[0162] (ii) an optional step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the nucleobase-containing moiety with the newly-added, terminal nucleoside); and

[0163] (iii) a deprotection step in which the NPPOC group of the product is removed to reveal a functional group (e.g., a 5’-hydroxy group) which can participate in a further coupling step.

[0164] In embodiments, a purification step (e.g., diafiltration, tangential filtration, precipitation, liquid-liquid extraction, or column chromatography) is carried out after step (i) but before 201393-PCT01-NP P78290WO step (iii), e.g. to remove any unreacted activated nucleoside and / or oxidation or sulfurisation reagents, but no purification step is carried out after step (iii), e.g. before a further round of chain extension. In embodiments, the purification step is carried out between step (ii) and (iii). In embodiments, no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the subsequent round of chain extension. In other embodiments, no purification step is carried out between steps (i) and (ii) or between steps (ii) and (iii).

[0165] As disclosed herein, the use of NPPOC is particularly advantageous because, after deprotection, it does not interfere with subsequent steps in the reaction cycle. As a consequence, there is no need for additional purification steps (e.g. diafiltration) to remove products of the deprotection reaction. However, there are certain situations in which limited purification by diafiltration may advantageously be used after other steps of the reaction cycle, i.e. after steps other than the deprotection step. For example, it may be advantageous for a purification step (e.g., diafiltration, tangential filtration, precipitation, liquid-liquid extraction, or column chromatography) to be carried out after a step of backbone modification (step (ii)). Optionally, it may be advantageous for a purification step (e.g., diafiltration, tangential filtration, precipitation, liquid-liquid extraction, or column chromatography) to be carried out between step (ii) and step (iii), and no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the sub sequent round of chain extension.

[0166] Thus, in embodiments, the process of the disclosure comprises:

[0167] (i) a coupling step in which a nucleobase-containing moiety (e.g., a 3 ’-hydroxylprotected nucleoside, or the growing oligonucleotide chain) is reacted with an activated nucleoside (e.g., a 3 ’-phosphoramidite activated nucleoside) which comprises an NPPOC-protected hydroxyl group (e.g., an NPPOC -protected 5’- hydroxyl group) to form a product having one nucleobase more than said nucleobase-containing moiety;

[0168] (ii) a step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the nucleobase-containing moiety with the newly-added, terminal nucleoside); and

[0169] (iii) a deprotection step in which the NPPOC group of the product is removed to reveal a functional group (e.g., a 5’-hydroxy group) which can participate in a further coupling step. 201393-PCT01-NP P78290WO

[0170] In embodiments, a purification step (e.g., diafiltration, tangential filtration, precipitation, liquid-liquid extraction, or column chromatography) is carried out after step (i) but before step (iii), e.g. to remove any unreacted activated nucleoside and / or oxidation or sulfurisation reagents, but no purification step is carried out after step (iii), e.g. before a further round of chain extension. In embodiments, the purification step is carried out between step (ii) and (iii). In embodiments, no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the subsequent round of chain extension. In embodiments, a diafiltration step is carried out between steps (ii) and (iii). In embodiments, a diafiltration step is carried out between steps (ii) and (iii) and no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the subsequent round of chain extension.

[0171] The process may comprise more than one round of chain extension, e.g. in accordance with the above embodiment. For example, where a single nucleobase -containing moiety is used in step (i) of the first cycle, and there are n cycles, each of which adds a nucleoside to the growing chain, the oligonucleotide product will comprise n+1 bases. It will be appreciated that a process of the present disclosure does not need to use NPPOC as a protecting group in each and every round of the chain extension cycle. In embodiments, however, NPPOC is used as a protecting group in each and every round of the chain extension cycle. In embodiments, the process comprises a plurality of coupling (chain extension) and deprotection steps.

[0172] Alternatively, the process may comprise only one round of chain extension. In one example, which is accordance with the above embodiment, the nucleobase -containing moiety of step (i) may comprise a plurality of nucleobases which are extended by a single nucleobase, i.e. where the nucleobase-containing moiety comprises n nucleobases then the oligonucleotide product will comprise n+1 bases. In a further example, a first nucleobase-containing moiety which comprises a plurality of nucleobases is reacted with an activated second nucleobase- containing moiety which comprises a plurality of nucleobases to form an oligonucleotide product, wherein the activated second nucleobase-containing moiety comprises an NPPOC- protected hydroxyl group. In this further example, where the first nucleobase-containing moiety comprises n nucleobases and the activated second nucleobase-containing moiety comprises m nucleobases, the oligonucleotide product will comprise n+m bases. Thus, in embodiments the process of the disclosure comprises (e.g. consists essentially of): 201393-PCT01-NP P78290WO

[0173] (i) a coupling step in which a first nucleobase-containing moiety (e.g., a 3 ’-hydroxylprotected nucleoside, or the growing oligonucleotide chain) is reacted with an activated second nucleobase-containing moiety (e.g., which comprises a 3’- phosphoramidite activated nucleoside moiety) which comprises an NPPOC- protected hydroxyl group (e.g., an NPPOC-protected 5 ’-hydroxyl group) to form a product, wherein the product has a number of nucleobases equal to the sum of the number of nucleobases in the first and second nucleobase-containing moieties;

[0174] (ii) an optional step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the first and second nucleobase- containing moieties); and

[0175] (iii) a deprotection step in which the NPPOC group of the product is removed to reveal a functional group (e.g., a 5 ’-hydroxy group).

[0176] In embodiments the process of the disclosure comprises (e.g. consists essentially of):

[0177] (i) a coupling step in which a first nucleobase-containing moiety (e.g., a 3 ’-hydroxylprotected nucleoside, or the growing oligonucleotide chain) is reacted with an activated second nucleobase-containing moiety (e.g., which comprises a 3’- phosphoramidite activated nucleoside moiety) which comprises an NPPOC- protected hydroxyl group (e.g., an NPPOC-protected 5 ’-hydroxyl group) to form a product, wherein the product has a number of nucleobases equal to the sum of the number of nucleobases in the first and second nucleobase-containing moieties;

[0178] (ii) a step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the first and second nucleobase-containing moieties); and

[0179] (iii) a deprotection step in which the NPPOC group of the product is removed to reveal a functional group (e.g., a 5 ’-hydroxy group).

[0180] In embodiments, the process consists essentially of the above steps which are preceded by one or more pre-chain extension steps (e.g., process steps prior to the chain extension cycle as defined herein) and / or which are followed by one or more post-chain extension steps (e.g., process steps after the chain extension cycle as defined herein).

[0181] In embodiments, no purification step is carried out during any of the chain extension steps in which NPPOC is used as a protecting group. In embodiments, NPPOC is used as a protecting group in each and every round of the chain extension cycle, and no purification step is carried out during any of the chain extension steps. In embodiments, NPPOC is used as a protecting 201393-PCT01-NP P78290WO group in each and every round of the chain extension cycle, and a purification step is only carried out after a step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the first and second nucleobase-containing moieties), i.e. step (ii), above. In embodiments, the purification step carried out after step (ii) is diafiltration. In embodiments, the oligonucleotide product (e.g., the final product) of the chain extension steps is purified, e.g. by diafiltration, tangential filtration, precipitation, liquid-liquid extraction or column chromatography. In embodiments, the oligonucleotide product is purified by diafiltration.

[0182] As described in more detail below, DMT may be used as a protecting group prior to the chain extension cycle, i.e. in one or more pre -chain extension steps. In embodiments, DMT is used as a protecting group in one or more pre-chain extension steps, NPPOC is used as a protecting group in each and every round of the chain extension cycle, and a purification step is only carried out before a first round of chain extension and after a step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the first and second nucleobase-containing moieties), i.e. after step (ii), above. In embodiments, the purification step is carried out between step (ii) and (iii). In embodiments, no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the subsequent round of chain extension. In embodiments, a diafiltration step is carried out between steps (ii) and (iii). In embodiments, a diafiltration step is carried out between steps (ii) and (iii) and no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the sub sequent round of chain extension. It will be appreciated that the purification step may be carried out after oxidation or sulfurisation of the phosphite which joins other nucleobase - containing moieties, and this is not limited to the first and second nucleobase-containing moieties. In embodiments, the purification step carried out before a first round of chain extension and between step (ii) and (iii) is diafiltration. In embodiments, the oligonucleotide product (e.g., the final product) of the chain extension steps is purified, e.g. by diafiltration, tangential filtration, precipitation, liquid-liquid extraction or column chromatography. In embodiments, the oligonucleotide product is purified by diafiltration.

[0183] Coupling step

[0184] The process of the disclosure may comprise a coupling step in which the deprotected oligonucleotide precursor is reacted with a nucleoside -containing moiety under conditions resulting in chain extension (i.e., forming the oligonucleotide or an extended precursor 201393-PCT01-NP P78290WO thereto). The nucleo side-containing moiety may be an activated nucleoside, or it may be an activated oligonucleotide. In embodiments, the nucleo side-containing moiety is NPPOC- protected such that the oligonucleotide or extended precursor thereto which is produced is also NPPOC-protected.

[0185] The coupling (chain extension) step may be carried out on the deprotected oligonucleotide precursor from the preceding deprotection step without any intervening purification (e. diafiltration). This is conveniently performed where the preceding deprotection step is a step of NPPOC deprotection as described herein.

[0186] In the coupling step (e.g., step (i) of the above embodiment), the activated nucleoside may be a phosphoramidite. In embodiments, the activated nucleoside is a nucleoside having an N,N- diisopropylphosphoramidite group attached to the 3’ position of the sugar. In embodiments, the activated nucleoside is a nucleoside having a (2-cyanoethyl)-N,N- diisopropylphosphoramidite group attached to the 3’ position of the sugar.

[0187] In embodiments, an activating agent is used in the coupling step. Suitable activating agents include those identified in “Coupling activators for the oligonucleotide synthesis via phosphoramidite approach” (Tetrahedron, (2013) 69(18):3615-3637, the contents of which are incorporated herein in their entirety). In embodiments, the activating agent comprises an agent selected from 4,5-dicyanoimidazole (DO), IH-tetrazole, 5 -ethylthio -IH-tetrazole (ETT), 5-benzylthio-lH-tetrazole (BTT), 5-[3,5-bis(trifluoromethyl)phenyl]-lH-tetrazole (Activator 42), benzimidazolium triflate (BIT), and saccharin 1 -methylimidazole (SMI). In embodiments, the activating agent comprises (or is) 4,5-dicyanoimidazole (DCI). In embodiments, a tertiary amine (e.g., 1 -methylimidazole) is present along with the activating agent. In embodiments, the activating agent comprises (or is) DCI and 1 -methylimidazole. In embodiments, the activating agent is DCI. In embodiments, the activating agent is filtered before use.

[0188] In embodiments, the activating agent is mixed with the activated nucleoside in a solvent before combining with the nucleobase-containing moiety. In embodiments, the solvent is MeCN. In embodiments, the MeCN is anhydrous. In embodiments, the MeCN is dried (i.e. made to be anhydrous) using AldraSORB™ water trapping packets prior to use. In embodiments, the solvent is a mixture of solvents, e.g. a mixture of MeCN and sulfolane.

[0189] Oxidation / sulfurization step 201393-PCT01-NP P78290WO

[0190] The coupled product may be subjected to oxidation / sulfurisation conditions prior to NPPOC deprotection (e.g., step (ii) of the above embodiment). Oxidation of a phosphite moiety in this step can result in a phosphate linkage between the newly-added, terminal nucleotide and the rest of the oligonucleotide. Sulphurisation of a phosphite moiety in this step can result in a phosphorothioate (or thiophosphate) linkage between the newly-added, terminal nucleotide and the rest of the oligonucleotide.

[0191] In embodiments, oxidation is effected using an activator in the presence of an oxygen-based nucleophile such as, e.g., an N-oxide (e.g., N-morpholine oxide, or N-pyridine oxide) or tertbutyl ammonium acetate. Suitable activators are described, e.g., in US patent publication No. 2024 / 0018180 (the contents of which are incorporated herein in their entirety). In embodiments, the activator is selected from a halosuccinimide (e.g., N-iodosuccinimide (NIS), N-chloro succinimide (NCS) or N-bromosuccinimide (NBS)), and 2,2’-dipyridyl disulfide (DPS) or a derivative thereof (e.g., 2,2’-dithiobis(benzothiazole)). In other embodiments, oxidation is effected using an oxaziridine (e.g., ( 1 S)-(+)-( 10- camphorsulfonyl)oxaziridine)) in a solvent such as MeCN. In embodiments, the solvent is substantially free of water, e.g. the solvent is anhydrous acetonitrile. In embodiments, oxidation is effected using (lS)-(+)-(10-camphorsulfonyl)oxaziridine in anhydrous MeCN.

[0192] In embodiments, sulfurisation is effected using 3-phenyl l,2,4-dithiazoline-5-one (POS) in acetonitrile. In other embodiments, sulfurisation is effected using xanthane hydride in pyridine, or using phenylacetyl disulfide in pyridine.

[0193] In embodiments, one or more occurrences of step (ii) in the process comprises a step independently selected from an oxidation step and a sulfurisation step. In embodiments, each occurrence of step (ii) in the process comprises a step independently selected from an oxidation step and a sulfurisation step.

[0194] In embodiments, the oligonucleotide or salt thereof which is produced by the chain extension cycle comprises one or more phosphate groups and / or one or more phosphorothioate groups. In embodiments, the oligonucleotide or salt thereof comprises one or more phosphorothioate groups. In embodiments, the oligonucleotide or salt thereof comprises one or more phosphorothioate groups and does not comprise any phosphate groups. In other embodiments, the oligonucleotide or salt thereof comprises one or more phosphate groups. In embodiments, the oligonucleotide or salt thereof comprises one or more phosphate groups and does not comprise any phosphorothioate groups. 201393-PCT01-NP P78290WO

[0195] Diafiltration

[0196] As described herein, an optional purification step may be carried out after a step of base and / or backbone modification (e.g., oxidation or sulfurisation of the phosphite which joins the first and second nucleobase-containing moieties), i.e. step (ii), above. In embodiments, the purification step is diafiltration. In embodiments, a diafiltration step is carried out between steps (ii) and (iii). In embodiments, a diafiltration step is carried out between steps

[0197] (ii) and (iii) and no purification step is carried out between steps (i) and (ii) or between step

[0198] (iii) and step (i) of the subsequent round of chain extension. As discussed above, a purification step (e.g. diafiltration) may be used prior to the chain extension cycle - this is particularly beneficial when DMT is used as a protecting group prior to the chain extension cycle, i.e. in one or more pre-chain extension steps. In embodiments, a diafiltration step is carried out before a first round of chain extension and between steps (ii) and (iii). In embodiments, a diafiltration step is carried out before a first round of chain extension and between steps (ii) and (iii) and no purification step is carried out between steps (i) and (ii) or between step (iii) and step (i) of the subsequent round of chain extension.

[0199] It will be appreciated that the diafiltration step may be carried out after oxidation or sulfurisation of the phosphite which joins other nucleobase-containing moieties, and this is not limited to the first and second nucleobase-containing moieties. In embodiments, a diafiltration step is used following sulfurisation of the phosphite which joins nucleobase- containing moieties. In embodiments, diafiltration is used after each sulfurisation step.

[0200] In embodiments, the diafiltration comprises the use of polybenzimidazole (PBI) membranes. In embodiments, the PBI is present in the membranes at a concentration of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt %, about 17 wt%, about 18 wt %, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, about 25 wt%, about 26 wt%, about 27 wt%, about 28 wt%, about 29 wt%, or about 30 wt%, e.g. about 17 wt%. In embodiments, MeCN is used as the diafiltration solvent. In embodiments, the diafiltration is performed at a pressure of between about 1 and about 40 bar, between about 5 and about 35 bar, between about 10 and about 30 bar, between about 15 and about 25 bar, e.g. between about 19 and about 21 bar. In embodiments, the diafiltration is performed at a temperature of between about 20 and about 28 °C. 201393-PCT01-NP P78290WO

[0201] In embodiments, the diafiltration comprises the use of polybenzimidazole (PBI) membranes wherein the PBI is present in the membranes at a concentration of about 17%, MeCN is used as the diafiltration solvent, the pressure is between about 19 and about 21 bar, and the temperature is between about 20 and about 28 °C.

[0202] Capping

[0203] An optional step of capping may be included in the process, e.g. after the sulfurisation / oxidation step of one or more rounds in the cycle, and before the subsequent deprotection step. This may be performed to control sequence-dependent impurities that can form during LPOS. In embodiments, capping is effected by reaction with a solution comprising a combination of 1 -methylimidazole, pyridine and acetic anhydride in a solvent such as MeCN. In embodiments, an acetonitrile solution of 1 -methylimidazole and pyridine is mixed with an acetonitrile solution of acetic anhydride in-line before being contacted with the growing oligonucleotide. In embodiments, lutidine is used in place of pyridine.

[0204] Deprotection step

[0205] The process of the disclosure employs NPPOC as a protecting group. NPPOC can conveniently be removed using irradiation. Thus, the process may comprise one or more deprotection steps in which an NPPOC-containing compound is irradiated. The NPPOC- containing compound may be an oligonucleotide or salt thereof which acts as a precursor to the oligonucleotide that is produced by the process of the disclosure. Alternatively, the NPPOC-containing compound maybe the oligonucleotide or salt thereof that is produced by the process of the disclosure. The deprotection step may be a step (iii) as set out in the above embodiment. In embodiments, the coupling step employs an activated nucleoside which comprises an NPPOC-protected hydroxyl group (e.g., an NPPOC -protected 5 ’-hydroxyl group). In such embodiments, the product of that coupling step is a growing oligonucleotide or a final oligonucleotide which also comprises an NPPOC-protected hydroxyl group (e.g., an NPPOC-protected 5 ’-hydroxyl group). Said product may be deprotected by irradiation as described herein. The amount of deprotected product and / or unreacted (i.e., NPPOC- protected) starting material after the deprotection step may be calculated, for example, using one or more internal standards (e.g., by HPLC), or by isolation using methods known to those of ordinary skill in the art. 201393-PCT01-NP P78290WO

[0206] In embodiments, one of the 3’ and 5 ’-protecting groups of the oligonucleotide is NPPOC and the other is a different protecting group which may be selectively deprotected. In embodiments, the 3 ’ and 5 ’-protecting groups have orthogonal deprotection conditions. In embodiments, the conditions used to deprotect the 5’ hydroxyl group do not deprotect the 3’ hydroxyl group.

[0207] In embodiments, the NPPOC deprotection step (or each NPPOC deprotection step) affords at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or at least about 99.9%, e.g. about 100% yield of the unprotected product (e.g., the unprotected primary hydroxyl group -containing product). In embodiments, the yield is an isolated yield. In other embodiments, the yield is calculated using an internal standard.

[0208] In embodiments, the NPPOC deprotection step (or each NPPOC deprotection step) affords less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about l%, less than about 0.5%, or less than about 0.1%, e.g. about 0%, of the NPPOC-protected starting material.

[0209] Thus, in embodiments the NPPOC deprotection step (or each NPPOC deprotection step) affords at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or at least about 99.9%, e.g. about 100% conversion.

[0210] In embodiments, the process comprises between 1 and 50 reaction cycles, e.g. between 2 and 40 or between 3 and 30 reaction cycles, each of which comprises a deprotection step. In embodiments, the process comprises between 1 and 30 reaction cycles, each of which comprises a deprotection step, wherein one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twenty three, twenty four, twenty five, twenty six, twenty seven, twenty eight, twenty nine, or thirty of the said deprotection steps comprise irradiating an NPPOC-protected oligonucleotide or salt thereof in solution. In embodiments, all of the said deprotection steps comprise irradiating an NPPOC-protected oligonucleotide or salt thereof in solution. 201393-PCT01-NP P78290WO

[0211] Irradiation

[0212] The process of the disclosure employs NPPOC as a protecting group. Thus, the process typically comprises one or more deprotection steps wherein the NPPOC protecting group is removed (e.g., step (iii) in the above embodiment). As explained above, NPPOC is a photolabile group which is conveniently removed using light in the UV to visible range.

[0213] Thus, in embodiments the process of the disclosure comprises a deprotection step, wherein an NPPOC-protected oligonucleotide or an NPPOC-protected precursor thereto (e.g., an NPPOC-protected growing oligonucleotide) is irradiated in solution to deprotect the oligonucleotide or precursor thereto (either in part or in full). In embodiments, every deprotection step which is performed on an NPPOC -containing oligonucleotide (or a precursor thereto) comprises irradiating the NPPOC-protected oligonucleotide (or the precursor thereto) in solution. The irradiation is typically carried out under conditions whereby a significant proportion of the NPPOC protecting groups are removed from the oligonucleotide. In embodiments, the irradiation results in at least about 90% deprotection, e.g. at least about 95%, at least about 99%, or about 100% deprotection. In embodiments, the deprotection step proceeds with a quantitative yield.

[0214] The scheme below illustrates the process of NPPOC-deprotection, showing an NPPOC- protected 5 ’-hydroxyl group (labelled “a”) as an example:

[0215] The NPPOC group is susceptible to selective photo -induced cleavage of the bond labelled “b” and may be cleaved by irradiation using the methods and conditions described herein (see also, e.g., Hasan et al., Tetrahedron (1997) 53(12):4247-4264). Cleavage of the bond labelled “b” results in liberation of the 5 ’-hydroxyl group (labelled “a”).

[0216] In embodiments, the irradiation is performed with light having a wavelength (e.g., having a wavelength of maximum intensity, Xm;L) of about 300 nm to about 420 nm. In embodiments, the irradiation is performed with light having a wavelength (e.g., a Xm;LX) of about 325 nm to about 410 nm, about 350 nm to about 400 nm, or about 365 nm to about 390 nm. In 201393-PCT01-NP P78290WO embodiments, the irradiation is performed with light having a wavelength (e.g., a km;«) of about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, or about 400 nm. In embodiments, the irradiation is performed with light having a wavelength of about 350 nm to about 400 nm, e.g. with light having a Xmaxof about 350 nm to about 400 nm. In embodiments, the irradiation is performed at a wavelength of about 360 nm to about 370 nm, e.g. with light having a Xmaxof about 365 nm. In embodiments, the irradiation is performed at a wavelength of about 380 nm to about 390 nm, e.g. with light having am;LXof about 380 nm. In embodiments, the irradiation is performed at a wavelength of about 380 nm to about 390 nm, e.g. with light having a Xmaxof about 385 nm.

[0217] In embodiments, the irradiation source is selected from an LED, a mercury lamp, a halogen bulb, an incandescent bulb, a CFLbulb, or the like. In embodiments, a filter is used to select a wavelength or a range of wavelengths of interest (e.g., as described above). In embodiments, the irradiation source is a LED, for example a blue LED such as the EvoluChem™ 365PF or 380PF LED. In embodiments, the irradiation source is a LED with light having a kmaxof about 385 nm.

[0218] In embodiments, the irradiation source is incorporated into a photoreactor. Accordingly, in embodiments, the irradiation is performed using a photoreactor, for example a Lucent360™ advanced photoreactor. It will be appreciated that the photoreactor may be configured to perform the irradiation at any of the wavelengths mentioned above. In embodiments, the irradiation is performed using an Iris Lab photoreactor e.g., with of about 385 nm.

[0219] In embodiments, the irradiation is a single period of continuous irradiation. In embodiments, the irradiation is more than one period of continuous irradiation. In embodiments, the irradiation is at a constant power. In embodiments, the irradiation power is increased over time to a maximum power. In embodiments, the irradiation power is increased over time to a maximum power, maintained at the maximumpower, and then terminated. In embodiments, the irradiation power is increased over time to a maximum power, maintained at the maximum power, and then decreased over time to zero. In embodiments, the irradiation comprises a pre-irradiation period. In embodiments, the pre -irradiation is performed with a lower power than the power used for the irradiation period.

[0220] As a person of ordinary skill in the art will appreciate, each cycle within the process is typically responsible for adding one nucleotide to the growing oligomer, and a key step in 201393-PCT01-NP P78290WO each cycle typically requires deprotection of a hydroxyl group (e.g., a 5 ’-hydroxyl group). As stated above, the hydroxyl group does not need to be NPPOC -protected in each cycle (it could be protected with a different protecting group in some cases), but the deprotection step should be rapid otherwise the time for the overall reaction can be significantly extended. The NPPOC deprotection step of the present process is rapid, which provides advantages in terms of faster process speeds, energy efficiency, reduced formation of impurities, and reduced costs.

[0221] In embodiments, the deprotection step (e.g., each deprotection step in which an NPPOC- protected compound is deprotected) involves irradiating the NPPOC-protected oligonucleotide or salt thereof for a period of time of up to about 3 hours, e.g. up to about 2 hours, up to about 1 hour, or up to about 30 minutes. Longer irradiation times are typically required when working at large scale, as compared to working on a laboratory scale. In embodiments, the irradiation time of the deprotection step (or of each deprotection step) is up to about 180 minutes, up to about 150 minutes, up to about 120 minutes, up to about 90 minutes, up to about 45 minutes, or up to about 30 minutes. In embodiments, the irradiation time of the deprotection step (or of each deprotection step) is about 180 minutes, about 150 minutes, about 120 minutes, about 90 minutes, about 45 minutes, or about 30 minutes. In other embodiments, the irradiation time of the deprotection step (or of each deprotection step) is up to about 20 minutes, up to about 10 minutes, up to about 5 minutes, up to about 2 minutes, or up to about 1 minute. In embodiments, the irradiation time is about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. It will be appreciated that, where the irradiation for a given deprotection step does not take place in a single period of continuous irradiation, the irradiation time can be determined by calculating the sum of the periods during which the NPPOC-protected oligonucleotide or salt thereof is exposed to the radiation. Thus, for example, an irradiation time of 2 minutes could be obtained by a single period of continuous radiation for 2 minutes, or by two separate periods of continuous radiation for 1 minute each, and so on.

[0222] In embodiments, the irradiation is performed at a temperature of from about 0 °C to about 50 °C, e.g. at a temperature of from about 5 °C to about 40 °C, about 10 °C to about 30 °C, about 10 °C to about 25 °C, or about 10 °C to about 20 °C. In embodiments, the temperature is maintained in the range of about 0 °C to about 30 °C. In embodiments, the temperature is maintained in the range of about 10 °C to about 20 °C. In embodiments, the temperature is 201393-PCT01-NP P78290WO maintained in the range of about 20 °C to about 25 °C. In embodiments, the temperature is maintained at about 25 °C.

[0223] In embodiments, the temperature of the liquid reaction mixture is monitored and / or controlled during the irradiation step. In embodiments, the temperature is controlled using a cooling means. In embodiments, the cooling means is selected from a heatsink, a fan, and a liquid cooling system. In embodiments, the cooling means is a liquid cooling system. In embodiments, the temperature is monitored (e.g., continually monitored) and the cooling is adjusted (e.g., by a computer) to maintain the temperature within predefined limits (e.g., as set out herein).

[0224] Reaction mixture - solvents, bases and concentrations

[0225] The deprotection step conveniently takes place in solution. Thus, the irradiation is typically performed on the NPPOC-protected compound in a solvent, which may include other components such as a base.

[0226] In embodiments, the solvent is an aprotic solvent. In embodiments, the solvent is selected from tetrahydrofuran (THF), sulfolane, 2-methyltetrahydrofuran, dioxane, ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), benzonitrile, dichloroethane (DCE), dichloromethane (DCM), chloroform, dimethyl sulfoxide (DMSO), toluene, and anisole. In embodiments, the solvent comprises (e.g., is) MeCN, e.g. a mixture of MeCN and sulfolane. Thus, in embodiments of the LPOS process of the disclosure, MeCN is used as a solvent. In embodiments of the LPOS process of the disclosure, a mixture of MeCN and sulfolane is used as a solvent.

[0227] In embodiments, the irradiation is performed in the presence of a base, e.g. an organic base. In embodiments, the irradiation is performed in the presence of a base selected from diisopropylamine (zP^NH), diisopropylethylamine (DIPEA), triethylamine (TEA), morpholine, imidazole, 1 -methylimidazole (NMI or A-methylimidazole), pyridine, pyrrolidine, piperidine, guanidine, l,4-diazabicyclo[2.2.2]octane (DABCO), and 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU). In embodiments, the base is selected from one or more of zP^NH, 1 -methylimidazole, and morpholine. In embodiments, the irradiation is performed in the presence of zP^NH. In embodiments, the irradiation is performed in the presence of 1 -methylimidazole. In embodiments, the base is 1 -methylimidazole. 201393-PCT01-NP P78290WO

[0228] In embodiments, the base is present in an amount of up to about 100 equivalents. In embodiments, the base is present in an amount of up to about 50 equivalents, e.g. up to about 40, about 35, about 30, about 25, about 20, about 18, about 16, about 14, about 12, about 10, about 8, about 6, about 5, or up to about 4 equivalents. In embodiments, the base is present in an amount of at least about 0.1 equivalents, e.g. at least about 0.2, about 0.5, about 1, about 2, about 3, about 4, or at least about 5 equivalents. In embodiments, the base is present in an amount between about 0.1 and 25 equivalents, e.g. between about 0.2 and 20, between about 0.5 and 16, or between about 1 and 10 equivalents. In embodiments, the amount of base is about 5 equivalents, about 3 equivalents, about 2 equivalents, about 1 equivalent, about 0.5 equivalents, about 0.2 equivalents or about 0.1 equivalents .

[0229] In embodiments, the base is present in an amount of up to about 100 equivalents, e.g. up to about 80, about 72, about 64, about 56, about 48, about 40, about 32, about 24, about 20, or up to about 16 equivalents. In embodiments, the base is present in an amount of up to about 64 equivalents. In embodiments, the amount of base is about 88 equivalents, about 80 equivalents, about 72 equivalents, about 64 equivalents, about 56 equivalents, or about 48 equivalents, or about 40 equivalents. In embodiments, the amount of base is about 64 equivalents.

[0230] The equivalents of a reagent, for example the base, in the deprotection step of the present process are to be understood as being presented relative to the amount (in moles) of the NPPOC-containing compound to be deprotected. Thus, for example, 1 equivalent of base would correspond to a 1 :1 molar ratio between the base and the NPPOC-containing compound. However, it will be appreciated that the equivalents of a reagent, for example the base, can be determined relative to another component present in the reaction mixture.

[0231] In embodiments, the base is iP^NH and the base is present in an amount from about 0.001 equivalents to about 3 equivalents, e.g. from about 0.01 equivalents to about 2 equivalents, from about 0.05 equivalents to about 1 equivalent, or from about 0.1 equivalents to about 0.4 equivalents. In embodiments, zPioNH is present in an amount of about 0.2 equivalents. In embodiments, the solvent is MeCN and zPi oNH is present in an amount of about 0.2 equivalents. In embodiments, the base is iP^NH and the base is present in an amount from about 10 equivalents to about 100 equivalents, e.g. about 88 equivalents, about 80 equivalents, about 72 equivalents, about 64 equivalents, about 56 equivalents, or about 48 equivalents, or about 40 equivalents. In embodiments, iP^NH is present in an amount of 201393-PCT01-NP P78290WO about 64 equivalents. In embodiments, the solvent is MeCN and iPr2NH is present in an amount of about 64 equivalents.

[0232] In embodiments, the base is 1 -methylimidazole and the base is present in an amount from about 0.01 equivalents to about 12 equivalents, e.g. from about 0.1 equivalents to about 10 equivalents, from about 0.2 equivalents to about 8 equivalents, from about 0.5 equivalents to about 6 equivalents, from about 1 equivalent to about 5 equivalents, or from about 2 equivalents to about 4 equivalents. In embodiments, 1 -methylimidazole is present in an amount of about 3 equivalents. In embodiments, the solvent is MeCN and 1 -methylimidazole is present in an amount of about 3 equivalents. In embodiments, the base is 1 - methylimidazole and the base is present in an amount from about 10 equivalents to about 100 equivalents, e.g. about 88 equivalents, about 80 equivalents, about 72 equivalents, about 64 equivalents, about 56 equivalents, or about 48 equivalents, or about 40 equivalents. In embodiments, 1 -methylimidazole is present in an amount of about 64 equivalents. In embodiments, the solvent is MeCN and 1 -methylimidazole is present in an amount of about 64 equivalents.

[0233] It will be appreciated that the NPPOC-protected oligonucleotide or salt thereof will be present at a certain concentration in the reaction solution, e.g. during irradiation. This may be described as the irradiation concentration.

[0234] In embodiments, the irradiation is performed at an oligonucleotide concentration of about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 5 mM to about 70 mM, or about 10 mM to about 60 mM. In embodiments, the irradiation is performed at an oligonucleotide concentration of about 20 mM to about 50 mM, about 30 mM to about 50 mM, or about 35 mM to about 45 mM, e.g. at a concentration of about 40 mM. In other embodiments, the irradiation is performed at an oligonucleotide concentration of about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM, e.g. at a concentration of about 20 mM. In other embodiments, the irradiation is performed at an oligonucleotide concentration of about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, or about 5 mM to about 15 mM, e.g. at a concentration of about 10 mM.

[0235] As will be appreciated, the concentration of the NPPOC-protected oligonucleotide or salt thereof in the deprotection (e.g., irradiation) step may be applied to other steps in the process 201393-PCT01-NP P78290WO of the disclosure. This is particularly the case where the same reaction solution is used in adjacent steps of the process, e.g. where there is no intervening step of purification and the reaction mixture can be moved directly to the next step in the process . In embodiments, the irradiation concentration is the same as the concentration usedin one or more of (e.g., all of) the other steps of the process.

[0236] In embodiments, the irradiation step is carried out under low oxygen conditions, e.g. wherein the reaction solution contains a low level of dissolved oxygen and / or in which any gaseous headspace has a low oxygen concentration. Low oxygen conditions may be promoted through measures such as the use of a solvent that has low oxygen solubility, the use of oxygen scavengers, sparging with a gas such as nitrogen, distillation of the solvent under inert atmosphere orboiling the solvent, and / or degassing by a freeze-pump-thaw method. The use of low oxygen conditions can improve the yield of the deprotected product and / or reduce the level of unwanted side -products. Dissolved oxygen can be measured in solution, for example, using commercially available O2 sensors.

[0237] In embodiments, the reaction mixture is sparged with a gas (other than oxygen) before irradiation. In embodiments, the solvent is sparged with a gas (other than oxygen) before the addition of one or more reagents such as, e.g., the base and / or growing oligonucleotide. In embodiments, the gas used for sparging is an inert gas. In embodiments, the gas is argon. In embodiments, the gas is nitrogen. In embodiments, the reaction mixture is sparged with nitrogen before irradiation. In embodiments, the reaction mixture is sparged with nitrogen before irradiation and before addition of the base. In other embodiments, especially where a non-volatile base (such as, e.g., NMI) is used, the reaction mixture is sparged with nitrogen before irradiation but after addition of the base. In embodiments, the sparging with nitrogen lasts for at least about 30 minutes before irradiation.

[0238] In embodiments, the reaction mixture is degassed by a freeze-pump-thaw method before irradiation. In embodiments, the solvent is degassed by a freeze -pump -thaw method before the addition of one or more reagents such as, e.g., the base and / or growing oligonucleotide. In embodiments, the freeze-pump-thaw method comprises back-filling with an inert gas, for example nitrogen or argon. In embodiments, the method comprises a plurality of freeze- pump-thaw cycles, e.g. at least three freeze-pump-thaw cycles.

[0239] The processes of the present disclosure can reduce solvent waste because washing out is not required between each cycle to remove byproducts and impurities from the deprotection step. 201393-PCT01-NP P78290WO

[0240] This may be contrasted with a conventional approach in which DMT is used as a protecting group. Accordingly, in embodiments, the processes uses less than about 90%, less than about 80%, less than about70%, less than about 60%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about20%, less than about20%, less than about 15%, less than about 10%, or less than about 5% of the solvent required by a conventional oligonucleotide synthesis process. In some embodiments, the conventional oligonucleotide synthesis process is a process which uses DMT as a 5 ’-hydroxyl protecting group, for example a process that exclusively uses DMT as a 5 ’-hydroxyl protecting group.

[0241] Process steps prior to the chain extension cycle

[0242] As illustrated herein, the process of the disclosure can include steps prior to the chain extension cycle. Such steps may include, for example, the loading of a nucleoside or oligonucleotide precursor on to a carrier moiety in solution (see, e.g., step B in Fig. 2 and step B in Fig. 4). The loading step may be associated with its own steps of protection and deprotection, e.g. (i) using NPPOC as a protecting group in accordance with the present disclosure (see, e.g., step A and “Deprotection” in Fig. 2), or (ii) using DMT as a protecting group in accordance with the present disclosure (see, e.g., step s A and “Deprotection” in Fig. 4). As will be apparent from the present disclosure, DMT may be used as a protecting group prior to the chain extension cycle. Advantageously, the use of DMT prior to the chain extension cycle (and typically not during the chain extension cycle) means that there are fewer DMT-deprotection steps in the process, and fewer additional purification steps to remove byproducts resulting from deprotection between cycles such as, e.g., diafiltration. In embodiments, DMT is used as a protecting group prior to the chain extension cycle. In embodiments, DMT is used as a protecting group prior to the chain extension cycle and DMT is not used as a protecting group in the chain extension cycle.

[0243] The product produced by the step(s) prior to chain extension will typically be a nucleobase- containing moiety having a free hydroxyl group which can participate in a first coupling step during chain extension. In embodiments, said nucleobase-containing moiety is a nucleoside having a free 5 ’-hydroxyl group.

[0244] Thus, in embodiments, the process of the invention comprises one or more of the following steps prior to coupling and chain extension (e.g., as described herein): 201393-PCT01-NP P78290WO a) a pre-loading step in which a nucleobase-containing moiety (e.g., a nucleoside) having a plurality of reactive functional groups (e.g., hydroxyl groups) is reacted in solution so as to protect one or more of said reactive functional groups with protecting groups, wherein at least one of said reactive functional groups remains unprotected and free to participate in further reactions; b) a loading step in which the product of step (a) is coupled to a carrier moiety via at least one of the unprotected reactive functional groups, wherein the product remains in solution; and c) a deprotection step in which one (or more than one) of the protecting groups of the product of step (b) is deprotected, thereby revealing a reactive functional group (e.g., a hydroxyl group) which is capable of participating in a coupling or chain extension reaction (e.g., as described herein).

[0245] In embodiments, the protecting group(s) employed in step (a) is (or are) NPPOC and step (c) involves irradiation of the product of step (b), e.g. using conditions as described herein. In embodiments, no purification step (e.g., diafiltration or tangential filtration) is carried out after step (c) and before a first round of chain extension.

[0246] As described above, DMT may be used as a protecting group prior to the chain extension cycle. Accordingly, in embodiments, the protecting group(s) employed in step (a) is (or are) DMT and step (c) involves deprotection of the product of step (b). Exemplary conditions for DMT deprotection include the use of (i) TFA and DDT, followed by (ii) 2 -picoline (see e.g. Example 4). In embodiments, the product of step (b) is contacted with TFA in deprotection step (c). In embodiments, the product of step (b) is contacted with TFA and DDT, followed by contact with 2-picoline, in deprotection step (c). Other conditions for the (selective) removal of DMT will be known to those of ordinary skill in the art. In embodiments, a purification step (e.g., diafiltration) is carried out after step (c) and before a first round of chain extension.

[0247] Pre-loading step of reactive group protection

[0248] The protecting group(s) employedin step (a) can include NPPOC. Thus, in embodiments, the protecting group(s) employed in step (a) is (or are all) NPPOC. In this case, the product of step (a) is an NPPOC-protected nucleobase-containing moiety which comprises at least one unprotected reactive functional group that is (or are) free to participate in further reactions such as loading as described herein. In embodiments, the NPPOC-protected nucleobase- 201393-PCT01-NP P78290WO containing moiety is an NPPOC-protected nucleoside, e.g. a nucleoside having a 5 ’ -hydroxyl group which is NPPOC-protected and having a 3 ’-hydroxyl group which is unprotected and free to participate in further reactions.

[0249] Free hydroxy groups on the nucleobase-containing moiety (e.g., the 5’ hydroxy group of a nucleoside) may be protected using an activated NPPOC derivative. As a person of ordinary skill in the art will appreciate, there are many activated NPPOC derivatives that may be used to effect step (a). For example, NPPOC-protected nucleosides may be prepared from the corresponding nucleoside by reacting a 5 ’ -unprotected nucleoside with an activated NPPOC derivative, for example 2-(2-nitrophenyl)propyl chloroformate (see., e.g., step A of Fig. 2, in which Y = Cl). Other suitable activated NPPOC derivatives are described in, e.g., Chinese patent publication No. CN 105968016 A (the entire content of which is incorporated herein by reference), including 4-nitrophenyl (2-(2-nitrophenyl)propyl) carbonate.

[0250] The protecting group(s) employed in step (a) can include DMT. Thus, in embodiments, the protecting group(s) employedin step (a) is (or are all) DMT. In this case, the product of step (a) is a DMT-protected nucleobase-containing moiety which comprises at least one unprotected reactive functional group that is (or are) free to participate in further reactions such as loading as described herein. In embodiments, the DMT-protected nucleobase- containing moiety is a DMT-protected nucleoside, e.g. a nucleoside having a 5 ’ -hydroxyl group which is DMT-protected and having a 3 ’-hydroxyl group which is unprotected and free to participate in further reactions.

[0251] Free hydroxy groups on the nucleobase-containing moiety (e.g., the 5’ hydroxy group of a nucleoside) may be protected using an activated DMT derivative. As a person of ordinary skill in the art will appreciate, there are many activated DMT derivatives that may be used to effect step (a). For example, DMT-protected nucleosides may be prepared from the corresponding nucleoside by reacting a 5 ’ -unprotected nucleoside with an activated DMT derivative, for example 4,4'-dimethoxytrityl chloride or 4,4'-dimethoxytrityl bromide (see, e.g., step A of Fig. 4, in which Z = Cl or Br, respectively). Other suitable activated DMT derivatives, and suitable conditions for DMT protection using said derivatives, will be known to a person of ordinary skill in the art.

[0252] Loading 201393-PCT01-NP P78290WO

[0253] Since the process of the disclosure is performed in liquid phase, a solid support is not required. However, the chain extension process which produces the oligonucleotide may conveniently be carried out with the growing oligonucleotide chain tethered to a carrier (also called a “hub” herein). The carrier can act, for example, as a protecting group for one of the hydroxyl groups on the oligo nucleoside that forms the first base in the desired oligonucleotide (e.g., to protect the 3 ’-hydroxyl group while the oligonucleotide is extended from the 5 ’-hydroxyl group). The use of a carrier in the liquid-phase process of the disclosure may also provide advantages in terms of overall yield, e.g. due to improved solubility and / or improved retention during purification steps within the process.

[0254] In embodiments, the carrier is capable of being tethered to more than one nucleobase- containing moiety, e.g. the carrier is a star macromolecule which can be tethered to a plurality of nucleobase-containing moieties. In embodiments, the carrier is a 2-star, 3-star, 4-star, 5- star, 6-star, 7-star, or 8-star macromolecule. In embodiments, the carrier is an n-star macromolecule which is tethered to n nucleobase-containing moieties. In embodiments, the carrier is a 4-star macromolecule which is tethered to four nucleobase-containing moieties. In embodiments, the arms of the carrier (e.g. the star macromolecule) comprise a derivative of PEG, e.g. a sarcosine derivative of PEG or a primary amine derivative of PEG. In embodiments, the carrier comprises a PEGylated pentaerythreitol or a derivative thereof, e.g. a sarcosine derivative or a primary amine derivative of PEG.

[0255] In embodiments, the carrier comprises at least one sarcosine derivative of PEG comprising

[0256] H the structure: wherein n is an integer from 0 to 500. In embodiments, the carrier is a 4-star macromolecule which has the structure: 4wherein n in each case is independently an integer from 0 to 500. In embodiments, eachn is independently an integer from 0 to 400, e.g. from 0 to 300, from 0 to 200, from 0 to 100, from 20 to 90, or from 40 to 80. In embodiments, each n is independently an integer from 60 to 70, e.g. about 64. In embodiments, each n is independently an integer fromO to 250, e.g. selected from about 8, about 25, about 53, about 110, and about 224. In embodiments, each n is the same. In embodiments, the carrier has a 201393-PCT01-NP P78290WO molecular mass between about 1 and 50 kDa, e.g. about 2, 5, 10, 20 or 40 kDa. In embodiments, the carrier has a molecular mass of at least about 10 kDa.

[0257] In embodiments, the carrier comprises at least one primary amine derivative of PEG comprising the structure: wherein n is an integer from 0 to 500. In embodiments, the carrier is a 4-star macromolecule which has the structure: wherein n in each case is independently an integer from 0 to 500. In embodiments, each n is independently an integer from 0 to 400, e.g. from 0 to 300, from 0 to 200, from 0 to 100, from 20 to 90, or from 40 to 80. In embodiments, each n is independently an integer from 60 to 70, e.g. about 64. In embodiments, each n is independently an integer from 0 to 250, e.g. selected from about 10, about 27, about 55, about 112, and about 225. In embodiments, each n is the same. In embodiments, the carrier has a molecular mass between about 1 and 50 kDa, e.g. about 2, 5, 10, 20 or 40 kDa. In embodiments, the carrier has a molecular mass of at least about 10 kDa.

[0258] In embodiments, the carrier is a 4-star macromolecule which has the structure: wherein n in each case is independently an integer from 0 to 250, e.g. about 55, and wherein the carrier has a molecular mass of about 10 kDa.

[0259] It will be appreciated that a single carrier molecule may contain PEG components of different length, and / or that the PEG components of the carrier material may contain a range of values. Thus, in embodiments the integer n represents the average value across the PEG components of the carrier.

[0260] In embodiments, the carrier is coupled to the product of step (a) (e.g., the NPPOC-protected nucleoside or the DMT-protected nucleoside) using a bifunctional linker. In embodiments, the linker is a dicarboxylic acid. In embodiments, the linker is succinic acid. In embodiments, the coupling is carried out under conditions in which a carboxylic acid of the dicarboxylic acid forms an activated ester. In embodiments, TBTU is used to form an HOBt active ester.

[0261] In embodiments, the linker (e.g. a bifunctional linker such as succinic acid) is reacted with the protected nucleobase-containing moiety (e.g. the NPPOC-protected nucleoside or the 201393-PCT01-NP P78290WO

[0262] DMT-protected nucleoside of step (a)) to form a conjugate between the linker and the nucleobase-containing moiety, wherein said conjugate contains a free carboxyl group which may be in the form of a salt (e.g., a triethylamine salt).

[0263] In embodiments, the free carboxyl group is activated (e.g., using TBTU to form an HOBt active ester) and reacted with the carrier to form a conjugate between the nucleobase- containing moiety and the carrier. In embodiments, the activating conditions comprise TBTU in the presence of DIPEA. In embodiments, the activating conditions comprise TBTU and DIPEA in MeCN. In embodiments, the coupling between the free carboxyl group of the nucleobase-containing moiety and the carrier is conducted as follows: (i) the nucleobase- containing moiety is contacted with TBTU in a first solvent in a first reaction vessel; (ii) the carrier and DIPEA are provided in a second solvent in a second reaction vessel; and (iii) the solutions of the first reaction vessel and the second reaction vessel are combined to form a reaction mixture. The reaction mixture may be quenched at the end of the reaction with hexylamine. In embodiments, the first solvent and the second solvent are both acetonitrile. An example of this embodiment may be found in e.g., Scheme 3 of Example 3.

[0264] Numerous activating agents may be used to couple an NPPOC-protected nucleoside or a DMT-protected nucleoside with a linker and / or a carrier. Examples of such agents include, without limitation, the following: dicyclohexylcarbodiimide (DCC); diisopropylcarbodiimide (DIC); ethyl-(N’,N’-dimethylamino)propylcarbodiimide hydrochloride (EDC); (benzotriazol- l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP); (benzo triazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP); (7-azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP); bromotripyrrolidinophosphoniumhexafluorophosphate; bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-CI); O-(benzo triazol- 1 -yl)-N,N,N’ ,N’ -tetramethyluronium hexafluorophosphate (HBTU); O-(benzotriazol-l-yl)-N,N,N’,N’ -tetramethyluronium tetrafluoroborate (TBTU); O-(7-azabenzotriazol-l-yl)-N,N,N’,N’ -tetramethyluronium hexafluorophosphate (HATU); O -(7 -azabenzo triazol- l-yl)-N,N,N’,N’ -tetramethyluronium tetrafluoroborate (TATU); O-(6-chlorobenzotriazol-l-yl)-N,N,N’,N’ -tetramethyluronium hexafluorophosphate (HCTU); O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetra methyluronium tetrafluoroborate (TOTU); O-(N-succinimidyl)- 1 , 1 ,3,3-tetramethyl-uronium tetrafluoroborate (TSTU); O-(5-norbornene-2,3-dicarboximido)-N,N,N’,N’- tetramethyluronium tetrafluoroborate (TNTU); O-(l,2-dihydro-2-oxo-l-pyridyl-N,N,N’,N’- 201393-PCT01-NP P78290WO tetramethyluronium tetrafluoroborate (TPTU); 3 -(diethylphosphoryloxy)- 1,2, 3 -benzo triazin - 4(3H)-one (DEPBT); carbonyldiimidazole (CDI); and propylphosphonic anhydride (T3P).

[0265] In alternative embodiments, the NPPOC-protected nucleoside or the DMT-protected nucleoside from step (a) may be protected (e.g., at the 3 ’-hydroxy group) using a protecting group that is not a carrier as described above. That compound may then be deprotected and participate in chain extension as described herein to yield an oligonucleotide.

[0266] Process steps after the chain extension cycle

[0267] As illustrated herein, the process of the disclosure may include additional steps after chain extension. Thus, for example, the mature oligonucleotide can be further reacted or elaborated to incorporate additional chemical functionality (e.g., a linker to attach the oligonucleotide to another moiety such as a targeting moiety) and / or to remove the oligonucleotide from any carrier.

[0268] Coupling with a linker moiety

[0269] In embodiments, the oligonucleotide which results from the chain extension step(s) comprises a functional group (e.g., a 5 ’-hydroxy group) which may be coupled to a linker. Thus, in embodiments, the process of the disclosure comprises a step of reacting the oligonucleotide with a linker to provide an oligonucleotide -linker conjugate. In embodiments, the linker comprises a chain of from 2 to 21 contiguous atoms selected from C, O, N and P. In

[0270] X

[0271] -APi O R2

[0272] O 1 embodiments, the linker has the structure R wherein: X is O or S; R1is either absent (in which case the adjacent oxygen may carry a negative charge) or is an optionally substituted alkyl group (e.g., cyanoethyl); and R2is a group selected from -(Ci-is)alkyl- Ra, -(Ci-is)alkenyl-Ra, and -(Ci-is)alkynyl-Ra, wherein Rais selected from -ORband -N(RC)2, wherein Rbis selected from hydrogen and a protecting group (e.g., a tosyl or benzyl group) , and each Rcis independently selected from hydrogen and a protecting group (e.g., a monomethoxytrityl (MMT) or benzyloxycarbonyl (Cbz)). The linker will typically be attached directly to a hydroxyl group on the oligonucleotide, e.g. to a 5’ hydroxyl group.

[0273] In embodiments, X is O. In other embodiments, X is S. As will be appreciated, compounds having O or S in this position may be achieved by coupling the mature oligonucleotide with a 201393-PCT01-NP P78290WO phosphite-containing reagent, followed by oxidation or sulfurisation, e.g. using reagents and conditions as described herein.

[0274] In embodiments, R1is cyanoethyl. In embodiments, R2is -(C4-s)alkyl-Ra, wherein Rais -NHRCwherein Rcis a protecting group (e.g., MMT). In embodiments, R2is a protected hexylamine linker, e.g. having the structure -(CH2)6NH-MMT. In embodiments, the linker has the structure , wherein X is S or O. In embodiments, the linker has the structure

[0275] Removal of the oligonucleotide from the carrier

[0276] In embodiments in which the growing oligonucleotide (and the mature oligonucleotide) are attached to a carrier in solution, a further step which may be carried out post-chain extension involves the cleavage of the oligonucleotide from the carrier. The skilled person will be aware of appropriate methods for removing the carrier from the oligonucleotide, depending on the nature of the carrier and its attachment to the oligonucleotide.

[0277] By way of example, where the carrier is a PEG-containing star macromolecule which is attached to the oligonucleotide via an ester linkage (e.g., from a dicarboxylic acid moiety) with a hydroxyl group of the oligonucleotide, the carrier may be removed and the hydroxyl group revealed using suitable hydrolytic conditions such as, e.g., aqueous ammonia solution and a secondary or tertiary amine such as diethylamine or triethylamine. In embodiments, the oligonucleotide is cleaved from the carrier using aqueous ammonia solution and diethylamine. The reaction mixture may be heated to promote cleavage ate.g., about 40 °C to about 70 °C, about 45 °C to about 65 °C, or about 50 °C to about 60 °C, such as about 55 °C.

[0278] Other post-chain-extension steps

[0279] The oligonucleotide product of the chain extension steps may participate in other reactions to yield the desired oligonucleotide. For example, where the oligonucleotide product comprises cyanoethanol groups, these may be removed using known conditions such as, e.g., the 201393-PCT01-NP P78290WO hydrolytic conditions described herein for cleavage of the oligonucleotide from the carrier. Likewise, protecting groups on the nucleobases of the oligonucleotide may be removed, including under the hydrolytic conditions described herein for cleavage of the oligonucleotide from the carrier. Thus, a step of hydrolysis (e.g., using diethylamine in aqueous ammonia solution) can perform multiple functions in removing protecting groups and cleaving the oligonucleotide from its carrier (see e.g., Scheme 4 of Example 4).

[0280] Further steps which would be apparent to the skilled reader include, e.g., coupling of the oligonucleotide to a targeting moiety. Targeting moieties include, for example: GalNAc- containing compounds (see, e.g., Springer et al., Nucleic Acid Ther. (2018) 28(3):109-l 18); antibodies (see, e.g., Toloue et al., Methods Mol Biol. (2011) 764:123-139); and lipid nanoparticles (see, e.g., Kalita et al., Pharmaceutics (2022) 14(11):2520). Conjugates formed between the oligonucleotide and the targeting moiety may be used in a therapeutic setting and can be administered using known routes.

[0281] Flow conditions

[0282] The process of the disclosure can conveniently be performed under continuous flow conditions for part (or all) of some (or all) of the steps. Thus, in embodiments one or more steps of the process are performed under continuous flow conditions (e.g., in whole or in part). In embodiments, substantially all of said one or more steps is performed under continuous flow conditions. In embodiments, said one or more steps comprises at least one deprotection step as described herein. In embodiments, said one or more steps comprises at least one NPPOC deprotection step as described herein. In embodiments, each NPPOC deprotection step in the process is performed under continuous flow conditions.

[0283] In embodiments, the residence time for the NPPOC deprotection step is from about 0.1 to about 60 minutes, e.g. from about 0.1 to about 30 minutes, from about 0.1 to about 20 minutes, or from aboutO.l to about 10 minutes. In embodiments, the residence time for the NPPOC deprotection step is from about 1 to about 10 minutes, e.g. from about 1 to about 9 minutes, from about 1 to about 8 minutes, from about 1 to about 7 minutes, from about 1 to about 6 minutes, or from about 1 to about 5 minutes. In embodiments, the residence time is about 1 minute. In embodiments, the residence time is about 2 minutes. In embodiments, the residence time is about 3 minutes. In embodiments, the residence time is about 4 minutes. In embodiments, the residence time is about 5 minutes. 201393-PCT01-NP P78290WO

[0284] As will be appreciated, the flow conditions can also be influenced by various parameters of the experimental setup such as, e.g., reactor volume, light flux, reaction concentration, etc. Modification of the flow conditions to account for such parameters would be within the routine capability of the skilled person, when guided by the present disclosure.

[0285] Oligonucleotides

[0286] The process of the disclosure produces an oligonucleotide (or a salt thereof). It will be appreciated that the disclosure is not limited in any way to naturally occurring oligonucleotides or even to naturally occurring nucleobases, but that numerous modifications are contemplated within the term “oligonucleotide”, including modifications to the nucleobase, the sugar or sugar-like moiety, and / or the backbone. The person of ordinary skill in the art will readily appreciate the scope of compounds and oligonucleotides which may be prepared in accordance with the disclosure. The following sections set out certain non- limiting examples of nucleotides, including variants of naturally occurring nucleotides, which may be incorporated into oligonucleotides in accordance with the disclosure.

[0287] Thus, in embodiments the oligonucleotide of the disclosure (e.g., prepared according to the process described herein) is an oligomeric compound which comprises linked nucleosides. Oligonucleotides may comprise unmodified oligonucleotides (e.g., comprising nucleosides of naturally occurring RNA or DNA) or may comprise modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA. That is, a modified nucleoside typically comprises a modified sugar moiety, a modified nucleobase, and / or a modified backbone (e.g., at least one modified nucleobase internucleoside linkage).

[0288] In embodiments, a modified oligonucleotide comprises one or more modified nucleosides comprising a modified sugar moiety. In embodiments, a modified oligonucleotide comprises one or more modified nucleosides comprising a modified nucleobase. In embodiments, a modified oligonucleotide comprises one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and / or intemucleoside linkages of a modified oligonucleotide define a pattern or motif. In embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and / or intemucleoside linkage motif (as used 201393-PCT01-NP P78290WO herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).

[0289] In embodiments, an oligonucleotide comprises one or more type of modified sugar and / or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or motif. In embodiments, such a sugar motif includes but is not limited to any of the sugar modifications discussed herein.

[0290] In embodiments, a modified oligonucleotide comprises (e.g., consists of) a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In embodiments, each nucleoside of the modified oligonucleotide comprises a modified sugar moiety. In embodiments, modified oligonucleotides comprise (e.g., consist of) a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In embodiments, each nucleoside of a uniformly modified nucleotide comprises the same 2’ - modification.

[0291] In embodiments, an oligonucleotide comprises modified and / or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In embodiments, each nucleobase is modified. In embodiments, none of the nucleobases are modified. In embodiments, each purine or each pyrimidine is modified. In embodiments, each adenine is modified. In embodiments, each guanine is modified. In embodiments, each thymine is modified. In embodiments, each uracil is modified. In embodiments, each cytosine is modified. In embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methyl cytosines. In embodiments, all of the cytosine nucleobases are 5-methyl cytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases.

[0292] In embodiments, a modified oligonucleotide comprises a block of modified nucleobases. In such embodiments, the block may be at the 3 ’-end of the oligonucleotide. In embodiments, the block is within 3 nucleosides of the 3 ’-end of the oligonucleotide. In other such embodiments, the block may be at the 5 ’-end of the oligonucleotide. In embodiments, the block is within 3 nucleosides of the 5 ’-end of the oligonucleotide. 201393-PCT01-NP P78290WO

[0293] In embodiments, an oligonucleotide comprises modified and / or unmodified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage (“-O-P(=O)(OH)-”). In embodiments, each intemucleoside linking group of a modified oligonucleotide is a phosphorothioate intemucleoside linkage (“-O-P(=O)(SH)-”). In embodiments, each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage and phosphodiester intemucleoside linkage. In embodiments, each phosphorothioate intemucleoside linkage is independently selected from a stereorandom phosphorothioate, a (Sp) phosphorothioate, and a (Rp) phosphorothioate.

[0294] Sugar moieties

[0295] The oligonucleotide typically comprises an oligomer which comprises a plurality of nucleosides or modified nucleosides, linked together by intemucleoside linkages (i.e., a backbone). Each nucleoside (or modified nucleoside) typically comprises a sugar moiety (or modified sugar moiety) bonded to a nucleobase (or modified nucleobase). In embodiments, each nucleoside independently comprises a sugar moiety or a modified sugar moiety (e.g., as described herein). In embodiments, each nucleoside independently comprises a modified sugar moiety (e.g., as described herein).

[0296] In embodiments, each sugar moiety is independently selected from ribose and 2’ -deoxyribose moiety (i.e., the sugar component of RNA and DNA, respectively). It will be appreciated that a sugar moiety may be protected at one or more of its hydroxyl groups (e.g., at the 2’ - hydroxyl group of a ribose moiety).

[0297] In embodiments, each modified sugar moiety is selected from a non-bicyclic modified sugar moiety, a bicyclic or tricyclic sugar moiety, and a sugar surrogate. Sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties. In embodiments, each modified sugar moiety is selected from a bridged nucleic acid (BNA) sugar moiety, a locked nucleic acid (LNA) sugar moiety, and a conformationally restricted nucleic acid (CRN) sugar moiety.

[0298] In embodiments, at least one (e.g., all) of the modified sugar moieties are non-bicyclic modified sugar moieties comprising a furano syl ring with one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non- 201393-PCT01-NP P78290WO bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2’, 4’, and / or 5’ positions. In embodiments one or more non -bridging substituent of non-bicyclic modified sugar moieties is branched. Examples of 2 ’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’ -F, 2'-OCH3(“OMe” or “O-methyl”), and 2'-O(CH2)2OCH3 (“MOE”). In embodiments, 2’- substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-Ci-Cio alkoxy, O-Ci-Cio substituted alkoxy, O-Ci-Cio alkyl, O-Ci-Cio substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn), where each Rmand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted Ci-Cio alkyl, and the 2’ -substituent groups described in Cook et al., U.S. 6,531,584; Cook et al., U.S. 5,859,221; and Cook et al., U.S. 6,005,087. Embodiments of these 2' -substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4’ -substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharanet al., WO 2015 / 106128. Examples of 5’- substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5’-methyl (R or S), 5'-vinyl, and 5’-methoxy. In embodiments, non-bicyclic modified sugar moieties comprise more than one non -bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008 / 101157 and Rajeev et al., US 2013 / 0203836.

[0299] In embodiments, a 2 ’-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’ -substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCHS, O(CH2)2SCHS, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)- N(Rm)(Rn)), where each Rmand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted Ci-Cio alkyl.

[0300] In embodiments, a 2 ’-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’ -substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3(“NMA”). 201393-PCT01-NP P78290WO

[0301] In embodiments, a 2 ’ -substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’ -substituent group selected from: F, OCH3, and

[0302] OCH2CH2OCH3. In embodiments, a modified oligonucleotide comprisesone or more of a 2’- MOE nucleoside, a 2’-OMe nucleoside, a 2’-F nucleoside, and a 2’-NMA nucleoside. In embodiments, the modified oligonucleotide comprises a stereo -non-standard sugar moiety.

[0303] In embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by isomeric configuration. For example, a 2’-deoxyfuranosyl sugar moiety may be in seven isomeric configurations other than the naturally occurring P-D-deoxyribosyl configuration. Such modified sugar moieties are described in, e.g., WO 2019 / 157531, incorporated by reference herein. A 2’ -modified sugarmoiety has an additional stereocentre at the 2’-position relativeto a 2’ -deoxyfurano syl sugar moiety; therefore, such sugar moieties have a total of sixteen possible isomeric configurations. 2’-modified sugar moieties described herein are typically in the P-D-ribosyl isomeric configuration unless otherwise specified.

[0304] In naturally occurring nucleic acids, sugars are linked to one another 3’ to 5’. In embodiments, oligonucleotides include one or more nucleoside or sugar moiety linked at an alternative position, for example at the 2’ or inverted 5’ to 3’. For example, where the linkage is at the 2’ position, the 2 ’-substituent groups may instead be at the 3’-position. A person of ordinary skill in the art would understand how to modify the processes of the invention to prepare these oligonucleotides.

[0305] Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. Nucleosides comprising such bicyclic sugar moieties have been referred to as bicyclic nucleosides (BNAs, or bridged nucleic acids), locked nucleosides, or conformationally restricted nucleotides (CRN). In embodiments, the oligonucleotide comprises a bicyclic sugar moiety such as a locked nucleoside. Examples of such compounds which may be employed in the process of the present disclosure are described in US Patent Publication No. 2013 / 0190383; and PCT publication WO 2013 / 036868. In embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (“LNA”), 4'-CH2-S-2', 4'-(CH2)2-O-2' (“ENA”), 4'-CH(CH3)-O-2' (referred to as “constrained ethyl” or “cEt”), 4’-CH2-O-CH2-2’, 4’-CH2-N(R)-2’, 4'-CH(CH2OCH3)-O-2' (“constrained 201393-PCT01-NP P78290WO

[0306] MOE” or “cMOE”) and analogues thereof (see, e.g., Seth et al., U.S. 7,399,845, Bhat et al., U.S. 7,569,686, Swayze et al., U.S. 7,741,457, and Swayze et al., U.S. 8,022,193), 4' - C(CH3)(CH3)-O-2' and analogues thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'-CH2- N(OCH3)-2' and analogues thereof (see, e.g., Prakash et al., U.S. 8,278,425), 4'-CH2-O- N(CH3)-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745), 4'- CH2-C(H)(CH3)-2' (see, e.g, Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2- C(=CH2)-2' and analogues thereof (see e.g., Seth et al., U.S. 8,278,426), 4’-C(RaRb)-N(R)-O- 2’, 4’-C(RaRb)-O-N(R)-2’, 4'-CH2-O-N(R)-2', and 4'-CH2-N(R)-O-2', wherein each R, Ra, and Rb is, independently, H, a protecting group, or C1-C12 alkyl (see, e.g. Imanishi et al., U.S. 7,427,672). In embodiments, the oligonucleotide comprises one or more constrained ethyl nucleotides (see, e.g., Seth et al., Nucleic Acids Symp Ser (Oxf) (2008) 52:553-554).

[0307] In embodiments, such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, -C(Ra)=C(Rb)-, -C(Ra)=N-, - C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)X-, and -N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Raand Rb is, independently selected from: H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ1J2, SJi, N3, COOJi, acyl (C(=O)- H), substituted acyl, CN, sulfonyl (S(=0)2-Ji). and sulfoxyl (S(=O)-Ji); and each Ji and J2 is, independently selected from: H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, and a protecting group.

[0308] In embodiments, the oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 locked nucleosides. In embodiments, the locked nucleoside is cEt. In embodiments, the oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 locked nucleosides wherein the locked nucleoside is cEt. In embodiments, the oligonucleotide comprises 6 locked nucleosides wherein the locked nucleoside is cEt.

[0309] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 201393-PCT01-NP P78290WO

[0310] 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362-8379; Wengel et a., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al. U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO 1999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131; Seth et al., U.S. 8,030,467; Seth et al., U.S.

[0311] 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008 / 0039618 and Migawa et al., US2015 / 0191727.

[0312] In embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the a-L configuration or in the P-D configuration.

[0313] LNA ( p-1 J-ccrn figuration ) a-H.NA (tt- / .-coiillguraiit)ii ) bridge 4-CI L-O-2’ bridge • ■ 4'-( H2-O-2' a-L-methyleneoxy (4’-CH2-O-2’) or a-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off -target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mai Cane Ther 6(3):833- 843; Grunweller, A. et al., (2003) Nucleic Acids Research 31 (12):3185 -3193). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g, LNA or cEt) are identified in exemplified embodiments herein, they are in the P-D configuration, unless otherwise specified. 201393-PCT01-NP P78290WO

[0314] In embodiments, the LNA nucleoside wherein Bx represents the nucleobase (or modified nucleobase).

[0315] In embodiments, the nucleoside i , wherein Bx represents the nucleobase (or modified nucleobase). In embodiments, the nucleoside wherein Bx represents the nucleobase (or modified nucleobase). In embodiments, the nucleoside is , wherein Bx represents the nucleobase (or modified nucleobase). In embodiments, the nucleoside wherein Bx represents the nucleobase (or modified nucleobase).

[0316] In embodiments, the oligonucleotide comprises at least one nucleoside of the formula: least one nucleoside of the formula: , wherein Bx represents the nucleobase (or modified nucleobase). 201393-PCT01-NP P78290WO

[0317] In embodiments, modified sugar moieties comprise one or more non -bridging sugar substituent and one or more bridging sugar substituent (e.g., 5’ -substituted and 4’-2’ bridged sugars).

[0318] In embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulphur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and / or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4’-sulphur atom and a substitution at the 2'-position (see, e.g., Bhat et ah, U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and / or the 5’ position.

[0319] In embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in embodiments, a sugar surrogate comprises a six -membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), altritol nucleic acid (“ANA”), mannitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ.

[0320] Bioorg. &Med. Chem. 2002, 10, 841-854), and fluoro HNA: F-HNA , wherein Bx is a nucleobase (or a modified version thereof).

[0321] (“F-HNA”, F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran)

[0322] In embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315;

[0323] Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure: , wherein Bx a nucleobase (or a modified version thereof). 201393-PCT01-NP P78290WO

[0324] In embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

[0325] In embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011 / 133876. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331 ; and 5,719,262. Additional PNA compounds suitable for use in the oligonucleotides of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497 -1500.

[0326] In embodiments, sugar surrogates are the “unlocked” sugar structure of UNA (unlocked nucleic acid) nucleosides. UNA is an unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked sugar surrogate. Representative U.S. publications that teach the preparation of UNA include, but are not limited to, US Patent No. 8,314,227; and US Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are hereby incorporated herein by reference.

[0327] In embodiments, sugar surrogates are the glycerol as found in GNA (glycol nucleic acid) nucleosides, e.g. (S)-GNA: , wherein Bx represents a nucleobase (or a modified version thereof).

[0328] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

[0329] Nucleobases 201393-PCT01-NP P78290WO

[0330] The nucleobase components of the oligonucleotide are typically chosen to participate in structure-specific interactions with a target, wherein the target may be a biological molecule such as a nucleic acid. Suitable unmodified (e.g., naturally occurring) nucleobases will be apparent to the skilled person, as will modified versions thereof.

[0331] In embodiments, the oligonucleotide comprises one or more unmodified nucleobases and / or one or more modified nucleobases. In embodiments, the oligonucleotide comprises one or more nucleosides comprising an unmodified nucleobase. In embodiments, the oligonucleotide comprises one or more nucleosides comprising a modified nucleobase. The oligonucleotide may comprise one or more nucleosides that does not comprise a nucleobase, referred to herein as an abasic nucleoside. In embodiments, the oligonucleotide comprises one or more inosine nucleosides (i.e., nucleosides comprising a hypoxanthine nucleobase).

[0332] In embodiments, each unmodified nucleobase is independently selected from guanine, adenine, cytosine, 5-methylcytosine, thymine, and uracil.

[0333] In embodiments, a modified nucleobase is selected from: 5-substituted pyrimidine, 6- azapyrimidine, alkyl or alkynyl substituted pyrimidine, alkyl substituted purine, andN-2, N-6 and 0-6 substituted purine. In embodiments, a modified nucleobase is selected from: 2- aminopropyladenine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- N-methylguanine, 6-N-methyladenine, 2-propyladenme, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl (-OC-CH3), uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5 -ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5- trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7 -methyladenine, 2-F- adenine, 2-aminoadenine, 7 -deazaguanine, 7 -deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5- methyl 4-N-benzoylcytosine, 5 -methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3 -diazaphenoxazine -2-one, 1,3- diazaphenothiazine -2-one and 9-(2-aminoethoxy)-l,3-diazaphenoxazine -2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7 -deaza-adenine, 7-deazaguanosine, 2- aminopyndine and2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. 3,687,808, those disclosed in The Concise Encyclopaedia Of Polymer Science And 201393-PCT01-NP P78290WO

[0334] Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858 -859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebien, B, Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163-166 and 442-443.

[0335] Publications that teach the preparation of certain of the above modified nucleobases as well as other modified nucleobases include without limitation, Manoharan et al.,

[0336] US2003 / 0158403; Manoharan et al., US2003 / 0175906; Dinh et al., U.S. 4,845,205; Spielvogel et al, U.S. 5,130,302; Rogers et al, U.S. 5,134,066; Bischofberger et al, U.S. 5,175,273; Urdea et al, U.S. 5,367,066; Benner et al, U.S. 5,432,272; Matteucci et al, U.S. 5,434,257; Gmeiner et al, U.S. 5,457,187; Cook et al, U.S. 5,459,255; Froehler et al, U.S. 5,484,908; Matteucci et al, U.S. 5,502,177; Hawkins et al, U.S. 5,525,711 ; Haralambidis et al, U.S. 5,552,540; Cooket al, U.S. 5,587,469; Froehler et al, U.S. 5,594,121 ; Switzer et al, U.S. 5,596,091; Cook et al, U.S. 5,614,617; Froehler et al, U.S. 5,645,985; Cook et al, U.S. 5,681,941; Cook et al, U.S. 5,811,534; Cook et al, U.S. 5,750,692; Cook et al, U.S. 5,948,903; Cook et al, U.S. 5,587,470; Cook et al, U.S. 5,457,191 ; Matteucci et al, U.S. 5,763,588; Froehler et al, U.S. 5,830,653; Cooket al, U.S. 5,808,027; Cook et al, 6,166,199; and Matteucci et al, U.S. 6,005,096.

[0337] Internucleoside linkages (the oligonucleotide backbone)

[0338] The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. In embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus- containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond (“-O-P(=O)(OH)”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, vinylphosphonates, phosphoramidates, phosphorothioates (“-O-P(=S)(OH)-”), and phosphorodithioates (“-O- P(=S)(SH)”). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N’ -dimethylhydrazine (- CH2-N(CH3)-N(CH3)-). Modified intemucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the 201393-PCT01-NP P78290WO oligonucleotide. In embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

[0339] In embodiments, an internucleoside linkage is any of those described in WO 2021 / 030778, incorporated by reference herein. In embodiments, an internucleoside linkage comprises the formula: wherein (independently for each such internucleoside linking group of the oligonucleotide):

[0340] X is selected from O or S;

[0341] Ri is selected from H, CpCe alkyl, and substituted CpCe alkyl; and

[0342] T is selected from SO2R2, C(=O)Rs, and P(=O)R4Rs, wherein:

[0343] R2 is selected from an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a Ci-Ce alkoxy, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Cr Ce alkyl, substituted C2-C6 alkenyl substituted C2-C6 alkynyl, and a conjugate group;

[0344] R3 is selected from an aryl, a substituted aryl, CH3, N(CHa)2, OCH3 and a conjugate group;

[0345] R4is selected from OCH3, OH, CpCe alkyl, substituted Ci-Ce alkyl and a conjugate group; and

[0346] Rs is selected from OCH3, OH, Ci-Ce alkyl, and substituted Ci-Ce alkyl.

[0347] In embodiments, an internucleoside linkage comprises a mesyl phosphoramidate linking group having a formula: 201393-PCT01-NP P78290WO

[0348] In embodiments, a mesyl phosphoramidate intemucleoside linkage may comprise a chiral centre. In embodiments, modified oligonucleotides comprising (Rp) and / or (Sp) mesyl phosphoramidates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:

[0349] Representative internucleoside linkages having a chiral centre include but are not limited to alkylphosphonates, mesyl phosphoramidates, and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral centre can be prepared as populations of modified oligonucleotides comprising stereorandom internucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate or other linkages containing chiral centres in particular stereochemical configurations. In embodiments, populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages wherein the phosphorothioate intemucleoside linkages are present in a mixture of stereoisomers. In embodiments, populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereorandom. In embodiments, populations of modified oligonucleotides comprise mesyl phosphoramidate intemucleoside linkages wherein all of the mesyl phosphoramidate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage or mesyl phosphoramidate. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate or mesyl phosphoramidate of each individual oligonucleotide molecule has a defined stereoconfiguration. In embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate or mesyl phosphoramidate intemucleoside linkages in a 201393-PCT01-NP P78290WO particular, independently selected stereochemical configuration. In embodiments, the particular configuration of the particular pho sphoro thio ate or mesyl phosphoramidate linkage is present in at least 65% of the molecules in the population. In embodiments, the particular configuration of the particular phosphorothioate or mesyl phosphoramidate linkage is present in at least 70% of the molecules in the population. In embodiments, the particular configuration of the particular phosphorothioate or mesyl phosphoramidate linkage is present in at least 80% of the molecules in the population. In embodiments, the particular configuration of the particular phosphorothioate or mesyl phosphoramidate linkage is present in at least 90% of the molecules in the population. In embodiments, the particular configuration of the particular phosphorothioate or mesyl phosphoramidate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017 / 015555. In embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate or mesyl phosphoramidate in the (Sp) configuration. In embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate or mesyl phosphoramidate in the (Rp) configuration. In embodiments, modified oligonucleotides comprising (Rp) and / or (Sp) phosphorothioates comprise one or more of the following formulas, respectively: wherein “B” independently indicates any nucleobase.

[0350] Unless otherwise indicated, chiral internucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration. 201393-PCT01-NP P78290WO

[0351] Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, vinylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2- C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research, Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2component parts.

[0352] In embodiments, modified oligonucleotides comprise one or more inverted nucleosides, as shown below: wherein each Bx independently represents a nucleobase (or a modified nucleobase).

[0353] In embodiments, an inverted nucleoside is terminal (i.e., the last nucleoside on one end of an oligonucleotide) and so only one inverted internucleoside linkage as depicted above will be present. In such embodiments, additional features (such as a conjugate group) may be attached to the inverted nucleoside. Such terminal inverted nucleosides can be attached to either or both ends of an oligonucleotide.

[0354] In embodiments, such groups lack a nucleobase and are referred to herein as inverted sugar moieties. In embodiments, an inverted sugar moiety is terminal (i.e., attached to the last nucleoside on one end of an oligonucleotide) and so only one internucleoside linkage above will be present. In such embodiments, additional features (such as a conjugate group) may be 201393-PCT01-NP P78290WO attached to the inverted sugar moiety. Such terminal inverted sugar moieties can be attached to either or both ends of an oligonucleotide.

[0355] In embodiments, nucleic acidscan be linked2’ to 5’ ratherthan the standard 3’ to 5’ linkage. Such a linkage is illustrated below. wherein each Bx independently represents a nucleobase (or a modified nucleobase).

[0356] Gapmer oligonucleotides

[0357] In embodiments, a modified oligonucleotide comprises (e.g., consists of) a sequence of nucleosides having a gapmer motif, which is defined by two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer motif (the 5’ -wing, the gap, and the 3 ’ -wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap region (the 3 ’ -most nucleoside of the 5 wing and the 5 ’-most nucleoside of the 3 ’-wing) differ from the sugar moiety of the neighbouring gap nucleosides, thus defining the boundary between the wings and the gap region (i.e., the wing / gap junction). In embodiments, the sugar moieties within the gap are the same as one another. In embodiments, the gap region includes one or more nucleoside h aving a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In embodiments, the second nucleoside from the 5 ’-most gap nucleoside comprises a 2’ -OMe sugar moiety, and all other gap nucleosides comprise 2’ -deoxy sugar moieties. In embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In embodiments, the sugar motif of the 5'-wing differs from the sugar motif of the 3 '-wing (asymmetric gapmer). 201393-PCT01-NP P78290WO

[0358] In embodiments, the gapmer is a deoxy gapmer, i.e., a gapmer that comprises a deoxy region. In embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2 ’-deoxynucleosides and the nucleosides on the wing sides of each wing / gap junction are modified nucleosides. In embodiments, each nucleoside of the gap comprises a 2’ -P-D- deoxyribosyl sugar moiety. In embodiments, each nucleoside of each wing of a gapmer comprises a modified sugar moiety. In embodiments, at least one nucleoside of the gap of a gapmer comprises a modified sugar moiety. In embodiments, one nucleoside of the gap comprises a modified sugar moiety and each remaining nucleoside of the gap comprises a 2’- deoxy sugar moiety. In embodiments, at least one, or exactly one, nucleoside of the gap of a gapmer comprises a 2’-OMe sugar moiety.

[0359] In embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap region are all modified. In such embodiments, some or all of the internucleoside linkages in the wings may be unmodified phosphodiester internucleoside linkages. In embodiments, the terminal intemucleoside linkages are modified. In embodiments, the sugar motif of a modified oligonucleotide is a gapmer, and the internucleoside linkage motif comprises at least one phosphodiester internucleoside linkage in at least one wing, wherein the at least one phosphodiester linkage is not a terminal internucleoside linkage, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. In certain such embodiments, all of the phosphorothioate linkages are stereorandom. In embodiments, all of the phosphorothioate linkages in the wings are (Sp) phosphorothioates, and the gap region comprises at least one Sp, Sp, Rp motif. In embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such intemucleoside linkage motifs.

[0360] In embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the gap region of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2’ -deoxyribosyl moiety. In embodiments, the modified nucleobase is selected from: a 2 -thiopyrimidine and a 5- propynylpyrimidine.

[0361] In embodiments, the above modifications (sugar, nucleobase, and / or intemucleoside linkage) are incorporated into a modified oligonucleotide. A modified oligonucleotide may be characterized by its modification motifs and overall lengths. In embodiments, the 201393-PCT01-NP P78290WO modifications are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the intemucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the intemucleoside linkages of the gap region of the sugar motif. Likewise, such sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Unless otherwise indicated, all modifications are independent of nucleobase sequence.

[0362] Populations of modified oligonucleotides

[0363] Populations of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be stereorandom populations or chirally enriched populations, or they can exist in a mixture of stereoisomers . All of the chiral centres of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral centre is not stereorandom in the modified oligonucleotides of the population. In a population having a mixture of stereoisomers, the relative amount of each stereoisomer at each position can be approximately equal, but it will typically be the case that one stereoisomer is present in a larger amount at each position (e.g., due to the influence of reaction parameters such as the 5 ’-nucleotide, the incoming amidite, and / or the coupling conditions). In embodiments, the modified oligonucleotides of a chirally enriched population are enriched for P -D ribosyl sugar moieties, and all of the phosphoro thioate internucleoside linkages are stereorandom. In embodiments, the modified oligonucleotides of a chirally enriched population are enriched for P-D ribosyl sugar moieties, and comprise at least one stereoenriched phosphorothioate internucleoside linkage (i.e., being in a particular stereochemical configuration).

[0364] Conjugates, linkers and terminal groups

[0365] The oligomeric compound produced by the process of the disclosure may consist of an oligonucleotide (modified or unmodified) and one or more conjugate groups and / or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and / or at any internal position. In embodiments, conjugate groups are attached to the 2' -position of a nucleoside of a modified oligonucleotide. 201393-PCT01-NP P78290WO

[0366] In embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3 ’ and / or 5 ’-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3’ -end of oligonucleotides. In embodiments, conjugate groups are attached near, e.g., one or two nucleobases from, the 3 ’-end of oligonucleotides. In embodiments, conjugate groups (or terminal groups) are attached at the 5 ’-end of oligonucleotides. In embodiments, conjugate groups are attached near, e.g., one or two nucleobases from, the 5 ’ -end of oligonucleotides. In embodiments, conjugate groups (or terminal groups) are attached at the 3 ’ -terminal nucleoside of an oligonucleotide. In embodiments, conjugate groups (or terminal groups) are attached at the 5 ’-terminal nucleoside of an oligonucleotide. The conjugate group(s) can modify one or more properties of the oligonucleotide, including but not limited to pharmacodynamic properties, pharmacokinetic properties, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.

[0367] Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

[0368] Conjugate groups may be attached to oligonucleotides through a linker (i.e., a conjugate linker). In other embodiments, the conjugate group is attached directly to the oligonucleotide, e.g. through a single covalent bond. In embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.

[0369] In embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group is cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In embodiments, the cleavable moiety is a cleavable bond. In embodiments, the cleavable moiety is a group of atoms comprising at least one cleavable bond. In embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In embodiments, the cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In 201393-PCT01-NP P78290WO embodiments, the cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

[0370] In embodiments, the cleavable bond is selected from: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In embodiments, the cleavable bond is one or both of the esters of a phosphodiester. In embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.

[0371] In embodiments, the cleavable moiety comprises (e.g., consists of) one or more linker- nucleosides. In certain such embodiments, the one or more linker-nucleosides are linked to one another and / or to the remainder of the oligomeric compound through cleavable bonds. In embodiments, such cleavable bonds are unmodified phosphodiester bonds. In embodiments, the cleavable moiety is a 2'-deoxynucleoside that is attached to either the 3' or 5 ’ -terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage, and is covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In such embodiments, the cleavable moiety may be 2'- deoxyadenosine.

[0372] In embodiments, oligomeric compounds comprise one or more terminal groups. In certain such embodiments, oligomeric compounds comprise a stabilized 5’ -phosphate. Stabilized 5’- phosphates include, but are not limited to 5 ’-phosphonates, including, but not limited to 5’- vinylphosphonates. In embodiments, terminal groups comprise one or more abasic nucleosides and / or inverted nucleosides. In embodiments, terminal groups comprise one or more 2’-linked nucleosides. In certain such embodiments, the 2’ -linked nucleoside is an abasic nucleoside.

[0373] Length

[0374] In embodiments, the oligonucleotide (or salt thereof) comprises between 2 and 100 nucleobases, e.g. between 3 and 60, between 4 and 40, between 5 and 30, or between 8 and 20 nucleobases. In embodiments, the oligonucleotide is a 2-mer, a 3-mer, a 4-mer, a 5-mer, a 6-mer, a 7-mer, an 8-mer, a 9-mer, a 10-mer, an 11-mer, a 12-mer, a 13-mer, a 14-mer, a 15- mer, a 16-mer, a 17-mer, an 18-mer, a 19-mer, a 20-mer, a 21-mer, a 22-mer, a 23-mer, a 24- mer, a 25-mer, a 26-mer, a 27-mer, a 28-mer, a 29-mer, or a 30-mer oligonucleotide. 201393-PCT01-NP P78290WO

[0375] Salts

[0376] The oligonucleotide may be obtained in the form of a free acid or base (or a zwitterion), or it may exist as an addition salt with one or more suitable acids or bases. In embodiments, the oligonucleotide is a basic compound which is provided as a pharmaceutically acceptable acid addition salt. In embodiments, the acid is selected from one or more of HC1, TFA, citric acid, and formic acid. In embodiments, the oligonucleotide is an acidic compound which is provided as a pharmaceutically acceptable base addition salt. In embodiments, the base is selected from one or more of EtsN, zPioNH, zPi'N, DIPEA, imidazole, pyridine, and N- methylpiperidine. In embodiments, the oligonucleotide is provided as an addition salt with a suitable base, for example EtaN. In embodiments, the oligonucleotide is provided as a sodium salt. Methods for forming salts are described herein and are also known in the art (see, e.g., Berge et al., J. Pharm Sci. (1977) 66:1-19).

[0377] Other features

[0378] Although sequences may be designated as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications (e.g., as described above). One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’ -OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2’ -OH in place of one 2’-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of a uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ ATCGATCG” encompasses any oligomeric compoundshaving such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified nucleobases, such as “AmCGAUCG,” whereinmC indicates a cytosine base comprising a methyl group at the 5 -position. 201393-PCT01-NP P78290WO

[0379] Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric centre and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that maybe defined, in terms of absolute stereochemistry, as (R) or (S), as a or P such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations are typically intended to represent the indicated stereochemistry. Compounds provided herein that are drawn or described with undefined stereochemistry are intended to include all possible isomers, including their stereorandom and optically pure forms, unless specified otherwise. Likewise, tautomeric forms of the compounds herein are also included unless otherwise indicated. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.

[0380] The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the m hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to:2H or3H in place of1H,13C or14C in place of12C,15N in place of14N,17O or18O in place of16O, and33S,34S,35S, or36S in place of32S. In embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.

[0381] Reaction yield and purity of the reaction products

[0382] It will be appreciated that the product of the process of the disclosure (e.g., the direct product of the final step of the chain extension cycle) may comprise a minor portion of oligonucleotides other than the desired (i.e., intended) oligonucleotide, as well as one or more other species such as reagents, reactants, impurities, etc. The product of the process of the disclosure may be in the form of a solution, e.g. containing some or all of the solvent(s) and / or other reagents from the steps in the process. Alternatively, the product of the process of the disclosure may be in solid form, e.g. in the form of a powder such as a lyophilized powder, which may be obtained by drying (e.g., freeze drying) the reaction mixture which results from the final synthetic step of the process. 201393-PCT01-NP P78290WO

[0383] The process of the disclosure, which uses NPPOC as a protecting group, has advantages over conventional processes (e.g., using DMT as a protecting group) in that fewer purification steps may be used, thereby reducing the amount of product lost during the process. The deprotection conditions used to remove the NPPOC protecting group are very mild, which can lead to fewer unwanted side reactions as compared to the harsh acidic conditions that have been used previously for, e.g., DMT deprotection. The product of the deprotection step of the process of the disclosure can thus contain reduced levels of unwanted side products and / or reaction impurities, improving the homogeneity and pharmaceutical acceptability of the product, while further reducing the need for additional purification steps. In addition, the deprotection step can proceed in high yield, thereby providing an oligonucleotide product having a high yield and / or purity.

[0384] In embodiments, the process of the disclosure provides an oligonucleotide product (comprising the desired oligonucleotide or salt thereof) having a low levels of impurities. In embodiments, the oligonucleotide product obtained by the process has low levels of phosphomannose isomerase inhibitors. In embodiments, said phosphomannose isomerase inhibitors comprise one or more of the trityl cation, monomethoxy trityl cation and dimethoxy trityl cation, and derivatives thereof such as, e.g., adducts with scavenging salts. Other impurities which may be reduced or avoided by the use of NPPOC as a protecting group in accordance with the present disclosure include, e.g., acid-mediated depurination impurities, products having too few nucleotides (e.g., n-1 impurities), products having too many nucleotides (e.g., n+1 impurities), products having improper backbone modifications (e.g., phosphate impurities in phosphoro thioate oligonucleotides, and dithioate impurities), 3 ’- and 5 ’-terminal phosphorothioates, CNET impurities, deaminated nucleobases, dehydro abasic nucleotides, guanine replacements (e.g., ADP and IDP impurities), TEA adducts, 3’- amino-2’ -deoxyribose impurities, and DMT C-phosphonate impurities.

[0385] In embodiments, the process of the disclosure provides an oligonucleotide product (e.g., a crude oligonucleotide product which is the result of the final synthetic step in the process) which does not comprise more than about0.01%, about0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, or about 10% of one or more phosphomannose isomerase inhibitors, for example trityl cation, monomethoxy trityl cation or dimethoxy trityl cation, or derivatives thereof. In embodiments, the one or more phosphomannose isomerase inhibitors are dimethoxy trityl cation. In embodiments, the one or more phosphomannose isomerase inhibitors are dimethoxy trityl cation derivatives. In embodiments, the one or more 201393-PCT01-NP P78290WO phosphomannose isomerase inhibitors are derivatives of dimethoxy trityl cation and one or more scavenging salts. Examples of scavenging salts include 2-mercaptoethane-l -sulfonic acid triethylamine salt and 2-mercaptoethane-l -sulfonic acid sodium salt.

[0386] In embodiments, the process of the invention provides an oligonucleotide product (comprising the desired oligonucleotide or the salt thereof) wherein the oligonucleotides of the oligonucleotide product have a low level of oligonucleotide base modification. In embodiments, the total oligonucleotides produced by the process have a low level of depurinated bases. In embodiments, the total oligonucleotides produced by the process do not comprise more than 5 mol% of oligonucleotides in which one or more nucleotides are depurinated. In embodiments, the total oligonucleotides produced by the process do not comprise more than 1 mol% of oligonucleotides in which one or more nucleotides are depurinated, e.g. not more than 0.8%, 0.6% or 0.4% of oligonucleotides in which one or more nucleotides are depurinated.

[0387] In embodiments, the process of the invention provides the desired oligonucleotide or salt thereof in high yield. In embodiments, the desired oligonucleotide or salt thereof represents at least 50% of the total oligonucleotides produced by the process, e.g. at least 55%, at least 60%, atleast 65%, at least70%, atleast75%, atleast 80%, atleast 85%, atleast 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% of the total oligonucleotides produced by the process. In embodiments, the desired oligonucleotide or salt thereof represents about 100% of the total oligonucleotides produced by the process. In embodiments, the amount of the desired oligonucleotide or salt thereof is determined by LCMS.

[0388] A person of ordinary skill in the art will be aware that there are different ways to express the purity of the desired oligonucleotide product of the process of the invention. One such method is “UV purity” as defined herein. In embodiments, the process of the invention provides an oligonucleotide product (comprising the desired oligonucleotide or the salt thereof) wherein the oligonucleotides of the oligonucleotide product have a UV purity of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 97%, at least 98%, or atleast 99%. In embodiments, the UV purity is at least about 80% . Another method of expressing purity is “full length product (FLP) purity” as defined herein. In embodiments, the process of the 201393-PCT01-NP P78290WO invention provides an oligonucleotide product (comprising the desired oligonucleotide or the salt thereof) wherein the oligonucleotides of the oligonucleotide product have a full length product (FLP) purity of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 97%, at least 98%, or at least 99%. In embodiments, the full length product (FLP) purity is at least about 75%. In embodiments, the process of the invention provides an oligonucleotide product (comprising the desired oligonucleotide or the salt thereof) wherein the oligonucleotides of the oligonucleotide product have both a UV purity and a full length product (FLP) purity of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 97%, at least 98%, or atleast 99%. In embodiments, both the UV purity and the full length product (FLP) purity are at least about 75%. In embodiments, the UV purity is at least about 80% and the full length product (FLP) purity is at least about 75%.

[0389] Synthesizer

[0390] In embodiments, one or more steps are conducted in a synthesizer. In an embodiment, the synthesizer is a continuous flow synthesizer which comprises: a reaction vessel; at least one flow path for temperature control, pressure control, and / or purification (e.g., diafiltration) of the reaction mixture; and at least one flow path for irradiation of the reaction mixture (see FIG. 3a and FIG. 3b). In another embodiment, the synthesizer comprises a jacketed reaction vessel which can interface with a photoreactor and a diafiltration unit. In embodiments, the photoreactor and / or diafiltration unit are detachable.

[0391] Uses of NPPOC, protected oligonucleotides and solutions comprising the same

[0392] As described in detail above, the disclosure provides a LPOS process in which NPPOC is used as a protecting group. Thus, the disclosure also provides the use of NPPOC as a protecting group in liquid phase oligonucleotide synthesis.

[0393] It will be appreciated that various embodiments set out above in connection with the process of the disclosure can also be applied to the aspect in which NPPOC is used as a protecting group for LPOS. Thus, in embodiments NPPOC is used as a protecting group in a process of the disclosure (as described herein). 201393-PCT01-NP P78290WO

[0394] The disclosure also provides an NPPOC-protected oligonucleotide or salt thereof, wherein the oligonucleotide or salt thereof is in solution.

[0395] It will be appreciated that various embodiments set out above in connection with the process of the disclosure can also be applied to the aspect which provides an NPPOC-protected oligonucleotide or salt thereof in solution. In embodiments, the NPPOC-protected oligonucleotide or salt thereof is an NPPOC-protected oligonucleotide as described herein.

[0396] The disclosure further provides a solution comprising an NPPOC-protected oligonucleotide or salt thereof. The solution comprising an NPPOC-protected oligonucleotide or salt thereof may be used in organic synthesis, for example in LPOS (e.g., in a process of the disclosure as defined herein).

[0397] Having been generally described herein, the following non- limiting examples are provided to further illustrate this disclosure.

[0398] EXAMPLES

[0399] Experimental techniques and general synthetic procedures

[0400] ' H NMR Spectra at 400 and 500 MHz were performed on a Bruker Avance DRX-400 and Bruker Avance DPX-500 spectrometer, respectively, with the chemical shifts (5 in ppm) in the solvent dimethyl sulfoxide -de (DMSO-de) referenced at 2.5 ppm at the quoted temperatures. Coupling constants (J) are given in Hertz.

[0401] The liquid chromatography / mass spectra (LC / MS) were obtained on a UPLC Acquity Waters instrument, light scattering detector Sedere and SQD Waters mass spectrometer using UV detection DAD 210<l<400 nm and column Acquity UPLC CSH Cl 8 1.7 pm, dimension 2.1x30 mm, mobile phase H2O + 0.1% HCO2H / CH3CN + 0.1% HCO2H.

[0402] Reaction solutions were analyzed by HPLC. Conversions were calculated using HPLC by UV absorbance relative to an internal standard (biphenyl). Yields were calculated using HPLC by UV absorbance relative to an internal standard (biphenyl), corrected by relative response factors (RREs) of reaction products.

[0403] In the following examples, when the source of the starting products is not specified, it should be understood that said products are known compounds (e.g., commercially available compounds from suppliers such as Sigma- Aldrich). 201393-PCT01-NP P78290WO

[0404] General Procedure A - Nucleotide coupling, sulfurisation and irradiation

[0405] To an 8 mL screw top vial equipped with a magnetic stirrer bar was added 5'-NPPOC-dT-3'- CE-Phosphoramidite 1 (58.9 mg, 0.0907 mmol - see Scheme 1) and stavudine 2 (13.4 mg, 0.0598 mmol - see Scheme 1), and sealed. The vial was inerted with 3 x vacuum / N2-refill cycles, followedby addition anhydrous MeCN (1.5 mL) and heated at 30 °C over 5 minutes with stirring at 300 rpm. A solution of 4,5-dicyanoimidazole (14.0 mg, 0.119 mmol) in anhydrous MeCN (0.60 mL) was then added and stirred for 30 minutes. A solution of 3- Hydroxypropionitrile (25 pL, 0.366 mmol) in anhydrous MeCN (0.30 mL) was then charged, and the reaction mixture held at 30 °C for a further 10 minutes, followed by addition of a solution of phenyl-3H-l,2,4-dithiazol-3-one (17.8 mg, 0.0912 mmol) in anhydrous MeCN (0.60 mL). The reaction mixture was stirred for a further 15 minutes at 33 °C, then transferred to a glovebox and split in to 4 x 0.750 mL portions in separate 2 mL screw cap vials. Once sealed, the vials were transferred to a Hepatochem Lucent 360 (immersion well set to 30 °C) and irradiated for 2 hours with stirring at 600 rpm. Reaction mixtures were sampled by UPLC and conversions calculated relative to an internal standard (biphenyl or m- terphenyl). In the case of irradiation atm;LX254 nm, a quartz vial was used.

[0406] General Procedure B -preparative scale synthesis of Stavudine (4)

[0407] To a 3-necked 250 mL round-bottomed flask equipped with a magnetic stirrer bar and bag of molecular sieves was added stavudine (456 mg, 2.034 mmol) and 5'-NPPOC-dT-3'-CE- Phosphoramidite 4 (2.00 g, 3.08 mmol - see Scheme 1) mL. The flask was inerted with 3 x vacuum / N2-refill cycles, followedby addition of anhydrous MeCN (50 mL) and heated at 30 °C over 5 minutes with stirring at 600 rpm. A solution of 4,5-dicyanoimidazole (492 mg mg, 4.17 mmol) in anhydrous MeCN (20.0 mL) was then added and stirred for 30 minutes. A solution of 3 -Hydroxypropionitrile (820 pL, 12.0 mmol) in anhydrous MeCN (10 mL) was then charged, and the reaction mixture held at 30 °C for a further 10 minutes, followed by addition of a solution of phenyl- 3H-1, 2, 4-dithiazol-3-one (595 mg, 3.02 mmol) in anhydrous MeCN (0.60 mL). The reaction mixture was stirred for a further 15 minutes at 33 °C. The reaction mixture was then evaporated under reduced pressure, re -dissolved in CH2Q2 (200 mL), and washed with deionized water (2 x 100 mL), dried over MgSCL, before evaporating under reduced pressure. The crude product was purified using column chromatography to yield a white crystalline solid (1.16 g, 1.44 mmol, 71% yield). 'H NMR (500 MHz, DMSO, 27°C) 11.37 - 11.45 (2H, m), 7.86- 7.92(1H, m), 7.71 - 7.79 (2H, m), 7.52 - 7.58 (1H, m), 201393-PCT01-NP P78290WO

[0408] 7.45 - 7.51 (1 H, m), 7.28 - 7.34 (1H, m), 6.87 - 6.92 (1H, m), 6.46 - 6.52 (1H, m), 6.19 - 6.26 (1H, m), 6.04 - 6.11 (1H, m), 5.09 - 5.15 (1H, m), 5.01 - 5.08 (1H, m), 4.21 - 4.43 (9H, m), 3.52 - 3.61 (1H, m), 3.23 (1H, s), 2.94 - 3.03 (2H, m), 2.41 - 2.52 (2H, m), 1.85 (3H, t), 1.78 (3H, dt), 1.35 (3H, d). m / z (ESI+) [M+H+] = 805.2.

[0409] General Procedure C - Sparging

[0410] Batch photochemistry reactions run in a 2 mL screw-cap vial were sealed and sparged with Nitrogen for a minimum of 10 minutes prior to irradiation.

[0411] Flow photochemistry reactions in which the input vessel was a suba-sealed conical flask, were sparged with Nitrogen for a minimum of 20 minutes prior to irradiation.

[0412] General Procedure D - Batch irradiation

[0413] 5'-NPPOC-dT-3'-CE-P(V) - Stavudine 4 (0.015 mmol - see Scheme 1) was weighed in to a 2 mL screw-cap vial equipped with a magnetic stirrer bar (NMI 0.2-3.0 equiv. also added at this point where applicable), followed by addition of a 20 mM solution of biphenyl in anhydrous MeCN (0.75 mL). The reaction was then sealed and subjected to the general sparge procedure (This was followedby non-NMI base addition (0.2-3 equiv) through an airtight Hamilton syringe where applicable), followed by transfer to a Hepatochem PhotoRedoxBox and irradiated for 1 hour with stirring at 600 rpm and fan cooling with the LED at full power. The reaction was sampled and yield calculated by UPLC relative to the internal standard.

[0414] General Procedure E - flow chemistry procedure

[0415] 5'-NPPOC-dT-3'-CE-P(V) - Stavudine (0.200-0.800 mmol) was weighed in to a 50 mL conical flask with a quick-fit neck, followedby addition of a 20 mM solution of biphenyl in MeCN (20 mL) (NMI 0.2-3.0 equiv. also added at this point where applicable). The reaction was then sealed and subjected to the general sparge procedure (This was followed by diisopropylamine or morpholine addition (0.2-3 equiv) through an air-tight Hamilton syringe where applicable). The reaction mixture was subjected to the flow conditions in a Snapdragon Iris Lab with a 11.2 mL flow cell (10-25 °C, tr1 - 5 minutes, km;LX385 nm), sampled upon reaching steady-state and yield calculated by UPLC relative to the internal standard.

[0416] General Procedure F - Diafiltration 201393-PCT01-NP P78290WO

[0417] Membranes used were polybenzimidazole (PBI, 17% weight) at a surface area of 33 cm2. Prior to use, membranes were subjected to at least 200 mL of washing via diafiltration with acetonitrile (MeCN). Unless otherwise stated, diafiltration of reaction media was performed into MeCN at a constant pressure of 19-21 bar and at ambient temperature (20-28 °C). The process involves gradual replacement of the original solvent with MeCN through continuous addition, ensuring efficient purging of low-molecular-weight impurities while maintaining oligonucleotide integrity. The diafiltration was monitored until the desired solvent exchange was achieved, and measuredin diavolumes (DV) relative to the fixed volume of retentate . After the desired diafiltration was complete, the reaction mixture was flushed out of the membrane loop with anhydrous MeCN (25 mL), and the mixture stored overnight in a freezer and dried with AldraSORB™ water trapping packets. The membrane loop was washed with anhydrous MeCN (at least 400 mL) between cycles.

[0418] General Procedure G - Coupling and Sulfurisation

[0419] Phosphoramidite solutions were prepared in anhydrous MeCN to a concentration of 0.4 M and dried fora minimum of 15 hours and maximum of 72 hours with AldraSORB™ water trapping packets prior to use. Dicyanoimidazole (DO, 0.9 M) and 3 -phenyl l,2,4-dithiazoline-5-one (POS, 0.7 M) were prepared in anhydrous MeCN and dried for a minimum of 15 hours with AldraSORB™ water trapping packets prior to use. Coupling reactions with constrained ethyl (cEt) amidites were performedby first pre -activating the amidite solution (2.0 equiv. per hub arm) with DCI (12 equiv. per hub arm) in a separate inerted round -bottomed flask for 20 minutes prior to addition to the on-hub solution in a jacketed vessel. Deoxy amidites (1.5 equiv. per hub arm) did not require pre -activation, and were added directly to a stirring solution of on-hub oligonucleotide and DCI (4.0 equiv. per hub arm). All coupling reactions were held for 12 minutes followed by quenching with cyanoethanol (3.0 equiv. per arm), held for an additional 5 minutes, and sulfurized with 3-phenyl 1, 2, 4-dithiazo line-5 -one (4.0 equiv. per arm).

[0420] General Procedure H - Deprotection

[0421] All reactions were performed on an Iris Lab photoreactor (irradiated volume = 9.2 mL) equipped with a 385 nm LED array under plug flow conditions at between 20 and 25 °C, with a backpressure of 2 bar, flow rate of between 1.6 and 1.7 mL / min and current of 2.8 A. Prior to use, the flow cell was flushed with a dry and degassed solution of anhydrous MeCN (at least 100 mL). To reaction solutions was added A- methylimidazole (64 equiv.) followedby sparging 201393-PCT01-NP P78290WO with nitrogen for at least 30 minutes prior to irradiation. Upon complete transfer of the bulk reaction mixture into the flow system via the pump, the flow cell was subsequently flushed with an additional 20 mL of anhydrous degassed MeCN. Reaction mixtures were sampled by UPLC after deprotection. General Procedure I - Cleavage and Deprotection

[0422] A sample of on-hub oligo (500 pL) was placed in a 20 mL boiling tube, followed by addition of diethylamine (200 pL) and ammonium hydroxide (10 mL). The tube was sealed and heated at 55 °C for 10 hours at 400 rpm in a shaker.

[0423] General Analytical Method A 201393-PCT01-NP P78290WO

[0424] UV purity was calculated by integrating the area under the main chromatographic peak detected at 260 nm by ion-pair HPLC.

[0425] Full length product (FLP) Purity: The full length product purity was calculated by identifying the species present (desired product and impurities) under the main chromatographic HPLC peak, followed by quantification through integration of the extracted ion chromatograms (EIC) of the identified species, and expressing it as a calibration -corrected percentage of the total oligonucleotide ion signal within the main chromatographic (HPLC) peak. 201393-PCT01-NP P78290WO

[0426] General Analytical Method B 201393-PCT01-NP P78290WO

[0427] UV purity was calculated by integrating the area under the main chromatographic peak detected at 260 nm by ion-pair HPLC.

[0428] Full length product (FLP) Purity: The full length product purity was calculated by identifying the species present (desired product and impurities) under the main chromatographic HPLC peak, followed by quantification through integration of the extracted ion chromatograms (EIC) of the identified species, and expressing it as a calibration -corrected percentage of the total oligonucleotide ion signal within the main chromatographic (HPLC) peak. Example 1 - Telescoped Nucleotide coupling, sulfurisation and deprotection process using NPPOC as a protecting group

[0429] Scheme 1 below shows a model reaction which was used for investigating process parameters of the LPOS reaction cycle of the disclosure.

[0430] 201393-PCT01-NP P78290WO

[0431] SCHEME 1

[0432] STEP 1 is a coupling step in which Compound 1 (an NPPOC-protected deoxyribonucleoside phosphoramidite) is reacted with Compound 2 (a model deoxyribonucleoside analogue) in the presence of DO. STEP 2 is a sulfuration step, in which Compound 3 is reacted with POS to form Compound 4. In STEP 3, Compound 4 is deprotected by irradiation with light in MeCN. STEP 3 may be carried out in the presence of a base. 201393-PCT01-NP P78290WO

[0433] Example 1A: The effect of irradiation wavelength on the overall yield

[0434] The impact of wavelength on the deprotection of Compound 4 was investigated using light sources with different maximum wavelengths. The process was carried out as described in Scheme 1 and General Procedure A. The results are presented in Table 1, below:

[0435] Table 1:

[0436] The NPPOC-protected nucleotides were stable under the coupling conditions used in Step 1 and also in the sulfurisation conditions used to convert the phosphite (III) triester to a phorphorus (V) species in Step 2. The NPPOC protecting group was readily removed by irradiation in a solvent with high conversion. The highest conversion over the 3 steps was achieved using light sources with maximum wavelengths of around 365 nm to 380 nm. Further investigations were typically carried out using a wavelength of 380 nm to decrease the risk of any impurity formation that might occur when irradiating at higher energies (i.e., at shorter wavelengths).

[0437] Example IB: The effect of base on the deprotection step

[0438] The impact of the presence of base in the deprotection step (Step 3) was investigated. In a first test, and according to general procedure D, Compound 4 (20 mM) was deprotected by irradiation in the presence of various different bases in MeCN as solvent. The reaction mixtures were irradiated for 60 minutes using a light source with Xmax= 380 nm. Reactions were performed without (Table 2) and with (Table 3) a nitrogen sparge prior to irradiation: 201393-PCT01-NP P78290WO

[0439] Table 2: Without N2 sparge before irradiation

[0440] Table 3: With N2 sparge before irradiation Bases were investigated that have different pKavalues in MeCN, namely: diisopropylamine (pKaH of 18.8); diisopropylethylamine (pKaH of 18.8); triethylamine (pKaH of 18.7); morpholine (pKaH of 16.1); 1 -methylimidazole (pKaH of 15.5); pyridine (pKaH of 12.5); and 3- methylpyrazole (pKaH of 9.1). The yield of the deprotection step decreases with increasing equivalents of iPr2NH (see Table 2), whereas it increases with increasing equivalents of NMI (see Table 3). The highest yields of Compound 5 were achieved using zPr2NH, NMI, or morpholine as the base with a N2 sparge prior to irradiation.

[0441] Example 1C: The effect of base on the deprotection step in a small scale batch process

[0442] A small scale batch process was setup to assess the impact of base type and concentration on the yield of the deprotected material. According to General Procedure D, Compound 4 (20 mM) in MeCN was deprotected by irradiation for 60 minutes using a light source with Xmax= 390 nm in the presence of a base. The reactions were sparged with N2 before irradiation. The results are presented in Table 4, below: 201393-PCT01-NP P78290WO

[0443] Table 4:

[0444] The yield in the deprotection step was close to quantitative (>98%) using zPr2NH and morpholine as base (0.2 equivalents) and also using 1 -methylimidazole as base (3.0 equivalents). Use of 0.2 or 1.1 equivalents of 1 -methylimidazole as base yielded around 80- 90% of deprotected material.

[0445] Example ID: The effect of residence time on the deprotection step

[0446] A 9.2 mL flow cell was used to assess the impact of residence time on the yield of the deprotection step. According to General Procedure E Compound 4 (10 mM or 40 mM) in MeCN was deprotected by irradiation using a light source with Xmax= 385 nm in the presence of a base. Reactions were performed at 25 °C unless stated otherwise. The residence time was varied as shown in Table 5, below:

[0447] Table 5:

[0448] '^Performed at 40 mM concentration of Compound 4. **Performed at 10-20 °C.

[0449] The deprotection reactions using zPr2NH (0.2 equivalents for 1 or 5 minutes) and 1- methylimidazole (3.0 equivalents for 5 minutes) afforded close to quantitative yield of the product. As a milder base, NMI can be used to decrease the likelihood of forming impurities such as, e.g., products resulting from cyanoethanol deprotection. 201393-PCT01-NP P78290WO

[0450] Example 2 - One pot deprotection and chain extension of dinucleotide model compound

[0451] Scheme 2 below shows the conversion of Compound 4 (see Scheme 1) into Compound 6 in a one pot process.

[0452] SCHEME 2

[0453] In STEP 1, Compound 4 (20 mM in MeCN, 0.2 eq. z‘Pr2NH with N2sparge prior to addition of the base) was irradiated atm;LX= 380 nm to remove the NPPOC protecting group. The resulting compound was then reacted in STEP 2 with Compound 1 in the presence of DO to form a protected trinucleotide intermediate. That intermediate was then treated in STEP 3 with POS (3 -phenyl l,2,4-dithiazoline-5-one) to convert it into Compound 6. The reaction mixture was transferred to an 8 mL vial prior to the coupling step to allow space for reagents to be added as solutions (see General procedures A and B above). These steps were carried out without any intervening purification or diafiltration steps and proceeded with quantitative conversion. Example 3 - Synthesis of Compound 7 using NPPOC as a protecting group

[0454] This Example describes the synthesis of a 5-mer oligonucleotide (Compound 7) using NPPOC as a protecting group. Compound 7 is shown below: 201393-PCT01-NP P78290WO

[0455] Loading

[0456] Scheme 3 below shows a loading step in which Compound 8 (a carrier moiety) is coupled with Compound 9 (an NPPOC-protected nucleobase-containing moiety comprising a succinate linker), in a loading step, to form Compound 10. As will be appreciated, Compound 9 may be prepared by protecting a nucleotide, cEt(MeC), with NPPOC, followed by coupling with succinic acid (a bifunctional linker).

[0457] SCHEME 3

[0458] To a 250 mL round-bottomed flask (A) was added Compound 9 (NPPOC cEt(MeC) succinate, 4.76 g, 6.65 mmol) followed by TBTU (2.34 g, 7.14 mmol). The flask was equipped with a stirrer bar and placed under an atmosphere of nitrogen. Anhydrous acetonitrile (100 mL) was added to the flask until near-complete dissolution was observed. The mixture in flask A was stirred at 800 rpm at room temperature

[0459] To a 250 mL round-bottomed flask (B) was added Compound 8 (sarcosine terminated 4-arm 10 kDA star PEG, 10.3 g, 1.10 mmol). The flask was equipped with a stirrer bar and placed under an atmosphere of nitrogen, followed by addition of anhydrous acetonitrile (40 mL). The mixture was stirred at 500 rpm until complete dissolution was observed. N- ethyldiisopropylamine (1.60 ml, 9.0 mmol) was added to flask B and the mixture stirred for 5 minutes at room temperature. 201393-PCT01-NP P78290WO

[0460] Flask B was added to flask A via a syringe, and the reaction mixture was stirred at 500 rpm at room temperature for 2 hours. The mixture was then quenched with hexylamine (1.20 mL, 9.1 mmol) and transferred to a 150 mL Duran flask equipped with a stirrer bar. A UPLC sample was taken.

[0461] The crude reaction mixture was subjected to diafiltration (according to General Procedure F) at approximately 40 mM for 6 diavolumes. UPLC samples were taken of the retentate and permeate during the diafiltration to monitor the purging of off-hub impurities, i.e. impurities which do not comprise the carrier moiety.

[0462] Chain Extension Cycles

[0463] The following reaction unit operations were then performed on Compound 10 in accordance with the above General Procedures in the sequence of:

[0464] 1 : Deprotection (General Procedure H), coupling (NPPOC cEt(G)iBu) followed by sulfurisation (General Procedure G), and diafiltration (6 DV) (General Procedure F).

[0465] 2: Deprotection (General Procedure H), coupling (NPPOC cEt(G)iBu) followed by sulfurisation (General Procedure G), and diafiltration (6 DV) (General Procedure F).

[0466] 3: Deprotection (General Procedure H), coupling (NPPOC dT) followed by sulfurisation (General Procedure G), and diafiltration (6 DV) (General Procedure F).

[0467] 4: Deprotection (General Procedure H), coupling (NPPOC dG(z‘Bu)) followed by sulfurisation (General Procedure G), diafiltration (6 DV) (General Procedure F), and deprotection (General Procedure H).

[0468] Cleavage and Deprotection

[0469] The resulting chain -extended product, Compound 11, was then subjected to cleavage and deprotection conditions to cleave the carrier and remove other protecting groups, to afford Compound 7, as shown in Scheme 4 below. 201393-PCT01-NP P78290WO

[0470] SCHEME 4

[0471] A 500 uL sample of product solution of Compound 11 was subjected to the standard cleavage and deprotection conditions as described in General Procedure I to yield the off-hub oligonucleotide product, Compound 7. Compound 7 was then analysed according to the method describedin General Analytical Method A. UV purity = 57.35%, full-length product purity = 42.11 %.

[0472] The process described above thus does not require additional purification steps to remove byproducts resulting from deprotection between cycles such as, e.g., diafiltration. Example 4 - Synthesis of Compound 12 using NPPOC as a protecting group

[0473] This Example describes the synthesis of a 5-mer oligonucleotide (Compound 12) using NPPOC as a protecting group. Compound 12 is shown below: 201393-PCT01-NP P78290WO

[0474] Loading

[0475] Scheme 5 below shows a loading step in which Compound 8 (a carrier moiety) is coupled with Compound 13 (a DMT-protectednucleobase-containing moiety comprising a succinate linker), in a loading step, to form Compound 14. As will be appreciated, Compound 13 may be prepared by protecting a nucleotide, 2'0-Me-A(Bz), with DMT, followed by coupling with succinic acid (a bifunctional linker).

[0476] SCHEME 5

[0477] To a 150 mL round-bottomed flask (A) was added Compound 13 (DMT-2'O-Me-A(Bz)-3'-O- succinate TEA salt, 1.5 g, 1.7 mmol) followed by TBTU (0.73 g, 2.20 mmol). The flask was equipped with a stirrer bar and placed under an atmosphereof Nitrogen. Anhydrous acetonitrile (20 mL) was added to the flask until dissolution was observed. The mixture in flask A was stirred at 600 rpm at room temperature.

[0478] To a 150 mL round-bottomed flask (B) was added Compound 8 (sarcosine terminated 4-arm 10 kDA star PEG, 2.58 g, 0.276 mmol). The flask was equipped with a stirrer bar and placed under an atmosphereof Nitrogen, followed by addition of anhydrous acetonitrile (40 mL). The mixture was stirred at 500 rpm until complete dissolution was observed. N- ethyldiisopropylamine (0.40 mL, 2.3 mmol) was added to flask B and the mixture stirred for 5 minutes at room temperature.

[0479] Elask A was added to flask B via a syringe, and the reaction mixture was stirred at 500 rpm at room temperature for 2 hours. The mixture was then quenched with hexylamine (0.30 mL, 2.0 mmol) and transferred to a 150 mL Duran flask equipped with a stirrer bar. A UPLC sample was taken.

[0480] The crude reaction mixture was subjected to diafiltration (according to General Procedure F) for 6 diavolumes at approximately 20 mM. UPLC samples were taken of the retentate and 201393-PCT01-NP P78290WO permeate during the diafiltration to monitor the purging of off-hub impurities, i.e. impurities which do not comprise the carrier moiety.

[0481] DMT Deprotection

[0482] Scheme 6 below shows a DMT deprotection step in which the DMT protecting group is removed from Compound 14 to afford Compound 15.

[0483] (14) (15)

[0484] SCHEME 6

[0485] The on-hub reaction solution of Compound 14 was added to a pre-inerted 250 mL jacketed vessel at 33 °C, followed by addition of 1 -dodecanethiol (DDT, 1.40 mL, 5.7 mmol). The reaction was held for 5 minutes with stirring at 600 rpm. Trifluoracetic acid (2.75 mL, 36.4 mmol) was then added and the reaction held for 40 minutes at 33 °C. 2 -Picoline (5.5 mL, 56 mmol) was added to the reactor and held for a further 5 minutes, followed by transfer into a 100 mL Duran flask equipped with a stirrer bar.

[0486] The crude reaction mixture was subjected to diafiltration under the standard conditions (according to General Procedure F) for 6 diavolumes at approximately 20 mM. UPLC samples were taken of the retentate and permeate during the diafiltration to monitor the purging of off- hub impurities, i.e. impurities which do not comprise the carrier moiety.

[0487] Chain Extension Cycles

[0488] The following reaction unit operations were then performed on Compound 15 in accordance with the above General Procedures in the sequence of:

[0489] 1 : Coupling (NPPOC dC(Ac)) followed by sulfurisation (General Procedure G), and diafiltration (6 DV) (General Procedure F). 201393-PCT01-NP P78290WO

[0490] 2: Deprotection (General Procedure H), coupling (NPPOC dA(Bz)) followed by sulfurisation (General Procedure G), and diafiltration (6 DV) (General Procedure F).

[0491] 3: Deprotection (General Procedure H), coupling (NPPOC dC(Ac)) followed by sulfurisation (General Procedure G), and diafiltration (6 DV) (General Procedure F).

[0492] 4: Deprotection (General Procedure H), coupling (NPPOC dA(Bz)) followed by sulfurisation (General Procedure G), diafiltration (6 DV) (General Procedure F), and deprotection (General Procedure H).

[0493] Cleavage and Deprotection

[0494] The resulting chain -extended product, Compound 16, was then subjected to cleavage and deprotection conditions to cleave the carrier and remove other protecting groups, to afford Compound 12, as shown in Scheme 7 below.

[0495] Scheme 7

[0496] A 500 pL sample of product solution of Compound 16 was subjected to the standard cleavage and deprotection conditions as described in General Procedure I to yield the off-hub oligonucleotide product, Compound 12. Compound 12 was then analysed according to the method describedin General Analytical Method A. UV purity = 91.77%, full-length product purity = 88.31 %.

[0497] Example 5 - Synthesis of Compounds 17, 18, and 19, using NPPOC as a protecting group

[0498] Three further oligonucleotides were synthesised from Compound 12 of Example 4 using NPPOC as a protecting group. Additional chain extension, deprotection, diafiltration and 201393-PCT01-NP P78290WO cleavage and deprotection steps were followed as described above to generate Compounds 17, 18, and 19. These three compounds were analysed according to the method described in General Analytical Method B. The results are shown in Table 6, below:

[0499] Table 6:

[0500] Example 6 - Synthesis of Compounds 20 and 21 using NPPOC as a protecting group

[0501] Two further oligonucleotides were synthesised using NPPOC as a protecting group - Compounds 20 and 21. The structure of Compound 20 (SEQ ID NO: 1) is shown below: 201393-PCT01-NP P78290WO

[0502] An siRNA fragment of the sequence 5’-fAo-mUo-fAo-mUo-fGo-mUs-fAs-mG-3’ (with a full length product purity of 78.7%) was extended using the additional chain extension, deprotection, diafiltration and cleavage and deprotection steps as described above to generate Compounds 20 and 21. These two compounds were analysed according to the method described in General Analytical Method B. The results are shown in Table 7, below:

[0503] Table 7:

[0504] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0505] In addition, where features or aspects are describedin terms of Markush groups, those skilled in the art will recognize that such features or aspects are also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0506] All publications, patent applications, patents, and other references mentioned herein arc expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

201393-PCT01-NP P78290WOCLAIMS1. A process for synthezising an oligonucleotide or a salt thereof in liquid phase, wherein 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) is used as a protecting group.

2. The process of claim 1, wherein NPPOC is used as a 5 ’-hydroxyl protecting group.

3. The process of claim 1 or claim 2 comprising a deprotection step, wherein an NPPOC-protected oligonucleotide or an NPPOC-protected precursor thereto (e.g., an NPPOC-protected growing oligonucleotide) is irradiated in solution to deprotect the oligonucleotide or precursor thereto (either in part or in full) .

4. The process of claim 3, wherein the irradiation results in at least about 90% deprotection, e.g. at least about 95%, at least about 99%, or about 100% deprotection.

5. The process of claim 3 or claim 4, wherein the irradiation is performed with light having a wavelength (e.g., am;«) of about 350 nm to about 400 nm.

6. The process of any one of claims 3 to 5, wherein the irradiation is performed in the presence of a base, e.g. wherein the base is selected from one or more of zPioNH, 1- methylimidazole, and morpholine, optionally wherein the base is 1 -methylimidazole.

7. The process of any one of claims 3-6 wherein the reaction mixture is sparged with nitrogen before irradiation.

8. The process of anyone of claims 3-7, further comprising a coupling step wherein the deprotected oligonucleotide precursor is reacted with a nucleoside -containing moiety under conditions resulting in chain extension (i.e., forming the oligonucleotide or an extended precursor thereto).

9. The process of claim 8, wherein the nucleoside-containing moiety is NPPOC- protected such that the oligonucleotide or extended precursor thereto which is produced is also NPPOC-protected.201393-PCT01-NP P78290WO10. The process of claim 8 or claim 9, wherein the chain extension step is carried out on the deprotected oligonucleotide precursor from the preceding deprotection step without any intervening purification (e.g., diafiltration).

11. The process of any one of claims 8-10, comprising a plurality of coupling (chain extension) and deprotection steps.

12. The process of any one of claims 1 -11, wherein one or more steps are performed under continuous flow conditions.

13. The process of any one of claims 1 -12, wherein MeCN is used as a solvent.

14. The process of any one of claims 1-13, wherein the oligonucleotide or salt thereof is a 7-mer or longer oligonucleotide, e.g. an 8-mer to 30-mer, such as a 16-mer oligonucleotide.

15. The process of any one of claims 1 -14 wherein the oligonucleotide or salt thereof comprises one or more phosphoro thioate groups.

16. The process of any one of claims 1 -15, wherein the oligonucleotide or salt thereof comprises DNA, RNA, or a derivative thereof.

17. The process of any one of claims 1 -16, wherein (i) the oligonucleotide product obtained by the process does not comprise more than 5 % by dry weight of phosphomannose isomerase inhibitors, for example trityl cation, monomethoxy trityl cation, dimethoxy trityl cation, and / or derivatives thereof; and / or (ii) the total oligonucleotides produced by the process do not comprise more than 5 mol% of oligonucleotides in which one or more nucleotides are depurinated.

18. The process of any one of claims 1 -17, wherein the oligonucleotide or salt thereof represents at least 50% of the total oligonucleotides produced by the process.

19. Use of NPPOC as a protecting group in liquid phase oligonucleotide synthesis.201393-PCT01-NP P78290WO20. An NPPOC-protected oligonucleotide or salt thereof, wherein the oligonucleotide or salt thereof is in solution.

21. The NPPOC-protected oligonucleotide or salt thereof of claim 20, wherein the oligonucleotide or salt thereof is as defined in any one of claims 14-19.

22. A solution comprising an NPPOC-protected oligonucleotide or salt thereof.

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