Improved Oligonucleotide Synthesis

Abstract

Targeted Oligonucleotide O T Disclosed is a method for the solid phase synthesis of a nucleoside or oligonucleotide comprising: a nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; a deprotection mixture, whereby the protecting group is cleaved from the nucleoside or oligonucleotide; the deprotection mixture is a liquid composition comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol of formula (D); and R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, OH, C1-C6-alkyl, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN. Such a method not only inhibits depurination but also results in acceptable product yields.

Description

[Technical Field]

[0001] The present invention generally relates to the field of industrial- or laboratory-scale oligonucleotide synthesis, particularly solid-phase oligonucleotide synthesis. An improved method for solid-phase synthesis of oligonucleotides is disclosed. The present invention is directed to a method for effectively removing (temporary) protecting groups, including optionally substituted triarylmethyl residues, preferably di(p-methoxyphenyl)phenylmethyl protecting groups, during solid-phase oligonucleotide synthesis. A further aspect of the present invention is directed to a composition and its use for removing protecting groups, including optionally substituted triarylmethyl residues, preferably di(p-methoxyphenyl)phenylmethyl protecting groups, from hydroxyl moieties during chemical synthesis of oligonucleotides, particularly solid-phase synthesis. [Background technology]

[0002] Oligonucleotide synthesis typically involves one or more coupling steps, during each of which the free (i.e., unprotected) hydroxyl group of a first nucleoside or oligonucleotide reacts with the phosphorus moiety of a second nucleoside or oligonucleotide, which typically contains a protected backbone hydroxyl moiety. Protection of this backbone hydroxyl moiety is necessary to prevent double insertion and / or multimerization of the second nucleoside or oligonucleotide. Prior to the next coupling step, this hydroxyl moiety is typically deprotected and then participates in the next condensation reaction with the phosphorus moiety of a third nucleoside or oligonucleotide, and so on. (Hydroxyl) protecting groups that are cleaved before each condensation reaction (i.e., once per coupling cycle) are generally referred to as temporary protecting groups. Typically, functional groups of nucleobases (e.g., exocyclic amino groups) and carbohydrate (e.g., ribose or 2'-deoxyribose) moieties or other functional groups within internucleoside linkage groups are also protected during chemical oligonucleotide synthesis. However, these protecting groups are generally referred to as permanent protecting groups because they are typically removed only after all coupling cycles have been performed.

[0003] Temporary and permanent protecting groups are typically orthogonal to one another, meaning that one type can be removed under conditions that do not affect the other type of protecting group. Typically, permanent protecting groups are cleavable under alkaline conditions (i.e., by treatment with a base), and temporary protecting groups are cleavable under acidic conditions (i.e., by treatment with an acid).

[0004] For ease of separation from the reagents after each step, the growing oligonucleotide chain is generally attached to a solid support (see below).

[0005] Typically, protecting groups containing optionally substituted triarylmethyl residues, particularly di(p-methoxyphenyl)phenylmethyl (DMT) groups, also referred to herein as triarylmethyl-type protecting groups, are used as temporary (hydroxyl) protecting groups. The process of removing (temporary) triarylmethyl-type protecting groups (also referred to as detritylation process) has proven surprisingly difficult for the synthesis of high-quality oligonucleotides. One side reaction associated with acid-induced cleavage of triarylmethyl-type (hydroxyl) protecting groups is acid-induced nucleobase cleavage (i.e., one or more nucleobases are cleaved from the backbone of the oligonucleotide). This side reaction is particularly pronounced in purine-type nucleobases such as guanine and adenine. Nucleobase cleavage of purine-type nucleobases (i.e., nucleobases containing optionally substituted purine residues) is typically referred to as depurination. Various strategies have been explored to avoid nucleobase cleavage, especially depurination. Nucleobase protecting groups that reduce basicity, alternative temporary protecting groups, and coupling schemes using di- and trinucleotides have been tested, but none of these approaches have proven viable for routine oligonucleotide synthesis.

[0006] Habus and Agrawal (Patent Document 1 and Non-Patent Document 1) teach that detritylation agents containing 2% dichloroacetic acid (DCA) and 0.1% lower alcohols (i.e., C1-C6 alcohols) such as ethanol or methanol and / or 0.1-1% 1H-pyrrole in dichloromethane (DCM) suppress depurination during solid-phase oligonucleotide synthesis. It was speculated that the lower alcohols and / or 1H-pyrrole serve a dual function: as proton scavengers to reduce the acidity of the acid and as carbocation scavengers to suppress the reaction of triarylmethyl cations, particularly the DMT cation, with deprotected hydroxyl groups (Patent Document 1). The use of methanol or ethanol with protic acids for detritylation of oligonucleotides has also been proposed, for example, by Krotz and Ravikumar (Patent Document 2) and Hargreaves and Leproust (Patent Document 3), but not in the context of undesired nucleobase cleavage. The polyfluorinated alcohols trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol have also been used in detritylation cocktails to suppress depurination (Patent Document 4).

[0007] Non-Patent Document 2 found that a detritylation agent consisting of 3% trichloroacetic acid (TCA) in nitromethane / methanol (95:5) caused less depurination than a detritylation agent consisting of 3% TCA in DCM.

[0008] Hirai and Katayama (Patent Document 5) investigated the cleavage of the DMT protecting group (a pseudo-solid-phase protecting group) from the 5'-hydroxyl moiety of nucleosides that are attached to a soluble support via the 3'-hydroxyl moiety using trifluoroacetic acid (TFA, 394 μmol) in DCM (350 μL) in the presence of various cation scavengers, including methanol and m-cresol (162 μmol each) (see, e.g., the references in this publication).

[0009] ~

[0010] ). m-Cresol, in contrast to methanol, was found to function poorly as a cation scavenger.

[0009] reported the synthesis of oligonucleotide-neomycin conjugates by solid-phase oligonucleotide synthesis (SPOS) using 4% trichloroacetic acid in DCM for 5'-O-detritylation in every coupling cycle. After cleavage from the support, the 5'-terminal DMT group was removed along with the tert-butyloxycarbonyl (Boc) amino protecting group of the neomycin moiety. In this specific reaction, a mixture of 1,4-dioxane, trifluoroacetic acid (TFA), and m-cresol (96:3:1, v / v / v, approximately 95 mmol / L m-cresol) was used.

[0010] Both Non-Patent Document 4 and Non-Patent Document 5 teach increasing the concentration of dichloroacetic acid in the detritylation reagent from 3% to 10% or 15%, respectively, to prioritize detritylation over depurination, and minimizing the contact time between the detritylation reagent and the oligonucleotide. This approach is still used for industrial-scale production of oligonucleotides, such as those that can be used as active pharmaceutical ingredients (APIs). However, the scalability of the process is severely limited by the necessity to limit the contact time between the detritylation reagent and the oligonucleotide due to the limited maximum pumping capacity of the liquid handling system.

[0011] There is a long-felt unmet need for a solid-phase synthesis method for deprotecting (backbone) hydroxyl groups of growing oligonucleotide chains while suppressing undesired nucleobase cleavage, particularly depurination. Preferably, this method is scalable and adaptable to large-scale production of oligonucleotides, for example, in column and / or batch reactors. To be useful for industrial production, such a method must not only suppress undesired nucleobase cleavage, particularly depurination, but also result in acceptable product yields. Preferably, such a method does not rely on the use of halogenated solvents such as DCM. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO96 / 03417 [Patent Document 2] US7273933B1 [Patent Document 3] US20080058511 [Patent Document 4] WO2022 / 195111A1 [Patent Document 5] EP2711370A1 [Non-patent literature]

[0013] [Non-Patent Document 1] Nucleic Acids Research 1994, 22(20), pp. 4350-4351 [Non-patent document 2] H. Takaku et al. (Chemistry Letters 1983, 12(11), pp. 1661-1664) [Non-patent document 3] Arya et al. (Bioconjugate Chemistry 2007, 18(1), pp. 160-169) [Non-patent document 4] Cheruvallath (Organic Process Research & Development 2003, 7, pp. 917-920) [Non-Patent Document 5] Septak (Nucleic Acids Research, 1996, 24(15), pp. 3053-3058) Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention provides a solution to this problem. [Means for solving the problem]

[0015] One aspect of the present invention is a target oligonucleotide O T the method comprising step (b) incubating a nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from the nucleoside or oligonucleotide, wherein the deprotection mixture Mb is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety.

[0016] One aspect of the present invention is a target oligonucleotide O T the method comprising step (b) of incubating a nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from the nucleoside or oligonucleotide, wherein the deprotection mixture Mb is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety, and wherein the nucleoside or oligonucleotide comprises at least one purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0017] One aspect of the present invention is a target oligonucleotide O Tthe method comprising step (b) of incubating a nucleoside or oligonucleotide covalently linked to a solid support and comprising backbone hydroxyl moieties protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from the nucleoside or oligonucleotide, wherein the deprotection mixture Mb is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety, and wherein the backbone hydroxyl moieties protected by PG-0 are part of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group of adenine and guanine, in particular adenine.

[0018] As used herein, a purine nucleobase is a nucleobase containing an optionally substituted purine residue. Preferred examples of purine nucleobases are adenine and guanine. The purine nucleobase carries one or more protecting groups. In particular, the exocyclic amino groups present in, for example, adenine and guanine are protected. Preferred examples of suitable amine protecting groups are summarized in Table T-1 (see below). In some embodiments of the method of the present invention, the nucleoside or oligonucleotide covalently linked to a solid support and containing a backbone hydroxyl moiety protected by a protecting group PG-0 containing an optionally substituted triarylmethyl residue is component C-0 as defined herein. This means that all definitions, explanations, and embodiments relating to component C-0 also apply to the nucleoside or oligonucleotide covalently linked to a solid support and containing a backbone hydroxyl moiety protected by a protecting group PG-0 containing an optionally substituted triarylmethyl residue.

[0019] In some embodiments of the methods of the present invention, the target oligonucleotide O Tcomprises a first cycle oligonucleotide O-1, and the method comprises the following steps (a) and the following steps (b) to (e): (a) providing a component C-0 selected from the group consisting of nucleosides and oligonucleotides, component C-0 being covalently linked to a solid support and comprising backbone hydroxyl moieties protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue; (b) incubating component C-0 of step (a) with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from component C-0 to produce component C-0 having a free backbone hydroxyl group; # obtaining the (c) providing a building block B-1 selected from the group consisting of nucleosides and oligonucleotides, building block B-1 comprising a backbone hydroxyl moiety protected by a protecting group PG-1 comprising an optionally substituted triarylmethyl residue, and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block B-1 via a phosphorus atom; (d) Component C-0 # and the phosphorus atom of the phosphorus moiety of component B-1. # with component B-1 of step (c), thereby obtaining oligonucleotide O-1 of the first cycle; (e) optionally incubating the first cycle oligonucleotide O-1 obtained in step (d) with an oxidizing or sulfurizing agent, thereby converting any P(III) atoms in said first cycle oligonucleotide O-1 to P(V) atoms. a first coupling cycle comprising: In step (b), the deprotection mixture Mb is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety.

[0020] In some embodiments of the methods of the present invention, the target oligonucleotide O T comprises an n-th cycle of oligonucleotide On, and the method further comprises performing (n-1) repetitions of a coupling cycle comprising the following steps (b') to (e'), where n is an integer ranging from 2 to 99 and indicates the total number of coupling cycles performed to obtain the n-th cycle of oligonucleotide On, and each individual coupling cycle comprising the following steps (b') to (e') is identified by a serial number x, which is performed one by one from 2 to n: (b') The (x-1)th cycle oligonucleotide O-(x-1) obtained in the previous coupling cycle is incubated with the deprotection mixture M-b', thereby cleaving the protecting group PG-(x-1) from the (x-1)th cycle oligonucleotide O-(x-1) to form the (x-1)th cycle oligonucleotide (O-(x-1)) having a free backbone hydroxyl group. # obtaining the (c') providing a building block Bx selected from the group consisting of nucleosides and oligonucleotides, wherein building block Bx comprises a backbone hydroxyl moiety protected by a protecting group PG-x comprising an optionally substituted triarylmethyl residue, and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block Bx via a phosphorus atom; (d') (x-1)th cycle oligonucleotide (O-(x-1)) # and the phosphorus atom of the phosphorus moiety of building block Bx, under conditions suitable for forming a covalent bond between the free backbone hydroxyl group of the (x-1)th cycle oligonucleotide (O-(x-1)) obtained in step (b'). # with the building block Bx of step (c'), thereby obtaining the xth cycle oligonucleotide Ox; (e') optionally incubating the xth cycle oligonucleotide Ox obtained in step (d') with an oxidizing or sulfurizing agent, thereby converting any P(III) atom in said xth cycle oligonucleotide Ox into a P(V) atom; wherein in at least one repetition of step (b'), the deprotection mixture M-b' is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety.

[0021] As used in the context of the present invention, "target oligonucleotide O" T " or "liquid composition C" refers to "target oligonucleotide (O T )" or "liquid composition (C)". T ", "(O T )," "C," and "(C)" are to be understood as reference marks and do not imply any further limitation. Similar expressions, e.g., the first cycle oligonucleotides O-1 and (O-1) # , the (x-1)th cycle of oligonucleotides O-(x-1) and (O-(x-1)) # , nth cycle oligonucleotide On, components C-0 and C-0 # The same applies for the building blocks B-1 and Bx, the protecting groups PG-0, PG-1, PG-x and PG-(x-1), and the deprotection mixtures Mb, M-b', Mg and M-g'.

[0022] As used herein, the indefinite articles "a" and "an" mean "one or more," unless otherwise indicated.

[0023] As used herein, the term "solid-phase synthesis of (target) oligonucleotides" is understood in the broadest sense to refer to any synthesis of oligonucleotides in which an oligonucleotide attached to a solid support, preferably covalently linked, undergoes at least one chemical reaction. Preferably, the growing oligonucleotide chain is covalently linked to the solid support throughout at least one, preferably all, coupling cycles. In this specification, the terms "solid-phase synthesis of oligonucleotides" and "solid-phase oligonucleotide synthesis" (SPOS) are used interchangeably.

[0024] As used herein, "target oligonucleotide O" T The term "target oligonucleotide O" refers to any particular oligonucleotide to be synthesized by the methods of the present invention. T " is generally the final oligonucleotide product of the method of the invention. For example, "target oligonucleotide O T has the same sequence as the nth cycle oligonucleotide or contains one or more additional nucleoside moieties and / or is obtained by conjugating the nth cycle oligonucleotide to another compound.

[0025] As used herein, the term "oligonucleotide" is used in the most general manner to refer to any oligomer comprising at least two nucleoside subunits interconnected via an internucleoside linking group of any one of formulae A and B: [ka] [In Formula A: X 1 is selected from the group consisting of O and S; X 2 is OR 1 , S.R. 1 and H; R 1is any possible residue, preferably H and a protecting group (preferably a protecting group removable under alkaline conditions, in particular a 2-cyanoethyl group, i.e., CH2-CH2-CN)); [ka] [In formula B: X 3 is selected from the group consisting of O and S; R 2 is a protecting group, preferably a protecting group removable under alkaline conditions, in particular a 2-cyanoethyl group; In Formula A and Formula B, * and ** each independently indicates a binding site (i.e., attachment point) of a nucleoside subunit, i.e., * and ** each represents an atom of a respective nucleoside subunit, said nucleoside subunit being attached to an internucleoside linking group, wherein: * and the atom represented by ** and the atoms represented by are both O.

[0026] It will be understood that the internucleoside linking group of Formula A or Formula B may be protonated (e.g., at a carbonyl or thiocarbonyl group) or deprotonated (e.g., an OH or SH group is deprotonated) even if not specifically shown in Formula A and Formula B. Similarly, it will be understood that Formula A and Formula B encompass all salts, stereoisomers, and tautomeric forms of the respective internucleoside linking group even if not specifically shown in Formula A and Formula B.

[0027] Internucleoside linking groups, also referred to as internucleoside linking groups or linking groups, are classified according to the oxidation state of their respective phosphorus (P) atoms. Internucleoside linking groups typically contain a P(III) or P(V) atom. Throughout this text, the terms P(III) or phosphorus(III) atom are used interchangeably to refer to a P atom in a particular oxidation state, i.e., oxidation state III (i.e., +3). Similarly, the terms P(V) or phosphorus(V) atom are used interchangeably to refer to a P atom in a particular oxidation state, i.e., oxidation state V (i.e., +5). Internucleoside linking groups in which the P atom is a P(III) atom are referred to herein as P(III) or phosphorus(III) linking groups. Internucleoside linking groups in which the P atom is a P(V) atom are referred to herein as P(V) or phosphorus(V) linking groups.

[0028] Examples of P(V) linking groups are: - a phosphate diester (i.e., phosphodiester) group (i.e., X 1 is O and X 2 is OH), - thiophosphate (i.e., phosphorothioate) groups (i.e., X 1 is S and X 2 is OH), - dithiophosphate diester (i.e., phosphorodithioate) groups (i.e., X 1 is S and X 2 is a group of formula A, - phosphate triester group (i.e., X 1 is O and X 2 OR 1 and R 1 is not H and is preferably a protecting group such as a 2-cyanoethyl group), - Thiophosphate triester group (i.e., X 1 is S and X 2 OR 1 and R 1 is not H and is preferably a protecting group such as a 2-cyanoethyl group), and - dithiophosphate triester groups (i.e., X 1 is S and X 2 OR 1 and R 1 is not H and is preferably a protecting group such as a 2-cyanoethyl group) is.

[0029] Examples of P(III) linking groups are: - phosphite triester groups (i.e., X 3 is O), - thiophosphite triester groups (i.e., X 3 a group of formula B in which is S), and - H-phosphonate diester group (i.e., X 1 is O and X 2 is H) is.

[0030] It will be understood by those skilled in the art that when an oligonucleotide contains two or more internucleoside linking groups, these internucleoside linking groups can be the same or different (i.e., have the same or different chemical structures). The oligonucleotide also contains one or more P(III) linking groups and one or more P(V) linking groups. As known to those skilled in the art, the internucleoside linking groups can be modified during oligonucleotide synthesis. For example, the P(III) linking group can be converted into a P(V) linking group by incubation with an oxidizing or sulfurizing agent, and / or the protecting group can be removed.

[0031] As used herein, the term "substituent" is understood in its broadest sense to refer to any chemical residue or moiety. Thus, as used herein, the terms "substituent," "residue," and "moiety" are used interchangeably, unless otherwise indicated.

[0032] The term "group" is also used to refer to certain substituents (i.e., residues or moieties). However, the terms "substituent," "moiety," or "residue" are broader than the term "group" when used in the following cases:

[0033] As used herein, the term hydroxyl group refers to an OH residue. In other words, a hydroxyl group is always a free hydroxyl group. However, the term hydroxyl moiety also encompasses hydroxyl residues resulting from the reaction of a hydroxyl group with another chemical group, such as hydroxyl residues involved in an internucleoside linking group or hydroxyl residues bound to a protecting group. The terms sulfhydryl group, SH, and sulfhydryl moiety are also used. As used herein, the terms amino group and amine group are used interchangeably to refer to a substituent attached to the respective chemical structure via a nitrogen atom, which is further attached to two or three residues selected from the group consisting of H, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, and heteroaryl. These residues are further substituted and further specified, for example, when an amino group is designated as a di(C1-C6-alkyl)amino group (e.g., N(C1-C6-alkyl)2). The di(C1-C6-alkyl)amino group is also formally a cyclic amino group in which two alkyl residues are bonded to each other to form a ring structure. Examples of such cyclic amino groups include pyrrolidine and piperidine groups. The term amine moiety also encompasses amine residues resulting from the reaction of an amine group with another chemical group, for example, an amine residue involved in the attachment of a protecting group.

[0034] As used herein, the term "optionally substituted" is understood in the broadest sense to mean that in the respective optionally substituted structure, such as said optionally substituted phenyl moiety or said optionally substituted triarylmethyl residue, one or more hydrogen residues are optionally replaced, independently of one another, by another residue, also called a substituent. If a "substituent" (i.e., a residue or moiety) is not further specified, the substituent is any possible stable atom or group of atoms, preferably selected from the group consisting of alkyl residues, O-alkyl, halogen residues (F, Cl, Br, I), cyano (i.e., CN) residues, heteroalkyl residues, alkenyl residues, heteroalkenyl residues, alkynyl residues, heteroalkynyl residues, aryl residues, heteroaryl residues, ketone residues (especially C(O)alkyl), aldehyde residues (CHO), carboxylic acid residues or esters (especially C(O)O-alkyl) or amides thereof, amine groups or moieties, boryl residues (i.e., a substituent bonded via a B atom), silyl residues (i.e., a substituent bonded via a silicon atom), hydroxyl groups or moieties, sulfhydryl groups or moieties, and combinations thereof. Unless otherwise indicated, these substituents are further substituted, i.e., one or more hydrogen atoms of these substituents are replaced by further substituents.

[0035] As used throughout this invention, the term "alkyl" is understood in its broadest sense and is used to refer to any aliphatic group (in other words, a residue, moiety, or substituent) composed of atoms of the chemical elements carbon (C) and hydrogen (H), but not containing any heteroatoms. Additionally, alkyl groups, as defined herein, are characterized in that they contain at least one carbon atom, and one or more carbon atoms are bonded to each other only through direct single bonds. Preferably, unless indicated otherwise in the context of a particular embodiment, alkyl groups contain 1 to 20, 1 to 6, or 1 to 5 carbon atoms. Unless otherwise stated, the term "alkyl" generally encompasses unbranched, branched, and cyclic groups. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl (propan-1-yl), isopropyl (propan-2-yl), n-butyl (butan-1-yl), sec-butyl (butan-2-yl), tert-butyl (2-methylpropan-2-yl), isobutyl (2-methylpropan-1-yl), cyclohexyl, and cyclopentyl.

[0036] As used throughout the present invention, the term "heteroalkyl" is understood in the broadest sense and is used to refer to any alkyl group in which one or more carbon or hydrogen atoms are replaced by heteroatoms, provided that the heteroalkyl group must contain at least one carbon atom. A heteroatom in this context is any atom of a chemical element other than carbon or hydrogen, and preferably, unless otherwise indicated in the context of a particular embodiment, is an atom independently selected at each occurrence from the group consisting of N (nitrogen), O (oxygen), S (sulfur), P (phosphorus), F (fluorine), Cl (chlorine), Br (bromine), I (iodine), and Si (silicon). Preferably, unless otherwise indicated in the context of a particular embodiment, a heteroalkyl group contains 1 to 19, 1 to 6, or 1 to 5 carbon atoms. Unless otherwise stated, the term "heteroalkyl" generally encompasses unbranched, branched, and cyclic groups. Non-limiting examples of heteroalkyl groups include halogenated alkyl groups such as trifluoromethyl, oxygenated alkyl groups (i.e., alkyloxy groups), aminated alkyl groups (i.e., alkylamine groups, such as dimethylamine groups), and cyclic groups such as piperidine, pyrrolidine, or morpholine. A heteroalkyl substituent is typically bonded through one or more of its carbon atoms.

[0037] As used throughout the present invention, the term "alkenyl" is understood in the broadest sense and is used to refer to any aliphatic group derived from an alkyl group by introducing one or more C≡C double bonds between carbon atoms. Similarly, the term "heteroalkenyl" is understood in the broadest sense and is used to refer to any aliphatic group derived from a heteroalkyl group by introducing one or more C≡C double bonds between carbon atoms. As used herein, unless otherwise stated in the context of a particular embodiment, the terms "alkenyl" and "heteroalkenyl" generally encompass both (E) and (Z) isomers, as well as mixtures thereof. As used throughout the present invention, the term "alkynyl" is understood in the broadest sense and is used to refer to any aliphatic group derived from an alkyl group by introducing one or more C≡C triple bonds between carbon atoms. Similarly, the term "heteroalkynyl" is understood in the broadest sense and is used to refer to any aliphatic group derived from a heteroalkyl group by introducing one or more C≡C triple bonds between carbon atoms. Alkenyl, heteroalkenyl, alkynyl, and heteroalkynyl groups are understood to contain at least two carbon atoms.

[0038] As used herein, the term "hydrocarbon" is understood in its broadest sense and is used to denote any compound or residue composed of atoms of the chemical elements carbon (C) and hydrogen (H), but does not contain any heteroatoms. The term "aliphatic hydrocarbon" encompasses alkyl, alkenyl, and alkynyl groups as defined herein, unless indicated otherwise in the context of a particular embodiment.

[0039] As used throughout the present invention, the term "aryl" is understood in the broadest sense and is used to refer to any monocyclic or polycyclic aromatic group, provided that all aromatic ring atoms are carbon atoms. Unless otherwise indicated in the context of a particular embodiment, an aryl group preferably contains 6 to 30, 6 to 20, or 6 to 15, especially 6, aromatic carbon atoms. Benzene (i.e., phenyl group, Ph) is a preferred example of a monocyclic aromatic (i.e., aryl) group. In this context, the term "polycyclic" will be understood by those skilled in the art to refer to a fused aromatic ring system in which two or more aromatic rings are fused together. That is, the fused aromatic rings share at least one bond between aromatic carbon atoms, and thus, this shared bond and the carbon atoms forming the bond are part of two or more monocyclic aromatic groups that constitute the polycyclic aromatic group. Non-limiting examples of polycyclic aromatic (ie, aryl) groups are naphthalene (ie, naphthyl), anthracene (ie, anthracenyl), and phenanthrene (ie, phenanthryl).

[0040] As used throughout this invention, the term "heteroaryl" is understood in its broadest sense and is used to denote any monocyclic or polycyclic heteroaromatic group that differs from an aromatic (i.e., aryl) group in that at least one, and preferably 1 to 5 or 1 to 3, aromatic ring atoms is a heteroatom. A heteroatom in this context is any atom of a chemical element other than carbon and hydrogen, and preferably is an atom independently selected from the group consisting of N, O, and S, at each occurrence, unless otherwise indicated in the context of a particular embodiment. Non-limiting examples of monocyclic heteroaromatic (i.e., heteroaryl) groups include pyridine (i.e., pyridyl), pyridazine (i.e., pyridazinyl), pyrimidine (i.e., pyrimidinyl), pyrazine (i.e., pyrazinyl), 1,2,3-triazine (i.e., 1,2,3-triazinyl), 1,2,4-triazine (i.e., 1,2,4-triazinyl), 1,3,5-triazine (i.e., 1,3,5-triazinyl), 1,2,3,4-tetrazine (i.e., 1,2,3,4-tetrazine). i.e., 1,2,3,4-tetrazinyl), 1,2,4,5-tetrazine (i.e., 1,2,4,5-tetrazinyl), pyrrole (i.e., pyrrolyl), pyrazole (i.e., pyrazolyl), imidazole (i.e., imidazolyl), 1,2,3-triazole (i.e., 1,2,3-triazolyl), 1,2,4-triazole (i.e., 1,2,4-triazolyl), thiophene (i.e., thiophenyl), and furan (i.e., furanyl). Non-limiting examples of polycyclic heteroaromatic (i.e., heteroaryl) groups include quinoline (i.e., quinolinyl), quinazoline (i.e., quinazolinyl), quinoxaline (i.e., quinoxalinyl), benzofuran (i.e., benzofuranyl), benzothiophene (i.e., benzothiophenyl), benzimidazole (i.e., benzimidazolyl), benzothiazole (i.e., benzothiazolyl), benzoxazole (i.e., benzoxazolyl), dibenzofuran (i.e., dibenzofuranyl), and acridine (i.e., acridinyl).

[0041] As used herein, the term "aliphatic" is used to refer to any compound or residue that is not aromatic, i.e., does not contain an aromatic or heteroaromatic ring system. Unless otherwise indicated in the context of a particular embodiment, such aliphatic compounds are understood to be unbranched, branched, or cyclic. For example, an aliphatic cyclic amine moiety is, for example, a piperidine, pyrrolidine, or morpholine moiety, but is not a pyridine moiety, but instead is a heteroaromatic amine moiety, or more generally a heteroaryl or heteroaromatic moiety.

[0042] In accordance with common practice, the names of substituents (i.e., residues or moieties, and groups) are typically derived herein from their chemical names and denoted by the final syllable "-yl." However, this term is used herein interchangeably with the chemical name of the respective chemical atom or group of atoms, common abbreviations, and element symbols themselves, as if they were not substituents. For example, a CH-substituent is also referred to as methyl or Me, a Cl-substituent is also referred to as chlorine or simply Cl, a phenyl substituent is also designated as Ph, and a cyano group is also designated as CN. When a substituent is designated using element symbols, e.g., CN, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), and contains two or more atoms, the point of attachment of the respective residue (i.e., the atom that is the origin of the chemical bond to the parent structure) is generally designated herein as the leftmost atom depicted, e.g., the carbon atom in CN and the oxygen atom in O(C1-C6-alkyl), unless otherwise indicated. When a substituent is designated as OR, z-1 The same rationale applies when a group is depicted using a generic residue R such as "butyl" and whose oxygen atom constitutes the point of attachment of the residue to the parent structure. As used herein, unless stated otherwise in the context of a particular embodiment, when a particular substituent is referred to by a generic term such as "butyl," this language encompasses all isomers, including, for example, positional isomers (e.g., n-butyl, sec-butyl, tert-butyl, etc.), diastereomers, and enantiomers, where applicable, having the chemical structure corresponding to the respective generic substituent name such as "butyl."

[0043] As used throughout the present invention, the number of carbon atoms in a residue (moiety or substituent or group) is sometimes indicated in subscript form, e.g., C1-C6-alkyl or C6-C 20 -aryl. These subscripts refer to the range of carbon atoms allowed for a particular residue in the context of a particular embodiment. When referring to an aryl or heteroaryl residue, these subscripts refer to the aromatic ring carbon atoms. For example, a C1-C6-alkyl group contains 1 to 6 carbon atoms, but not less than 1 or more than 6 carbon atoms. Similarly, a C6-C 20 -Aryl groups contain from 6 to 20 aromatic ring carbon atoms, but never less than 6 or more than 20 aromatic ring carbon atoms.

[0044] In this specification, unless otherwise stated in the context of specific embodiments, when referring to oligonucleotide, said oligonucleotide generally exists as a mixture of isomers, particularly as a mixture of stereoisomers.Those skilled in the art will understand that oligonucleotide molecules (typically) only exist in a single (stereo)isomeric form at a certain time.Therefore, when oligonucleotide is sometimes referred to as existing as a mixture of (stereo)isomers, this refers to a group of oligonucleotide molecules that have essentially the same nucleoside sequence, and the molecules of said group exist in different (stereo)isomeric forms.Therefore, said group of oligonucleotide molecules is a mixture of many distinct stereoisomers, or is enriched in one specific stereoisomeric form, or is essentially composed of molecules of specific stereoisomeric form.

[0045] It is understood by those skilled in the art that certain groups in oligonucleotides, such as OH, SH, and SeH groups, are deprotonated.Similarly, it is understood that certain groups in oligonucleotides, such as amino groups, are protonated.It is understood by those skilled in the art that oligonucleotides defined herein may optionally carry any counterion known in the art, particularly cations such as sodium cations, potassium cations, magnesium cations, ammonium cations, and generally cations derived from amines such as trimethylamine (TEA), diisopropylamine (DIPEA), or heteroaromatic cations such as pyridine or collidine, and / or anions such as chloride anions, bromide anions, acetate anions, trifluoroacetate anions, carbonate anions, hydrocarbon anions, phosphate anions, hydrogen phosphate anions, dihydrogen phosphate anions, perchlorate anions, and combinations thereof.Deprotonation or protonation of oligonucleotides and (non-covalent) binding of one or more cations and / or anions occur, for example, during the synthesis, isolation, and purification of oligonucleotides. In general, it will be understood by those skilled in the art that oligonucleotides as defined herein are non-covalently bound or associated with species with which the oligonucleotide comes into contact during its synthesis, isolation, or purification, such as cations and / or anions, or residues of protecting groups, and one or more trace amounts of cation scavengers, such as thiols or silanes. It will also be understood that such non-covalently bound or associated species are typically not depicted in the oligonucleotide or in the chemical formulas, particularly generic formulas, that refer to the oligonucleotide.

[0046] Non-limiting examples of oligonucleotides of the present invention are 2'-deoxynucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), constrained ethyl nucleic acid analogs (cET), bridged nucleic acid (BNA), tricyclo DNA, unlocked nucleic acid (UNA), small interfering RNA (siRNA), microRNA, antisense oligonucleotide (ASO), gapmer, glycerol nucleic acid, phosphorothioate oligonucleotide, phosphorodithioate oligonucleotide, and derivatives and analogs thereof, all of which are known to those skilled in the art.

[0047] Oligonucleotides are preferably linear sequences of nucleoside subunits, and any two adjacent nucleoside subunits are interconnected by an internucleoside linking group as defined above. Such linear sequences are also called oligonucleotide chains. According to common practice, the number of nucleoside subunits of an oligonucleotide is indicated by a number (an integer of 2 or greater) followed by the syllable "mer" or by using a suitable prefix (e.g., di for 2, tri for 3, etc.). For example, an oligonucleotide that contains exactly two nucleoside subunits is referred to as a 2mer or 2mer oligonucleotide or dinucleotide, and an oligonucleotide that contains exactly 20 nucleoside subunits is referred to as a 20mer or 20mer oligonucleotide.

[0048] Such an oligonucleotide chain comprises a first terminal nucleoside subunit and a second terminal nucleoside subunit, each of which has exactly one adjacent nucleoside subunit. In the case of a dinucleotide (i.e., a 2-mer oligonucleotide), the two terminal nucleoside subunits are interconnected only by a first internucleoside linking group. An oligonucleotide comprising more than two nucleoside subunits comprises exactly two terminal nucleoside subunits and one or more non-terminal nucleoside subunits, each of which is characterized by having exactly two adjacent nucleoside subunits (i.e., one preceding nucleoside subunit and one succeeding nucleoside subunit). The term "adjacent," when referring to the nucleoside subunits of an oligonucleotide (chain), refers to any two nucleoside subunits interconnected by an internucleoside linking group.

[0049] It is understood that the nucleoside moiety that forms part of an oligonucleotide is referred to herein as the nucleoside subunit of said oligonucleotide.In this specification, the term nucleoside moiety includes both the nucleoside itself and the nucleoside subunit of the oligonucleotide defined herein.However, the term "nucleoside" (without the addition of "moiety" or "subunit") is only used to indicate mononucleoside, i.e., the nucleoside moiety that is not the nucleoside subunit of an oligonucleotide.The possible chemical structures of nucleoside moiety are known to those skilled in the art.

[0050] The nucleoside moiety is: a carbohydrate moiety (in other words, a sugar moiety), preferably a monosaccharide moiety, more preferably a pentose moiety, in particular a ribose moiety (e.g., typically present in RNA) or a 2'-deoxyribose moiety (e.g., typically present in DNA); and - Nucleic acid bases wherein the carbohydrate moiety and the nucleobase are typically covalently linked to each other via a direct covalent bond, typically an N-glycosidic bond. The properties and chemistry of carbohydrates and nucleobases form part of the general knowledge of those skilled in the art, and they can select the carbohydrate moiety and nucleobase of the nucleoside moiety depending on the structure of the oligonucleotide to be synthesized.

[0051] As used herein, unless otherwise indicated in the context of a particular embodiment, the term "nucleoside moiety" includes naturally occurring nucleoside moieties as well as non-natural nucleoside moieties in which the carbohydrate moiety and / or nucleobase are chemically modified, or even in which the nucleobase is absent (i.e., abasic site). Non-limiting examples of naturally occurring nucleoside moieties include adenosine, 2'-deoxyadenosine, guanosine, 2'-deoxyguanosine, cytidine, 2'-deoxycytidine, uridine, (2'-deoxy)thymidine, ribothymidine, inosine, and methylated derivatives thereof, all of which are known to those skilled in the art. Further examples include queusine, archaeosine, wybutosine, lysidine, and N. 6 -threonylcarbamoyl adenosine.

[0052] The term "derivative", as used herein, is understood in the broadest sense and refers to a compound that can be obtained from an original compound (i.e., the parent compound) by one or more, preferably one, chemical reactions. As a result, a derivative differs from the original (parent) compound, for example, with respect to substitution pattern or with respect to the presence or absence of one or more atoms, groups of atoms, functional groups or protecting groups.

[0053] For example, chemical modifications of (unnatural) nucleoside moieties include modifications of the carbohydrate, particularly the ribose or 2'-deoxyribose moiety. Such carbohydrate modifications include, by way of example: - introducing an O-CH3 (i.e., O-methyl or OMe) group, an O-CH2-CH2-O-CH3 (i.e., O-methoxyethyl or O-MOE) group, or an F-substituent (i.e., a fluorine substituent) at the 2'-carbon atom; - introducing a methylene or ethylene bridge between the 2'-oxygen and the 4'-carbon of the ribose moiety ("locked" derivatives); - interconnected between the 3'- and 5'-carbon atoms of ribose or 2'-deoxyribose moieties (e.g., in tricyclic DNA, tcDNA); - replacing the ribose or 2'-deoxyribose moiety with another pentose, such as arabinose, or a hexose, such as mannose; and - Using L-enantiomers of carbohydrates instead of the naturally occurring D-enantiomers; and / or combinations of these modifications is selected from the group consisting of:

[0054] As used herein, the term "nucleobase" encompasses both unnatural nucleobases and naturally occurring nucleobases such as adenine, guanine, cytosine, thymine and uracil.Those skilled in the art know how to select suitable unnatural nucleobases to replace the naturally occurring nucleobases of nucleoside moieties, so that unnatural nucleobases can still carry out specific interactions with complementary nucleobases.The interaction between complementary nucleobases is mediated by hydrogen bonds (see Watson-Crick base pair).Unnatural nucleobases are preferably purine or pyrimidine derivatives that can carry out specific interactions with other nucleobases.

[0055] Non-natural nucleoside moieties are formed, for example, from naturally occurring carbohydrates and non-natural nucleobases, or from non-natural carbohydrates and naturally occurring nucleobases, or from non-natural carbohydrates and non-natural nucleobases.

[0056] An example of a nucleoside moiety is a nucleoside moiety of Formula C: [ka] [In formula C, B N is a nucleobase bearing one or more protecting groups; Q 1 is OR 3 (when the nucleoside moiety is the 3'-terminal nucleoside moiety of an oligonucleotide or when the nucleoside moiety is a nucleoside) and an oxygen atom covalently bound to an internucleoside linking group (when the respective nucleoside moiety is part of an oligonucleotide and is not the 3'-terminal nucleoside moiety thereof); R 3 is selected from the group consisting of H, a protecting group, and a conjugate moiety that is not a nucleoside, nucleotide, or oligonucleotide; Q 2 is OR 4 (when the nucleoside moiety is the 5'-terminal nucleoside moiety of an oligonucleotide or when the nucleoside moiety is a nucleoside) and an oxygen atom covalently bound to an internucleoside linking group (when the respective nucleoside moiety is part of an oligonucleotide and is not the 5'-terminal nucleoside moiety thereof); R 4 is selected from the group consisting of H, a protecting group, and a conjugate moiety that is not a nucleoside, nucleotide, or oligonucleotide; R I H, F, and OR 5 and R 5 is H, a protecting group, any possible substituent, or a conjugate moiety that is not a nucleoside, nucleotide, or oligonucleotide; R III is H or R III and R I are bonded together to form a cyclic structure; R II , R IV, and R V are, independently of one another, all possible substituents, and two or three of these residues are optionally joined together to form a cyclic structure.

[0057] As noted above, unless otherwise indicated in the context of a particular embodiment, oligonucleotides herein generally exist as mixtures of isomers. No stereochemical information can be inferred from Formula C. In some preferred embodiments, the nucleoside moiety of Formula C is a nucleoside moiety of the following formula Ca: [ka] [In formula Ca, the carbon atoms are numbered 1' to 5' (according to common practice), but this serves merely for illustrative purposes and should not be construed as limiting in any way. In formula Ca, Q 1 , Q 2 , B N , R I , R II , R III , R IV , R V , R 3 , R 4 , and R 5 is defined as in formula C, it being understood that the number of carbon atoms is the same in either the ribose or 2'-deoxyribose based nucleoside moiety].

[0058] From the foregoing, it will be understood that any nucleoside subunits containing a ribose or 2'-deoxyribose moiety, e.g., nucleoside moieties of formula C and / or Ca, are preferably incorporated into an oligonucleotide either all in the 3'→5' direction or all in the 5'→3' direction (all in the same direction). Such nucleoside subunits are linked to one (if a terminal nucleoside subunit) or two (if a non-terminal nucleoside subunit) internucleoside linking groups, but preferably, linkage to the internucleoside linking groups occurs exclusively via the 3'-hydroxyl moiety (i.e., the hydroxyl moiety attached to the 3'-carbon atom) and / or the 5'-hydroxyl moiety (i.e., the hydroxyl moiety attached to the 5'-carbon atom). Since all nucleoside subunits are preferably incorporated in the same direction, the internucleoside linking group between the nucleoside subunits containing a ribose or 2'-deoxyribose moiety is preferably bonded to the 5'-hydroxyl moiety of one nucleoside subunit and the 3'-hydroxyl moiety of another nucleoside subunit. Obviously, the 3'-hydroxyl moiety of the 3'-terminal nucleoside subunit is not involved in bonding to the internucleoside linking group. Similarly, the 5'-hydroxyl moiety of the 5'-terminal nucleoside subunit is not involved in bonding to the internucleoside linking group.

[0059] From the foregoing, it is understood that an oligonucleotide comprises two or more nucleoside subunits (nucleoside moieties). It is further understood that these two or more nucleoside subunits of an oligonucleotide may be the same or different (i.e., have the same or different chemical structures).

[0060] As used herein, the term "nucleotide" is understood in the broadest sense and preferably refers to a conjugate of a nucleoside moiety and a phosphate group or a derivative thereof, wherein a hydroxyl moiety of said nucleoside moiety is linked through its oxygen atom to the phosphorus atom of the phosphate moiety or its derivative.

[0061] As understood by those skilled in the art, the internucleoside linking group and any carbohydrate moiety, for example, ribose or 2'-deoxyribose moiety, are referred to herein as the "backbone" of oligonucleotide.Therefore, the term "backbone" of oligonucleotide excludes nucleic acid base.It should also be understood that in (mono)nucleoside, the backbone is the carbohydrate moiety, for example, ribose or 2'-deoxyribose moiety, because the nucleoside does not contain an internucleoside linking group.

[0062] It is common knowledge for those skilled in the art that oligonucleotides are conjugated to other moieties that are not nucleosides, nucleotides, or oligonucleotides as defined herein for various purposes.In particular, the free OH-group (for example, the 3'-OH and / or 5'-OH group of ribose or 2'-deoxyribose moiety) at the terminal nucleoside of oligonucleotide chain is preferably involved in this conjugation.For example, the 5'-OH group of the antisense strand of siRNA (small interfering RNA known to those skilled in the art) is modified with vinylphosphonate to prevent cellular degradation and improve efficacy.Another example is conjugation to N-acetylgalactosamine (GalNAc), which is interesting for targeting oligonucleotide delivery to hepatocytes. The GalNAc structure is often branched to accommodate three or four GalNAc moieties, for example, conjugated to the 5'-OH group (see, e.g., WO2016055601), the 3'-OH group (see, e.g., WO2009073809) of an oligonucleotide, or a monovalent GalNAc moiety is conjugated via a linker to the 2' position of a subsequent ribose moiety within the oligonucleotide chain (see, e.g., WO2019075419). Tis an oligonucleotide conjugate unless otherwise indicated in the context of a particular embodiment. As used herein, the term "oligonucleotide conjugate" refers to any oligonucleotide comprising at least one nucleoside subunit covalently linked to another moiety that is not a nucleoside, nucleotide, or oligonucleotide, such as a peptide, protein, lipid, carbohydrate, or hydrocarbon moiety, with carbohydrate moieties being preferred. In some embodiments of the methods of the present invention, the target oligonucleotide O T is not an oligonucleotide conjugate.

[0063] "Target oligonucleotide O, including oligonucleotide O-1 in the first cycle T " (in other words, the target oligonucleotide O T (including oligonucleotide O-1 in the first cycle) is the target oligonucleotide O T "The target oligonucleotide O-1 contains the nucleoside sequence of the oligonucleotide O-1 in the first cycle." T " (in other words, the target oligonucleotide O T (including the nth cycle oligonucleotide On) is the target oligonucleotide O T contains the nucleoside sequence of the oligonucleotide On in the nth cycle.

[0064] As used herein, the term "nucleoside sequence" refers to a series of nucleoside subunits within an oligonucleotide. For linear oligonucleotides, a nucleoside sequence typically begins with a first terminal nucleoside subunit, optionally followed by one or more non-terminal nucleoside subunits, and ends with a second terminal nucleoside subunit. As used herein, the term "nucleoside sequence" does not specify one or more internucleoside linking groups, and does not take into account the presence or absence of protecting groups. Thus, two oligonucleotides containing the same nucleoside subunits in the same order are considered herein to contain the same nucleoside sequence, regardless of whether the internucleoside linking groups interconnecting these nucleoside subunits are the same (i.e., have the same chemical structure), and regardless of whether the carbohydrate moieties, nucleobases, and any atoms or functional groups within the internucleoside linking groups are protected. Solid supports are considered herein as (permanent) protecting groups and are therefore included in the general term "protecting group" unless otherwise indicated in the context of a particular embodiment. For example, internucleoside linking groups differ in terms of the oxidation state of the phosphorus atom and / or the presence or absence of a protecting group such as a 2-cyanoethyl group. As another example, the exocyclic amino groups of nucleic acid bases such as cytosine, 5-methylcytosine, guanine, and adenine can be protected or unprotected, and the 5'-terminal hydroxyl moiety can be protected or unprotected without affecting what is referred to herein as the nucleoside sequence.

[0065] Unless otherwise indicated in the context of a particular embodiment, for example, when an initial coupling cycle comprising steps (b) through (e) is followed by a further step comprising a further coupling cycle, for example, a further coupling cycle comprising steps (b') through (e'), the target oligonucleotide O TIt will be understood that the target oligonucleotide O-1 contains more nucleoside subunits than the oligonucleotide O-1 of the first cycle. Unless otherwise indicated in the context of a particular embodiment, for example, when (n-1) repetitions of the coupling cycle comprising steps (b') through (e') are followed by further steps comprising one or more condensation reactions with another nucleoside or oligonucleotide, the target oligonucleotide O-1 T It will be understood that the target oligonucleotide O contains more nucleoside subunits than the nth cycle oligonucleotide O n. Additionally or alternatively, the target oligonucleotide O T is also a conjugate as defined herein, i.e., a conjugate covalently attached to a moiety that is not a nucleoside, nucleotide, or oligonucleotide, such as a carbohydrate moiety. Such conjugation is achieved, for example, after the final iteration of the coupling cycle.

[0066] In some embodiments of the methods of the present invention, the target oligonucleotide O T In such an embodiment, the target oligonucleotide O T In some embodiments of the method of the present invention, the target oligonucleotide O-1 differs from the oligonucleotide O-1 of the first cycle only with respect to the presence or absence of an internucleoside linking group and a protecting group. T In such an embodiment, the target oligonucleotide O T The nth cycle oligonucleotide O-1 differs from the nth cycle oligonucleotide O-1 only with respect to the presence or absence of internucleoside linking groups and protecting groups. For example, the target oligonucleotide preferably contains only phosphorus(V) linking groups, while the first cycle oligonucleotide O-1 or the nth cycle oligonucleotide O-1 contains one or more phosphorus(III) linking groups. For example, the target oligonucleotide O-1 preferably contains only phosphorus(V) linking groups. Tpreferably does not contain any protecting groups, whereas the oligonucleotide O-1 of the first cycle and the oligonucleotide On of the nth cycle typically contain some protecting groups and are covalently linked to at least a solid support, which are considered herein as (permanent) protecting groups.

[0067] In some embodiments of the methods of the present invention, the target oligonucleotide O T In some embodiments of the methods of the invention, the target oligonucleotide O comprises 2 to 200, 2 to 150, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, or 2 to 20 nucleoside subunits. T comprises at least one purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0068] As used herein, the terms "removing a protecting group," "cleaving a protecting group," and "deprotecting" a functional group are used interchangeably. Similarly, as used herein, the terms "cleaving a protecting group," "removing a protecting group," and "deprotecting" a functional group are used interchangeably. The adjective "free" is used to emphasize that a particular atom or functional group does not carry a protecting group, but this does not imply that any group not designated "free" carries a protecting group. As used herein, the process of removing protecting groups, including optionally substituted triarylmethyl residues such as DMT groups, is referred to as "detritylation," and the reaction mixture in which such detritylation occurs is referred to as a detritylation cocktail.

[0069] As used herein, the term "protecting group" is understood in its broadest sense as any group that is introduced into a molecule by chemical modification (i.e., one or more, typically one, chemical reaction) of an atom or functional group (i.e., the atom or functional group to be protected), and that, once introduced, prevents said atom or functional group from participating in a chemical reaction in a subsequent process step. The terms "protected" and "bearing a protecting group" are used interchangeably herein to refer to an atom or functional group that is covalently bonded to a protecting group. Protecting groups used to protect amine moieties are referred to herein as amine protecting groups or amino protecting groups. Similarly, protecting groups used to protect hydroxyl moieties are referred to herein as hydroxyl protecting groups. It will be understood by those skilled in the art that a protecting group replaces a hydrogen residue from the atom or functional group to be protected. For example, a hydroxyl protecting group covalently bonds to the oxygen atom of the hydroxyl moiety to be protected, thereby replacing a hydrogen residue.

[0070] As used herein, the two interchangeable terms "protecting group containing an optionally substituted triarylmethyl residue" and "triarylmethyl-type protecting group" are understood in the broadest sense as any protecting group covalently bonded to the atom or functional group to be protected (e.g., the oxygen atom of a hydroxyl moiety) via carbon atoms to which three optionally substituted aryl moieties are attached. The basic triarylmethyl-type protecting group is a triphenylmethyl group (i.e., a trityl group). As known to those skilled in the art, substituents capable of stabilizing the triarylmethyl cation formed during the acid-catalyzed removal of the triarylmethyl-type protecting group, such as alkyl or alkoxy substituents, facilitate the acid-catalyzed cleavage of the respective protecting group.

[0071] Examples of the triarylmethyl-type protecting group include a trityl group, a (p-methylphenyl)diphenylmethyl group (i.e., a 4-methyltrityl group), a di(p-methylphenyl)phenylmethyl group (i.e., a 4,4'-dimethyltrityl group), a tri(p-methylphenyl)methyl group (i.e., a 4,4',4''-trimethyltrityl group), a (p-methoxyphenyl)diphenylmethyl group (i.e., an MMT group), a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group), a tri(p-methoxyphenyl)diphenylmethyl group (i.e., a DMT group), a tri(p-methoxyphenyl)diphenylmethyl group (i.e., a 4,4',4''-trimethyltrityl group), a (p-methoxyphenyl)diphenylmethyl group (i.e., a ... tri(p-methoxyphenyl)diphenylmethyl group (i.e., a DMT group), a tri(p-methoxyphenyl)diphenylmethyl group (i.e., a DMT group), a tri(p-methoxyphenyl)diphenylmethyl group (i.e., a DMT group), a tri(p-methoxyphenyl )methyl group (i.e., TMT group), 4,4'-dimethoxy-3"-[N-(imidazolylmethyl)]trityl group (i.e., IDT group), 4,4'-dimethoxy-3"-[N-(imidazolylethyl)carbamoyl]trityl group (i.e., IET group), bis(4-methoxyphenyl)-1'-pyrenylmethyl group (i.e., Bmpm group), and 4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4',4"-dimethoxytrityl group (i.e., Tbf-DMT group). Trityl group, MMT group, and DMT group are preferred. The DMT group is most preferred herein as the temporary hydroxyl protecting group. Thus, in some embodiments, a protecting group comprising an optionally substituted triarylmethyl residue is a DMT protecting group in each occurrence.

[0072] As used herein, the term "liquid composition" is understood in the broadest sense to refer to any composition comprising a solvent (whether a single solvent or a mixture of solvents) and one or more components dissolved in said solvent. A liquid composition may be further categorized, for example, as liquid composition C, if it meets certain conditions.

[0073] As used herein, the term "coupling cycle" is understood in the broadest sense and refers to a series of two or more process steps, including a deprotection step used to provide a nucleoside or oligonucleotide with a free hydroxyl group, and a subsequent coupling step (also called a condensation step) in which the free hydroxyl group participates in a bond-forming reaction with a phosphorus moiety of another nucleoside or oligonucleotide. Multiple iterations (i.e., multiple times) of the coupling cycle are performed. During each of these iterations, an extended oligonucleotide is obtained. In embodiments of the present invention, a coupling cycle may be further characterized, for example, by describing it as including steps (b) through (e) or steps (b') through (e'). In the aforementioned coupling cycles comprising steps (b) through (e), i.e., steps (b), (c), (d), and (e), these steps are performed in this order, and step (e) is optional unless otherwise indicated in the context of a particular embodiment. It should be understood that describing a coupling cycle as including steps (b) through (e) does not alter the fact that step (e) is optional and may or may not be performed. In the aforementioned coupling cycles comprising steps (b') through (e'), i.e., steps (b'), (c'), (d'), and (e'), these steps are performed in that order, with step (e') being optional unless otherwise indicated in the context of a particular embodiment. It will be understood that describing a coupling cycle as comprising steps (b') through (e') does not alter the fact that step (e') is optional and may or may not be performed. Furthermore, it will be understood that the coupling cycle may include additional steps inserted before, between, or after the identified steps.

[0074] The method of the present invention includes an initial coupling cycle carried out after step (a) and comprising steps (b) to (e). Optionally, this initial coupling cycle is followed by (n-1) additional repetitions of the coupling cycle comprising steps (b') to (e'), where n is an integer ranging from 2 to 99 and represents the total number of coupling cycles carried out to obtain the nth cycle of oligonucleotide On. The total number of coupling cycles carried out includes the initial coupling cycle comprising steps (b) to (e) as well as (n-1) repetitions of the coupling cycle comprising steps (b') to (e'), but does not include any coupling cycles optionally carried out, for example, to prepare component C-0 or any of the building blocks used. Each coupling cycle comprising steps (b') to (e') is identified herein by a sequential number x, which runs from 2 to n, where n is the integer representing the total number of coupling cycles carried out to obtain the nth cycle of oligonucleotide On.

[0075] In some embodiments of the methods of the present invention, n is an integer ranging from 2 to 89, from 2 to 79, from 2 to 69, from 2 to 59, from 2 to 49, from 2 to 39, from 2 to 29, or from 2 to 19.

[0076] For example, when n=2, it will be understood that the method according to the invention comprises a total of two coupling cycles: an initial coupling cycle comprising steps (b) to (e) and (n-1)=1, i.e., 1 iteration, of a coupling cycle comprising steps (b') to (e'), the latter numbered as x=2=n. When n=30, it will be understood that the method according to the invention comprises a total of 30 coupling cycles: an initial coupling cycle comprising steps (b) to (e) and (n-1), i.e., 29 iterations, of a coupling cycle comprising steps (b') to (e'), the latter numbered as consecutive integers x, and performed 2 to 30 times.

[0077] The final repetition of the coupling cycle including steps (b') to (e') is called the nth cycle, i.e., x = n. Therefore, the serial number x is used herein to identify any particular coupling cycle including steps (b') to (e'), the components used in the coupling cycle, and the oligonucleotide obtained from the coupling cycle. For example, the component Bx is understood to be, for example, component B-5 in the fifth coupling cycle (x = 5). Furthermore, the oligonucleotide Ox in the xth cycle is understood to be, for example, oligonucleotide O-5 in the fifth coupling cycle. Since the first coupling cycle including steps (b) to (e) is counted in the total number of coupling cycles, it is readily understood that the fifth coupling cycle is the fourth repetition of the coupling cycle including steps (b') to (e'). Therefore, the serial number x does not indicate the number of repetitions of the coupling cycle including steps (b') to (e'). Instead, x is simply used to consecutively number the coupling cycles including steps (b') to (e'). In other words, x is used to consecutively number the coupling cycles from the second coupling cycle onwards.

[0078] Furthermore, since x starts from 2 and is performed one by one, it is understood that the coupling cycle before any given coupling cycle x is called coupling cycle (x-1), and the oligonucleotide obtained in this previous coupling cycle is called oligonucleotide O-(x-1) of the (x-1)th cycle. Thus, the xth coupling cycle begins with removing the protecting group PG-(x-1) from oligonucleotide O-(x-1) of the (x-1)th cycle of the previous coupling cycle. Next, oligonucleotide Ox of the xth cycle of coupling cycle x is obtained by removing the protecting group PG-(x-1) from oligonucleotide O-(x-1) of the (x-1)th cycle. #with building block Bx. For example, in the second coupling cycle, oligonucleotide O-(x-1) of the (x-1)th cycle becomes oligonucleotide O-1 of the first cycle of step (d) or (e). As another example, in the third coupling cycle, oligonucleotide O-(x-1) of the (x-1)th cycle becomes oligonucleotide O-2 of the second cycle of step (d') or (e'). It will further be understood that the nth cycle is the final coupling cycle to generate oligonucleotide On of the nth cycle.

[0079] Step (a) of the method of the present invention is providing a component C-0 selected from the group consisting of nucleosides and oligonucleotides, where component C-0 is covalently linked to a solid support and comprises a backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue.

[0080] The terms "nucleoside" and "oligonucleotide" are explained above. In some embodiments of the methods of the present invention, component C-0 is a nucleoside. In some embodiments of the methods of the present invention, component C-0 is an oligonucleotide. In some embodiments of the methods of the present invention, component C-0 is an oligonucleotide comprising 50, 40, 30, 25, 20, 15, 10, or 5 or less nucleoside subunits. In some embodiments of the methods of the present invention, component C-0 is selected from the group consisting of nucleosides and oligonucleotides comprising 50, 40, 30, 25, 20, 15, 10, or 5 or less nucleoside subunits.

[0081] The term "backbone hydroxyl moiety" refers to a hydroxyl moiety that is part of the backbone of component C-0, and will be understood based on the above explanation of the terms "hydroxyl moiety" and "backbone." Preferably, the hydroxyl moiety is a hydroxyl moiety of a carbohydrate moiety, in particular an optionally substituted and / or protected ribose or 2'-deoxyribose moiety. Preferably, the backbone hydroxyl moiety is contained in the terminal nucleoside subunit of component C-0. In some preferred embodiments, component C-0 comprises exactly one backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue. In some preferred embodiments, component C-0 comprises exactly one protecting group comprising an optionally substituted triarylmethyl residue, which is the protecting group PG-0.

[0082] In some embodiments of the methods of the present invention, the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0, is a compound of formula I: [ka] [In Formula I: Each oxygen atom (O) depicted within each nucleoside subunit x-0 through xm represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; Each of the nucleoside subunits x-0 through xm is the same or different (i.e., has the same or different chemical structure); PG-0 is a protecting group containing an optionally substituted triarylmethyl residue, preferably a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; m is an integer greater than or equal to 0; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1and S.R. z-1 independently selected from the group consisting of: R z-1 is a protecting group that is the same or different for each repeat unit m; CA is a capping moiety or covalent chemical bond; L is a linker moiety or a covalent chemical bond; SM is a solid support].

[0083] In some embodiments of the methods of the present invention, the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 of formula I, is a compound of formula Ia: [ka] [In formula Ia: m, PG-0, Y 1 , Z 1 , R z-1 , CA, L, and SM are defined as in Formula I; B N are nucleobases which are the same or different in each occurrence; R VI is, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R VIII is independently in each occurrence H or R of the same nucleoside subunit VIII and R VI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R VIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R VIis the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R VII , R IX , and R X are all H or are linked together, so that each nucleoside subunit has the structure of formula Ia-tc: [ka] In formulas Ia-tc: The oxygen atom from which the dashed line originates is the 3'-carbon atom, i.e., R in formula Ia. VII represents an oxygen atom bonded to a carbon atom to which is bonded; The dashed lines represent the connection between each oxygen atom of the nucleoside subunit of formula Ia-tc and the CA-L-SM or P(Y 1 )(Z 1 ) indicates a covalent chemical bond interconnecting one of the following; The oxygen atom from which the wavy line originates is the oxygen atom attached to the 5'-carbon atom, i.e., C(R IX )(R X ) represents the carbon atom to which it is attached; The wavy lines represent the connection between each oxygen atom of the nucleoside subunit of formula Ia-tc and PG-0 or P(Y 1 )(Z 1 ) indicates a covalent chemical bond interconnecting one of the

[0084] In some embodiments of the methods of the present invention, the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 of formula I, particularly formula Ia, is a compound of formula Ib: [ka] [In formula Ib: m, PG-0, Y 1 , Z 1 , R z-1 , B N, C.A., L., S.M., R. VI , and R VIII is defined as in formula Ia, Independently for each nucleoside subunit, R VII , R IX , and R X are all H or are linked together, so that each nucleoside subunit has the structure of formula Ib-tc: [ka] In formula Ib-tc: The oxygen atom from which the dashed line originates is the 3'-carbon atom, i.e., R in formula Ib. VII represents an oxygen atom bonded to a carbon atom to which is bonded; The dashed lines represent the connection between each oxygen atom of the nucleoside subunit of formula Ib-tc and the CA-L-SM or P(Y 1 )(Z 1 ) indicates a covalent chemical bond interconnecting one of the following; The oxygen atom from which the wavy line originates is the 5'-carbon atom, i.e., C(R IX )(R X ) represents an oxygen atom bonded to a carbon atom to which it is bonded; The wavy lines represent the connection between each oxygen atom of the nucleoside subunit of formula Ib-tc and PG-0 or P(Y 1 )(Z 1 ) indicates a covalent chemical bond interconnecting one of the

[0085] In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 of any one of Formulas I, Ia, and Ib, CA is a covalent chemical bond and L is a linker moiety. In such embodiments, it will be understood that CA of any one of Formulas I, Ia, and Ib is a covalent chemical bond interconnecting the 3'-O atom of each nucleoside subunit (e.g., nucleoside subunit x-0 of Formula I) and the linker moiety L. In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 of any one of Formulas I, Ia, and Ib, L is a covalent chemical bond and CA is a capping moiety. In such embodiments, it will be understood that L of any one of Formulas I, Ia, and Ib is a covalent chemical bond interconnecting the capping moiety CA and the solid support SM. In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by protecting group PG-0, e.g., component C-0 of any one of Formulas I, Ia, and Ib, CA and L are covalent chemical bonds. In such embodiments, it will be understood that both CA and L of any one of Formulas I, Ia, and Ib together represent the same covalent chemical bond interconnecting the 3'-O atom of the respective nucleoside subunit (e.g., nucleoside subunit x-0 of Formula I) and the solid support SM. Thus, in such embodiments, CA and L do not represent two adjacent covalent chemical bonds.

[0086] As used herein, the term "solid support" refers to a macroscopically insoluble solid or gel-like substrate, and the term "insoluble" refers to the solvent used throughout oligonucleotide synthesis. In accordance with common practice, the terms "solid support," "insoluble support," and "solid phase" or "solid-phase" are used interchangeably herein. These terms encompass the terms "resin" and "resin," which are understood in the broadest sense as particulate structures that can be used in solid-phase oligonucleotide synthesis. Widely used examples of suitable solid supports are controlled pore glass (CPG), such as long-chain alkylamine-CPG bead supports, cross-linked polystyrene beads, and cross-linked polystyrene-PEG composites, such as Tentagel™ resin. Because solid supports are insoluble in the solvents used in oligonucleotide synthesis, the conjugates of the solid support and the growing oligonucleotide chain are similarly insoluble, and therefore are isolated from the reaction mixture (liquid phase) by simple filtration, e.g., by draining the solvent through a filter, membrane, or frit that retains the solid support and oligonucleotide conjugate in the reaction vessel. In oligonucleotide synthesis, the components of the reaction mixture typically vary for different steps of the coupling cycle, e.g., for each coupling cycle including steps (b) through (e). Therefore, being able to drain the reaction mixture while retaining the growing oligonucleotide chain in the reaction vessel greatly facilitates the process. In addition, washing steps are typically performed between coupling cycle steps, in which the conjugates of the solid support and the growing oligonucleotide chain are treated with a pure solvent, followed by draining the solvent, to remove excess reagents or reactants, as well as soluble by-products. The growing oligonucleotide chain is typically attached (directly or via a linker) to the solid support via a functional group, typically a hydroxyl moiety, for which the solid support serves as a permanent protecting group.Thus, as used herein, the terms "protecting group" and "permanent protecting group" (but not the term "temporary protecting group") encompass solid supports, unless otherwise indicated in the context of a particular embodiment. Oligonucleotide synthesis using solid supports is also referred to herein as solid-phase oligonucleotide synthesis (SPOS). Any solid support used in solid-phase oligonucleotide synthesis can be used in the methods of the present invention.

[0087] A "linker moiety" is used to establish a link between a solid support and a nucleoside subunit of an oligonucleotide to be synthesized or a capping moiety attached thereto. As used herein, the term "linker moiety" refers to a moiety exhibiting at least two functional groups, one of which can participate in a covalent chemical bond with a functional group on the solid support, and the other of which can participate in a covalent chemical bond with a functional group of a nucleoside moiety of an oligonucleotide to be synthesized or a capping moiety attached thereto. Thus, the phrase "a nucleoside or oligonucleotide (e.g., component C-0) is covalently linked to a solid support" is understood to encompass both a direct covalent bond between the nucleoside or oligonucleotide and the solid support, and a covalent interconnection between the nucleoside or oligonucleotide and the solid support in the form of a linker moiety.

[0088] Typically, the solid support bears amino groups (e.g., aminomethyl groups of a polymer resin obtained by copolymerization of poly(styrene) with divinylbenzene followed by introduction of amino groups, e.g., by using an aminomethylation reaction), and the linker has a functional group capable of forming an amide bond with such an amino group on the support. The functional group of the nucleoside or capping moiety involved in the covalent chemical bond with the linker moiety is, for example, a hydroxyl or amino group, preferably a hydroxyl group, particularly the 3'-hydroxyl group of a ribose or 2'-deoxyribose moiety. For example, succinic acid-type linker moieties or hydroquinone-O,O'-diacetic acid linker moieties (also called "Q-linkers") are used, both of which are known to those skilled in the art. Other commonly used linkers are so-called universal linkers, which typically contain a DMT-protected hydroxyl moiety, to which the first nucleoside subunit is coupled (after initial detritylation). A non-limiting example of a suitable universal linker is the "Unylinker" type linker described in US Pat. No. 7,202,264.

[0089] In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 according to any one of formulas I, Ia, and Ib, when L is a linker moiety, L is a moiety according to any one of the following formulas LI, L-II, L-III, and L-IV: [ka] [In formulas LI and L-II: One of the two dashed lines indicates a covalent chemical bond to a hydroxyl moiety of the capping moiety CA or to a respective hydroxyl moiety of a respective nucleoside subunit (e.g., nucleoside subunit x-0 of Formula I); The other of the two dashed lines indicates a covalent chemical bond with a functional group of the solid support, preferably an amine or hydroxyl moiety; [ka] In formulas L-III and L-IV: Y 1 and Z 1 is defined as in formula I; The wavy lines indicate covalent chemical bonds between the hydroxyl moiety of the capping moiety CA or the respective hydroxyl moiety of the respective nucleoside subunit (e.g., nucleoside subunit x-0 of Formula I); The dashed line indicates a covalent chemical bond with a functional group, preferably an amine or hydroxyl moiety, of the solid support; X L-1 is selected from the group consisting of O, S, and a nitrogen atom (N) to which is attached a residue selected from the group consisting of H, a phenyl moiety (Ph) and a C1-C6-alkyl group; X L-2 is selected from the group consisting of a carbon atom (C) to which two residues independently selected from the group consisting of O, S, H and C1-C6-alkyl groups are bonded, and a nitrogen atom (N) to which a residue selected from the group consisting of H, C1-C6-alkyl groups and a phenyl (Ph) moiety is bonded.

[0090] In such embodiments, when CA is a covalent chemical bond, it will be understood that CA is one of the dashed lines in formulas LI and L-II or the wavy lines in formulas L-III and L-IV.

[0091] In some embodiments, in the linker moiety of any one of formulas L-III and L-IV: Y 1 is selected from the group consisting of O and S; Z 1 is OR z-1 , S.R. z-1 selected from the group consisting of: R z-1 is a protecting group removable under alkaline conditions, preferably a 2-cyanoethyl group (i.e., CH2-CH2-CN); X L-1is selected from the group consisting of O and a nitrogen atom (N) to which is attached a residue selected from the group consisting of a phenyl moiety (Ph) and a C1-C6-alkyl group; X L-2 is selected from the group consisting of O, S, a methylene group (CH2), and a nitrogen atom (N) having attached thereto a residue selected from the group consisting of CH3 and phenyl (Ph).

[0092] In some embodiments, in the linker moiety of any one of formulas L-III and L-IV: Y 1 is selected from the group consisting of O and S; Z 1 is OR z-1 and; R z-1 is a 2-cyanoethyl group (i.e., CH2-CH2-CN); X L-1 is selected from the group consisting of O and a nitrogen atom (N) having attached thereto a residue selected from the group consisting of a phenyl moiety (Ph) and CH3; X L-2 is O.

[0093] In some embodiments, in the linker moiety of any one of formulas L-III and L-IV: Y 1 is selected from the group consisting of O and S; Z 1 is OR z-1 and; R z-1 is a 2-cyanoethyl group (i.e., CH2-CH2-CN); X L-2 is the nitrogen atom (N) to which the phenyl moiety (Ph) is attached; X L-2 is O.

[0094] In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support, e.g., component C-0 according to any one of formulas I, Ia, and Ib, when L is a linker moiety, L is a moiety according to any one of formulas LI, L-II, L-III, and L-IV, and CA is a covalent chemical bond (i.e., is not a capping moiety).

[0095] As used herein, the term "capping moiety" refers to any moiety conjugated to the terminal nucleoside of an oligonucleotide chain, which is neither a nucleoside nor an oligonucleotide moiety, and preferably comprises a carbohydrate moiety, examples of which are the carbohydrate-containing moieties disclosed in WO2009073809.

[0096] In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 of any one of Formulas I, Ia, and Ib, the integer m is an integer ranging from 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1. Reference to an integer ranging from 0 to 5 will be understood to mean that the integer is 0, 1, 2, 3, 4, or 5. In some embodiments, in the nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., component C-0 of any one of Formulas I, Ia, and Ib, the integer m is 0. It will be understood that when the integer m in any one of Formulas I, Ia, and Ib is 0, the protecting group PG-0 is attached to the hydroxyl moiety of each of the remaining nucleoside subunits bearing -CA-L-SM (e.g., nucleoside subunit x-0 of Formula I).

[0097] In some embodiments, in the nucleoside or oligonucleotide, e.g., component C-0, of any one of Formulas I, Ia, and Ib, which is covalently linked to a solid support and which comprises a backbone hydroxyl moiety protected by a protecting group PG-0, the protecting group PG-0 is selected from the group consisting of a triphenylmethyl group (i.e., a trityl group), a (p-methoxyphenyl)diphenylmethyl group (i.e., an MMT group), and a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group). In some preferred embodiments, in the nucleoside or oligonucleotide, e.g., component C-0, of any one of Formulas I, Ia, and Ib, which is covalently linked to a solid support and which comprises a backbone hydroxyl moiety protected by a protecting group PG-0, the protecting group PG-0 is a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group).

[0098] In some embodiments, in Formulas I, Ia, and Ib, Y 1 is O for every m repeat units. In some embodiments, in Formulas I, Ia, and Ib, Y 1 is S for each repeat unit m.

[0099] In some embodiments, in Formulas I, Ia, and Ib, R z-1 is a protecting group removable under alkaline conditions, and R z-1are the same or different in each occurrence. As used herein, a protecting group is "removable under alkaline conditions" if it is cleaved by treatment with a base ("base" should not be construed to mean any base, but rather a specific base compatible with the protecting group being removed based on the common knowledge and routine experimentation of one of ordinary skill in the art). In this context, the term "base" is understood as a proton acceptor in the sense of the Bronsted-Lowry theory. Examples of such bases include ammonia, particularly aqueous ammonia (i.e., aqueous ammonium hydroxide), methylamine, tert-butylamine, diethylamine, triethylamine, diisopropylethylamine, and the like. In some embodiments, in any one of Formulas I, Ia, and Ib, R z-1 is, independently for each repeat unit m, a protecting group of the chemical structure CH2-CH2-EWG, where EWG is an electron-withdrawing group, preferably a cyano group. The electron-withdrawing group may be selected from the group consisting of, for example, a cyano group, a halogen atom, such as a chlorine, fluorine, or bromine atom, an aldehyde group, a keto group, a carboxyester group, or a carboxamide group. In some preferred embodiments, in any one of Formulas I, Ia, and Ib, R z-1 is a 2-cyanoethyl group (i.e., CH2-CH2-CN) for every m repeat units.

[0100] In some embodiments, in any one of formulas I, Ia, and Ib, Z 1 For each repeat unit m, OR z-1 and S.R. z-1 In some embodiments, in any one of formulas I, Ia, and Ib, Z 1 For each repeat unit m, OR z-1 and S.R. z-1 and R is independently selected from the group consisting of z-1 is a 2-cyanoethyl group (i.e., CH2-CH2-CN) for every m repeat units. In such embodiments, Z 1is independently selected from the group consisting of O-CH2-CH2-CN and S-CH2-CH2-CN for each repeat unit m. In some embodiments, in any one of Formulas I, Ia, and Ib, Z 1 For each repeat unit m, OR z-1 and R z-1 is a 2-cyanoethyl group (i.e., CH2-CH2-CN) for every m repeat units. In such embodiments, Z 1 is O-CH2-CH2-CN for every m repeat units.

[0101] In some embodiments, in any one of Formulas I, Ia, and Ib, the terminal nucleoside subunit having a hydroxyl moiety attached to a PG-0 protecting group is a nucleoside subunit that includes a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, particularly adenine.

[0102] Those skilled in the art can easily identify any kind of nucleobase B in any one of formulas Ia and Ib. N It will be understood that B may be present. See, for example, the discussion of nucleobases above. In some embodiments, in any one of formulas Ia and Ib, B N is a nucleobase, each occurrence independently selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil. N It will be understood by those skilled in the art that, even if not specifically shown, B is optionally protected, i.e., carries one or more protecting groups. Thus, for example, B N is described as adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil, this encompasses the aforementioned nucleobases in protected and free form (i.e., with or without protecting groups). The same rationale applies to nucleobases in general.

[0103] Those skilled in the art are familiar with nucleic acid base protecting groups and know how to select, introduce and remove them.In particular, the exocyclic amino group of nucleic acid bases such as adenine, guanine, cytosine and 5-methylcytosine is protected.Non-limiting examples of the exocyclic amino group protecting group of nucleic acid base include acetyl group, benzoyl group, isobutyryl group, pivaloyl group, pivaloyloxymethyl group, trifluoroacetyl group, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, 4-tert-butylphenoxyacetyl group and dimethylformamidinyl group. Carbonyl groups present in nucleobases such as thymine, uracil, and guanine can also be protected by reaction with, for example, phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, diphenylcarbamoyl chloride, etc. Examples of preferred nucleobase protecting groups are summarized in Table T-1 below.

[0104] [Table 1]

[0105] In some embodiments of the methods of the invention, the nucleoside or oligonucleotide is covalently linked to a solid support and comprises a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., each nucleobase of component C-0 of any one of formulas I, Ia, and Ib, particularly each nucleobase B N teeth, - adenine with a protected exocyclic amino group; - guanine with a protected exocyclic amino group; - cytosine with a protected exocyclic amino group; - 5-methylcytosine with a protected exocyclic amino group; - thymine; and - Uracil are independently selected from the group consisting of:

[0106] In some embodiments of the methods of the invention, the nucleoside or oligonucleotide is covalently linked to a solid support and comprises a backbone hydroxyl moiety protected by a protecting group PG-0, e.g., each nucleobase of component C-0 of any one of formulas I, Ia, and Ib, particularly each nucleobase B N teeth, - adenine whose exocyclic amino group is protected by a benzoyl, isobutyryl or phenoxyacetyl group; - guanine whose exocyclic amino group is protected by an isobutyryl group, a 4-isopropylphenoxyacetyl group or a dimethylformamidino group; - cytosine in which the exocyclic amino group is protected by an acetyl or benzoyl group; - 5-methylcytosine, in which the exocyclic amino group is protected by an acetyl or benzoyl group; - thymine; and - Uracil are independently selected from the group consisting of:

[0107] In some embodiments, in any one of formulas Ia and Ib: R VIis, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R VIII is independently in each occurrence H or R of the same nucleoside subunit VIII and R VI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R VIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R VI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R VII , R IX , and R X are all H or are linked together, and thus each nucleoside subunit has the structure of formula Ia-tc (of formula Ia) or formula Ib-tc (of formula Ib) above.

[0108] In some embodiments, in any one of formulas Ia and Ib: R III is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), and O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy); R IV is H in each occurrence.

[0109] In some embodiments, in Formula I: PG-0 is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; m is an integer in the range of 0-150, 0-100, 0-75, 0-50, 0-35, 0-30, 0-25, 0-20, 0-15, 0-10, 0-5, or 0-1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and S.R. z-1 independently selected from the group consisting of: R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety or a covalent chemical bond; SM is a solid support.

[0110] In some embodiments, in Formula I: PG-0 is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and S.R. z-1 independently selected from the group consisting of: R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety according to any one of the foregoing formulae LI, L-II, L-III, and L-IV, or a covalent chemical bond; SM is a solid support.

[0111] In some embodiments, in Formula I: PG-0 is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and; R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety according to any one of formulas LI, L-II, L-III, and L-IV above; SM is a solid support.

[0112] In some embodiments, in any one of formulas Ia and Ib: m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and S.R. z-1 independently selected from the group consisting of: R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety or a covalent chemical bond; SM is a solid support; B N are nucleobases which are the same or different in each occurrence; R VIis, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R VIII is independently in each occurrence H or R of the same nucleoside subunit VIII and R VI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R VIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R VI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R VII , R IX , and R X are all H or are linked together, and thus each nucleoside subunit has the structure of formula Ia-tc (of formula Ia) or formula Ib-tc (of formula Ib) above.

[0113] In some embodiments, in any one of formulas Ia and Ib: m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and S.R. z-1 independently selected from the group consisting of: R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety according to any one of the foregoing formulae LI, L-II, L-III, and L-IV, or a covalent chemical bond; SM is a solid support; B N are nucleobases which are the same or different in each occurrence; R VI is, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R VIII is independently in each occurrence H or R of the same nucleoside subunit VIII and R VI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R VIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R VI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R VII , R IX , and R X are all H or are linked together, and thus each nucleoside subunit has the structure of formula Ia-tc (of formula Ia) or formula Ib-tc (of formula Ib) above.

[0114] In some embodiments, in any one of formulas Ia and Ib: m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1For each repeat unit m, OR z-1 and; R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety or a covalent chemical bond; SM is a solid support; B N are nucleobases which are the same or different in each occurrence; R VI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R VIII is independently in each occurrence H or R of the same nucleoside subunit VIII and R VI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R VIII is the point of attachment to the carbon atom to which RVI is attached, and ++ is the point of attachment to the 2'-carbon (i.e., the carbon atom to which RVI is attached); Independently for each nucleoside subunit, R VII , R IX , and R X are all H or are linked together, and thus each nucleoside subunit has the structure of formula Ia-tc (of formula Ia) or formula Ib-tc (of formula Ib) above.

[0115] In some embodiments, in any one of formulas Ia and Ib: m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and; R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety according to any one of the foregoing formulae LI, L-II, L-III, and L-IV, or a covalent chemical bond; SM is a solid support; B N are nucleobases which are the same or different in each occurrence; R VI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R VII , R VIII , R IX , and R X is H in each occurrence.

[0116] In some embodiments, in any one of formulas Ia and Ib: m is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 to 1; Y 1is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and; R z-1 is a 2-cyanoethyl group for every m repeat units; CA is a covalent chemical bond; L is a linker moiety according to any one of formulas LI, L-II, L-III, and L-IV above; SM is a solid support; B N are nucleobases which are the same or different in each occurrence; R VI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R VII , R VIII , R IX , and R X is H in each occurrence.

[0117] The term "providing component C-0" in step (a) is understood in the broadest sense. Thus, "providing" means making available or introducing component C-0. For example, nucleoside-loaded solid supports for many standard nucleosides can be easily obtained from commercial suppliers such as Merck and Cytiva, among others. When the linker moiety is, for example, a succinate-type linker of formula L-1 or a hydroquinone-type linker of formula L-II, the first nucleoside subunit, if commercially available, is typically already attached to the solid support via the linker. Alternatively, an appropriately protected nucleoside is loaded onto a given solid support. For example, when using a universal linker such as that of formula L-III or L-IV, the first nucleoside subunit is typically not loaded onto a commercially available support, and therefore step (a) then involves coupling the first nucleoside subunit (e.g., as a phosphoramidite) to a universal linker support (from which the hydroxyl protecting group has been previously removed, see, e.g., US Pat. No. 7,202,264). All of this is common practice in the field of oligonucleotide synthesis and is well known to those skilled in the art. Those skilled in the art are aware of the many immobilization strategies available for this purpose and will routinely select one of them. Although the solid support-bound oligonucleotide is typically not commercially available, it is preferred that it be synthesized by some means. Those skilled in the art are also well aware of the many ways to obtain component C-0 by oligonucleotide synthesis, for example, by the means disclosed in US Pat. No. 7,202,264 B2 and RTPon et al., Nucleic Acids Research, 1997, 25(18), pp. 3629-3635.

[0118] Step (a) of the method of the present invention involves conditioning the solid support by washing with various solvents. These steps typically involve treating the solid support to which component C-0 is covalently linked with the respective solvent, for example DMF, followed by draining off said solvent.

[0119] Step (b) of the method of the present invention involves incubating a nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from the nucleoside or oligonucleotide, wherein said deprotection mixture Mb is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently attached to an optionally substituted phenyl moiety.

[0120] When step (b) is carried out after step (a) as part of the first coupling cycle comprising steps (b) to (e), step (b) comprises incubating component C-0 of step (a) with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from component C-0 to yield component C-0 having a free backbone hydroxyl group. # This is the process for obtaining the above.

[0121] Step (b') of the method of the present invention is to incubate the (x-1)th cycle oligonucleotide O-(x-1) obtained in the coupling cycle, i.e., the (x-1)th coupling cycle, with a deprotection mixture M-b', thereby cleaving the protecting group PG-(x-1) from the (x-1)th cycle oligonucleotide O-(x-1) to obtain the (x-1)th cycle oligonucleotide (O-(x-1)) having a free backbone hydroxyl group. # This is the process for obtaining the above.

[0122] The "nucleoside or oligonucleotide comprising a backbone hydroxyl moiety covalently linked to a solid support and protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue" is defined. This is also the defined component C-0. The term "cleaving a protecting group" is defined. The protecting group PG-0 is described above. The protecting group PG-(x-1) is also a "protecting group comprising an optionally substituted triarylmethyl residue" as defined above. In some embodiments of the method of the present invention, the protecting group PG-(x-1) is selected from the group consisting of a triphenylmethyl group (i.e., a trityl group), a (p-methoxyphenyl)diphenylmethyl group (i.e., an MMT group), and a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group) for each repetition of the coupling cycle. In some preferred embodiments of the method of the present invention, the protecting group PG-(x-1) is a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group) for each repetition of the coupling cycle.

[0123] In some embodiments of the methods of the present invention, each protecting group that comprises an optionally substituted triarylmethyl residue is a di(p-methoxyphenyl)phenylmethyl protecting group.

[0124] The oligonucleotide O-(x-1) of the (x-1)th cycle is described as referring to the oligonucleotide obtained in the previous coupling cycle, i.e., the (x-1)th coupling cycle.For example, in step (b') of the fifth iteration of the coupling cycle, x=5, the oligonucleotide O-(x-1) of the (x-1)th cycle is the oligonucleotide O-4 of the fourth cycle.The serial number x is the same for each oligonucleotide and its protecting group PG.For example, the oligonucleotide O-4 of the fourth cycle contains protecting group PG-4, and the oligonucleotide of the fifth cycle contains protecting group PG-5.

[0125] The terms "backbone" and "free hydroxyl group" are explained above. As used herein, a free backbone hydroxyl group is a free hydroxyl group located on the backbone of each nucleoside or oligonucleotide, e.g., the free 5'-OH group of a ribose or 2'-deoxyribose moiety. For ease of understanding, the products resulting from step (b) or (b'), which all contain free backbone hydroxyl groups according to the definition, are denoted by the superscript " # ". Thus, the component obtained from subjecting component C-0 to step (b) is designated by the number sign (i.e., hashtag) "Component C-0." # Similarly, the oligonucleotide obtained by subjecting the (x-1)th cycle oligonucleotide (O-(x-1)) obtained in the previous coupling cycle to step (b') is called the (x-1)th cycle oligonucleotide (O-(x-1)). # It is called.

[0126] In some embodiments of the methods of the present invention: - Ingredient C-0 # differs from component C-0 only in that the protecting group PG-0 is absent and therefore the hydroxyl moiety previously protected by the protecting group PG-0 is now present as a free hydroxyl group; - oligonucleotide (O-(x-1)) of each (x-1)th cycle # differs from the corresponding (x-1)th cycle oligonucleotide O-(x-1) only in that the protecting group PG-(x-1) is not present and therefore the hydroxyl moiety previously protected by the protecting group PG-(x-1) is now present as a free hydroxyl group.

[0127] The term "incubating" in the context of step (b) refers to any process in which the nucleoside or oligonucleotide, e.g., the component C-0, which is covalently linked to a solid support and comprises a backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue, and the deprotection mixture Mb are combined in a reaction vessel or reactor (e.g., a batch reactor or a column reactor). Typically, the reaction vessel or reactor already contains, e.g., the nucleoside or oligonucleotide, e.g., the component C-0, to which the deprotection mixture Mb is subsequently added.

[0128] The term "incubating" in the context of step (b) refers to any process in which the nucleoside or oligonucleotide, e.g., the component C-0, which is covalently linked to a solid support and includes a backbone hydroxyl moiety protected by a protecting group PG-0 containing an optionally substituted triarylmethyl residue, and the deprotection mixture Mb are combined in a reaction vessel or reactor (e.g., a batch reactor or a column reactor). Typically, the reaction vessel or reactor already contains the nucleoside or oligonucleotide, e.g., the component C-0, and then the deprotection mixture Mb is added. The term "incubating" in the context of step (b') refers to any process in which the oligonucleotide O-(x-1) of the (x-1)th cycle obtained in the previous coupling cycle and the deprotection mixture M-b' are combined in a reaction vessel or reactor (e.g., a batch reactor or a column reactor). Typically, the reaction vessel or reactor already contains the oligonucleotide O-(x-1) of the (x-1)th cycle, and then the deprotection mixture M-b' is added.

[0129] Solid-phase oligonucleotide synthesis is generally carried out using a column reactor. As used herein, the term "column reactor" is understood in its broadest sense as any reactor in which a column (e.g., made of stainless steel, glass, or another material) is packed with a solid support to which a growing oligonucleotide chain is tethered, and the column preferably does not have a mechanical stirrer or other agitation device. Instead, any reaction solution and solvent for washing typically passes through the column, usually from top to bottom, but may alternatively pass from top to bottom and bottom to top to achieve fluidization of the packed support. The reaction solution and solvent for washing may be circulated within the column reactor (i.e., passed through the column two or more times). The column reactor typically has a bottom frit and an upper frit, between which the solid support (to which the growing oligonucleotide chain is tethered) is packed. The size and dimensions of the reaction column are selected according to the intended scale of synthesis. For example, column reactors with a minimum internal volume of 5, 10, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280, or 300 liters are used. In certain embodiments, column reactors with a maximum internal volume of 1 to 500 liters, 5 to 450 liters, 10 to 400 liters, 50 to 300 liters, or 100 to 300 liters are used. The terms "fixed bed," "tight bed," and "packed bed" are coined interchangeably with respect to column reactors, in which the solid supports carrying the growing oligonucleotide chains are not agitated and liquid is pumped unidirectionally through a settled bed of solid supports (also called a resin bed), including, for example, unidirectional circulation of liquid through a loop containing the column. This fixed-bed approach is the current gold standard in automated solid-phase oligonucleotide synthesis.Fixed-bed reactors have been known for decades (see, e.g., US Pat. No. 6,623,703 B1 and US Pat. No. 6,825,339 B2) and are commercially available, for example, from Cytiva (see, e.g., OligoPilot™ Oligonucleotide Synthesizer and OligoProcess™ Oligonucleotide Synthesizer). Fixed-bed reactors are suitable for carrying out the method of the present invention. However, the method of the present invention is not limited to such reactors. Column reactors can also be operated as stirred-bed reactors, which means herein that the suspension of solid supports in the column is not tightly packed and is agitated by some means at least once during oligonucleotide synthesis. Suitable means for agitation include sparging with an inert gas such as nitrogen (i.e., bubbling), and directing liquid flow up and down within the reactor (i.e., bidirectional flow), as disclosed, for example, in EP 0 130 739 A2. Although less common in column reactors, agitation, e.g., mechanical stirring, constitutes an alternative means of agitation.

[0130] When a column reactor is used, step (b) involves passing or circulating the deprotection mixture Mb through the column at a constant temperature, for a constant time, and at a constant flow rate. The inventors have surprisingly found that, particularly when the deprotection mixture Mb is liquid composition C, the flow rate of the deprotection mixture Mb through the column reactor can be quite low in some cases, for example, less than 300 cm / hr, less than 290 cm / hr, less than 280 cm / hr, less than 270 cm / hr, less than 260 cm / hr, less than 250 cm / hr, less than 240 cm / hr, less than 230 cm / hr, less than 220 cm / hr, less than 210 cm / hr, less than 200 cm / hr, less than 190 cm / hr, less than 180 cm / hr, or less than 160 cm / hr. This significantly improves the scalability of the process. The same rationale applies to step (b') and the deprotection mixture M-b'.

[0131] In some embodiments of the process of the present invention, the process is carried out in a column reactor and in step (b), the flow rate of said liquid composition C through the column reactor is less than 300 cm / hr. In some embodiments of the process of the present invention, the process is carried out in a column reactor and in steps (b) and (b'), the flow rate of liquid composition C through the column reactor is less than 300 cm / hr.

[0132] Alternatively, the inventors have discovered that a batch reactor can be used in the method of the present invention. As used herein, the term "batch reactor" is understood in its broadest sense as any reaction vessel (e.g., any type of tank) that includes an agitation device (i.e., a mechanism or device that allows for agitation of the contents of the vessel). Thus, the reactor allows for the suspension of a solid support in a solution or liquid composition. Any means for agitating the contents of the reaction vessel can be used. On a large scale, for example, a mechanical agitation device is used. Alternatively, for example, in automated synthesizers for solid-phase oligonucleotide synthesis, agitation is achieved by bubbling an inert gas (e.g., nitrogen) through a vessel containing the reaction mixture. A batch reactor for solid-phase oligonucleotide synthesis preferably includes a membrane or filter that allows for the evacuation of any liquid from the reactor while retaining the solid support, the conjugate of the solid support, and the growing oligonucleotide chain within the reactor. One type of batch reactor is a so-called stirred-bed reactor. As used herein, the term "stirred-bed reactor" refers to a batch reactor equipped with a mechanical agitation device. Batch reactors for oligonucleotide synthesis are commonly known to those skilled in the art. The term "stirred bed reactor" refers to a reactor in which the agitation of the solid support is achieved at least in part by agitation, typically mechanical agitation. Such reactors are known to those skilled in the art and are commercially available, for example, from Buchi AG (Uster, Switzerland) (see, for example, https: / / www.buchiglas.ch / fileadmin / buchiglas_international / download / dvs / Brochures_and_Flyers / G_filter_reactors_flyer.pdf). Carrying out the method of the present invention in a stirred bed reactor is a preferred option. As another example, CN107881102A discloses a stirred bed reactor that can be used in the method of the present invention. As another example, WO00 / 66258A2 discloses a stirred bed reactor that can be used in the method of the present invention. In this reactor, the agitation of the suspension of the solid support is achieved by mechanical agitation and, optionally, by bubbling nitrogen.The term "sparged bed reactor" is coined for a reactor in which agitation of the solid support is achieved at least in part by sparging with an inert gas, typically nitrogen. Usually, the inert gas is bubbled through the suspension of solid support from bottom to top, preferably through a bottom frit, which has the added benefit of preventing clogging of the bottom frit. Sparged-bed reactors have been used for solid-phase oligonucleotide synthesis for decades (see, e.g., AAPadmapriya et al., Antisense Research and Development 1994, 4, 185-199, https: / / doi.org / 10.1089 / ard.1994.4.185; NDSinha, "Large-Scale Oligonucleotide Synthesis Using the Solid-Phase Approach," in: S. Agrawal, "Protocols for Oligonucleotides and Analogs," Methods in Molecular Biology 1993, Vol. 20, Humana Press. https: / / doi.org / 10.1385 / 0-89603-281-7:437) and are suitable for the method of the present invention. A combination of mechanical stirring and inert gas sparging may also be used. As disclosed in Padmapriya et al., agitation can be achieved simply by shaking the reaction vessel. Such reactors are also suitable for the method of the present invention. Agitation in a batch reactor can also be achieved by inverting the reaction vessel relative to a stationary position, as disclosed, for example, in US Pat. No. 1,524,976 B2. Such reactors are also suitable for the method of the present invention. The method of the present invention can be used for large-scale synthesis (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 mol of target oligonucleotide). T), stirred-bed reactors and sparged-bed reactors are preferred, with stirred-bed reactors being most preferred. Those skilled in the art will appreciate that, after some obvious adaptations, such as replacing peptide synthesis reagents with oligonucleotide synthesis reagents and replacing peptide synthesis supports with oligonucleotide synthesis supports, reactors and devices for solid-phase peptide synthesis will generally also be suitable for solid-phase oligonucleotide synthesis according to the method of the present invention. For example, automated peptide synthesizers such as Symphony®, Symphony® X, or PurePep® Sonata®+ by Gyros Protein Technologies can be used in the method of the present invention.

[0133] Batch reactors are typically cylindrical with a top and bottom housing. The top housing usually contains one or more inlets for adding solid supports, washing solvents, and reagents. When a mechanical stirring device is used, the stirring assembly is usually located above the reactor with a stirring shaft extending from an opening (usually a central port) in the top housing. The bottom housing usually contains some type of filtration means, such as a membrane or frit, that can retain the solid supports and the oligonucleotide strands attached to them within the reactor when the liquid is drained from the reactor. Liquid drainage is sometimes facilitated or accelerated by applying inert gas pressure (e.g., nitrogen pressure) to the reactor and / or by applying a vacuum from the bottom of the reactor (i.e., below the filtration means). The bottom housing is also designed to allow for inert gas sparging, i.e., bubbling an inert gas such as nitrogen through the solid support suspension from the bottom of the reactor. Batch reactors are fabricated from any suitable material, such as stainless steel and glass, although stainless steel is preferred on an industrial scale. In a batch reactor, the reaction solution and washing solvent are added, for example, through an inlet located at the top of the reactor. One or more additional inlets and outlets are used to add and remove gas, particularly to create an inert gas atmosphere, such as a nitrogen atmosphere. Nitrogen pressure may be used to assist in the evacuation of the liquid. The washing solvent can be added from the top of the reactor to rinse off any material adhering to the reactor walls. The inlet may be designed so that the added liquid rinses the reactor walls. The inlet may take the form of, for example, a nozzle or spray cone, especially when it is desired to add the washing solvent or reagent without disturbing the resin bed. The arrangement of the inlets is not particularly limited. Separate inlets may be used for the washing solvent and some or each of the reagents. Using separate inlets is beneficial because it reduces the risk of cross-contamination. Alternatively, the washing solvent and all or some of the reagents are added through the same inlet. In this case, it is useful to flush the inlet with pure solvent after adding the reagents to prevent cross-contamination.The reaction solution and washing solvent may be added, for example, through an inlet located at the bottom of the reactor, preferably below the filter, membrane, or frit. In such cases, the added liquid typically enters the vessel through the filter, membrane, or frit. In this manner, the added liquid causes agitation of the solid support suspension, potentially preventing clogging of the filter, membrane, or frit. The reaction solution and washing solvent can also be added alternately from the top and bottom of the reactor. Reactors for solid-phase oligonucleotide synthesis are designed to allow the temperature of their contents to be controlled, for example, by heating or cooling. Heating is typically known to accelerate chemical reactions. Heating or cooling is achieved, for example, by using a jacketed (i.e., double-walled) reactor in which a heating or cooling medium, such as water, circulates through the gap between the reactor walls. Other heating means include microwave irradiation, infrared irradiation, and conduction heating. In such cases, the reactor material must be adapted accordingly, for example, to be transparent to the respective irradiation. Alternatively, or in addition, the temperature of the reaction solution and optionally the wash solvent is adjusted before entering the reaction vessel, for example, by heating or cooling the storage tank and / or the line (i.e., tubing) that transports the reaction solution or wash solvent to the reaction vessel. Particularly on an industrial scale, it is preferable to control the temperature of the storage tank and / or tubing.

[0134] The method of the present invention can also be carried out using the reactors (column reactors and batch reactors) disclosed in WO2021 / 1100773A1. As another example, WO2021 / 094518A1 discloses a reactor that can be used in the method of the present invention.

[0135] Circulation loops are a useful feature of column and batch reactors. While circulation loops are routinely used in conjunction with column reactors, they are also advantageously applied to batch reactors. For example, CN107881102A discloses a batch reactor utilizing a circulation loop. Such reactors are suitable for the process of the present invention. A circulation loop typically includes a first port through which the reaction medium or liquid can exit the reactor and enter the loop, and a second port through which the reaction medium or liquid can exit the loop and re-enter the reactor. Thus, the reaction solution and / or wash solvent circulate through the loop, thereby repeatedly passing through the reaction vessel. Typically, a circulation loop includes one or more pumps or the like for forcing the reaction solution and / or wash solvent through the loop. The advantages of a circulation loop are known to those skilled in the art and include additional agitation and the option of including certain desirable features in the loop, for example, instead of directly into the reaction vessel. For example, one or more measuring cells or detectors or probes of any kind are installed in the loop to allow real-time monitoring of the reaction and / or washing steps, typically the coupling and / or deprotection steps. As another example, a microwave irradiation center is incorporated in the loop to expose the passing reaction medium or liquid to microwave irradiation, thereby accelerating the reaction.

[0136] Online monitoring, also known as real-time monitoring, is known to those skilled in the art. In this specification, these terms are used interchangeably and are understood in the broadest sense to refer to any process of analyzing any reaction medium, liquid, or washing solvent during oligonucleotide synthesis, preferably to extract information about the progress of the reaction or washing. Two general strategies are distinguished, both of which are suitable for the method of the present invention. In the first strategy, analysis is performed after the sample to be analyzed, or even the entire reaction medium, liquid, or washing solvent, is removed from the reactor, for example, by draining it through a filter, membrane, or frit. In the second strategy, the reaction medium, liquid, or washing solvent is analyzed within the reactor and / or in the circulation loop. The location of a suitable detector, measurement cell, or probe is adjusted to suit the desired strategy. In the first strategy, the detector, measurement cell, or probe is typically installed outside the reaction vessel or column, usually below the filter, membrane, or frit, preferably between the filter, membrane, or frit and a waste or storage tank. In the second strategy, the detector or measuring cell or probe is typically installed in the reaction vessel or column and / or circulation loop. When the reactor used includes a circulation loop, it is most convenient to position the detector or measuring cell or probe within the loop rather than directly within the reaction vessel. However, it is also possible to position the detector or measuring cell or probe directly within the reaction vessel, as exemplified, for example, in T. Heilmann et al., Org. Process Res. Dev. 2023, 27, 65-77, https: / / doi.org / 10.1021 / acs.oprd.2c00203. Examples of suitable detectors include, among others, UV detectors, UV-vis detectors, infrared (IR) detectors, conductivity detectors, NMR detectors, and sensors or probes for measuring density and / or refractive index. UV(-vis) spectroscopy (i.e., UV detectors or UV-vis detectors) is particularly suitable for monitoring the progress of the detritylation reaction, such as, for example, step (b) or (b') of the method of the present invention.

[0137] In solid phase oligonucleotide synthesis, it is generally desirable to minimize or reduce solvent consumption. Those skilled in the art understand that reducing the volume of wash solvents is a promising approach to reducing the overall solvent consumption of oligonucleotide synthesis. In solid phase oligonucleotide synthesis, washing steps are typically performed between reaction steps, for example between step (a) and step (b), and / or between step (b) and step (c), and / or between step (c) and step (d), and / or between step (d) and step (e) or (b'), and / or between step (e) and step (b'), and / or between step (b') and step (c'), and / or between step (c') and step (d'), and / or between step (d') and step (e') or (b'), and / or between step (e') and step (b'), and / or between step (e') and step (b'), and / or between step (e') and step (b'), and / or between step (e') and step (b'), and / or between step (e') and step (b'), and / or between step (e') and step (b'), and / or between step (b') and step (b'). The washing step includes one or more washes, during each of which the solid support carrying the growing oligonucleotide chain is treated with (i.e., incubated with) a washing solvent, followed by draining the washing solvent. Additionally or alternatively, the washing solvent may be passed through a settled bed (settled resin bed) of the solid support carrying the growing oligonucleotide chain. The washing solvent may also be a mixture of solvents.

[0138] Various methods for reducing the volume of washing solvent are known, and these methods can be used in combination. As a first method, for example, the volume of washing solvent per washing step and / or the number of washing steps per coupling cycle are reduced, as disclosed in L. Xiao et al., Green Chem. 2023, 25, 4292-4301, https: / / doi.org / 10.1039 / D3GC00881A. This strategy can be used with the method of the present invention. Those skilled in the art will understand that this method is not limited to column reactors, but also applies to batch reactors such as stirred-bed reactors and sparged-bed reactors disclosed in this specification and the references cited herein. When carrying out the method of the present invention and wanting to reduce overall solvent consumption, it is advantageous to apply the strategy disclosed in L. Xiao et al., regardless of the type of reactor (e.g., column reactor or batch reactor) used. As a second method, the washing solvent is (partially) recycled, i.e., reused. For example, BI Andrews et al., J. Org. Chem. 2021, 86, 49-61, https: / / dx.doi.org / 10.1021 / acs.joc.0c02291, teaches collecting the cleaner solvent at the end of a wash step and reusing it at the beginning of the next equivalent wash step. In this context, the term "equivalent wash step" refers to the corresponding wash step of a subsequent coupling cycle (e.g., a wash step after detritylation, e.g., between steps (b) and (c) or between steps (b') and (c')). As will be apparent to those skilled in the art, the reactor configuration must be adjusted for such solvent recycling, for example, by introducing an additional storage tank to store the cleaner portion of the solvent to be reused. For example, online monitoring is used to determine whether a portion of the solvent should be reused. Those skilled in the art understand that the wash solvent can be reused not only for the corresponding wash step of a subsequent coupling cycle, but also potentially for another wash step of the same or a subsequent coupling cycle.As an example, a cleaner portion of the wash solvent used in the wash step after the coupling step (e.g., step (d) or (d') of the process of the invention) is reused in the wash step after the oxidation or sulfidation step (e.g., step (e) or (e') of the process of the invention) within the same coupling cycle. When carrying out the process of the invention and wishing to reduce the overall consumption of solvent, it is advantageous to apply a solvent recycle strategy, regardless of what type of reactor (e.g., column reactor or batch reactor) is used.

[0139] As a third approach, the washing step is carried out as a displacement wash. As used herein, the term "displacement wash" refers to a process in which the washing solvent is passed through the settled bed of the solid support in one direction without further agitation. Therefore, displacement wash is different from dilution wash. As used herein, the term "dilution wash" refers to a process in which the solid support to be washed is agitated in the washing solvent by any means, such as stirring, nitrogen sparging, or shaking, where the addition of washing solvent, agitation, and draining of the washing solvent are typically carried out multiple times, usually with a new batch of washing solvent each time. It will be understood that the solid support carries the growing oligonucleotide chain. Those skilled in the art will understand that displacement wash is routinely used in fixed-bed column reactors and typically requires less solvent than dilution wash (see, for example, US 6,623,703 B1 and US 6,825,339 B2). Dilution washes are frequently used in stirred-bed column reactors and batch reactors in general, and in sparged-bed and stirred-bed reactors in particular, simply because the scale of synthesis is typically quite small and therefore solvent consumption is not significant. However, it is clear that displacement washes are equally applicable to such reactors, especially when it is desired to reduce overall solvent consumption on an industrial scale. Also, some wash steps may be performed as dilution washes, while others may be performed as displacement washes. Displacement washes and dilution washes may also be combined within a single wash step. For example, a wash step may include a dilution wash portion followed by a displacement wash portion, or vice versa. For displacement washes in stirred-bed column reactors or batch reactors in general, and in stirred-bed and sparged-bed reactors in particular, agitation of the solid support is typically stopped, allowing the support to settle into a so-called resin bed. The wash solvent is then passed in one direction through the undisturbed settling resin bed. Fresh solvent is added in such a way that the settling resin bed is not disturbed, for example, via a spray cone or nozzle at the top of the reactor. Typically, it is desirable to continuously flow the wash solvent through the settling resin bed. This is accomplished by continuously draining the solvent from the reactor. It is desirable not to deplete the resin bed.Therefore, fresh wash solvent is usually added continuously or discontinuously, with a higher flow rate being used in the latter case. Those skilled in the art are aware of several strategies for adjusting the addition and discharge of solvent so as not to deplete the precipitation resin bed. For example, the liquid height above the precipitation resin bed is monitored periodically or even continuously, for example, manually by an operator, or preferably by a suitable detector, sensor, or probe, such as a radar sensor, typically installed in the reaction vessel. The end point of such a displacement wash is determined by online monitoring, for example, using a suitable detector at the reactor outlet, where the reactor monitors the concentration of one or more specific species in the discharged solvent. Alternatively, such a displacement wash is stopped when a specified volume of solvent has been discharged from the reactor. When carrying out the method of the present invention and wanting to reduce overall solvent consumption, it is advantageous to apply a displacement wash, regardless of the type of reactor (e.g., column reactor or batch reactor) used. As a fourth approach, if an additional process step (commonly referred to as a capping step), such as the blocking step (f) or (f') of the method of the present invention, is not performed, the overall solvent consumption is significantly reduced (or not increased). WO 2017 / 223258 A1 teaches that a capping step, such as step (f) or (f') of the method of the present invention, is not required in a coupling cycle that includes a sulfurization step as step (e) or (e'), particularly when xanthan hydride (5-amino-3H-1,2,4-dithiazole-3-thione), phenylacetyl disulfide (PADS), 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent), or 3-(N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione (CASRN: 1192027-04-5, DDTT) is used as the sulfurization agent. This approach is compatible with the methods of the present invention.

[0140] For example, batch reactors having a maximum internal volume of 1 to 1000 liters, 5 to 650 liters, 5 to 300 liters, 10 to 600 liters, 50 to 500 liters, or greater than 1000 liters may be used in the methods of the present invention. As a further example, batch reactors having a minimum internal volume of 5, 10, 30, 40, 50, 60, 70, 75, 80, 90, 100, 150, 200, 250, 300, 450, 600, 650, 700, 750, or 800 liters may be used in the methods of the present invention.

[0141] In some embodiments of the method of the present invention, at least step (b) is carried out in a batch reactor. In some embodiments of the method of the present invention, at least steps (b) and (b') are carried out in a batch reactor. In some embodiments of the method of the present invention, each repetition of a coupling cycle comprising steps (b) through (e) or steps (b) through (e') is carried out in a batch reactor. In some embodiments of the method of the present invention, at least step (b) is carried out in a stirred-bed reactor. In some embodiments of the method of the present invention, at least steps (b) and (b') are carried out in a stirred-bed reactor. In some embodiments of the method of the present invention, each repetition of a coupling cycle comprising steps (b) through (e) or steps (b') through (e') is carried out in a stirred-bed reactor.

[0142] When a batch reactor is used, step (b) involves adding the deprotection mixture Mb, i.e., liquid composition C, to the reactor containing component C-0 from step (a), followed by stirring, shaking, or agitation by any means at a constant temperature for a certain period of time, and then draining the deprotection mixture Mb, i.e., liquid composition C, and any other liquids (and dissolved components) from the reactor through a filter or membrane or frit, thereby removing the support-bound component C-0. #When a batch reactor is used, step (b') involves adding the deprotection mixture M-b', e.g., liquid composition C, to the reactor containing the (x-1)th cycle oligonucleotide O-(x-1), followed by stirring, shaking, or agitation by any means at a constant temperature for a period of time, followed by draining the deprotection mixture M-b', e.g., liquid composition C, and any other liquids (and dissolved components) from the reactor through a filter or membrane or frit, thereby releasing the support-bound (x-1)th cycle oligonucleotide O-(x-1). # in a reactor.

[0143] In some embodiments of the process of the present invention, at least steps (b) and (b') are carried out in a batch reactor, or at least steps (b) and (b') are carried out in a column reactor, and the flow rate of liquid composition C through the column reactor is less than 300 cm / hr.

[0144] As used herein, the terms "deprotection mixture Mb" and "deprotection mixture M-b'" refer to any mixture used to perform cleavage of the PG-0 or PG-(x-1) protecting group, respectively. As known to those skilled in the art, such protecting groups, including optionally substituted triarylmethyl residues, are typically removed by treatment with a protic acid or Lewis acid (i.e., an electron pair acceptor such as zinc bromide), preferably a protic acid. As used herein, the term "protic acid" is understood in its broadest sense and refers to a Brønsted acid, also known as a Brønsted-Lowry acid. The terms "protic acid" and "Brønsted acid" (or "Brønsted-Lowry acid") are well known to those skilled in the art. It is also well known that acid strength, or simply stated, the tendency of a Brønsted-Lowry acid to donate a proton, is expressed in terms of a pKa value, with stronger acids having lower (i.e., smaller) pKa values ​​than weaker acids. The term "pKa value" itself, as well as the means for determining the pKa value of a protic acid, form part of the general knowledge of those skilled in the art. Additionally, pKa values ​​for commonly used Bronsted-Lowry acids (and many others) are available in tabular form in the literature. As used herein, pKa values ​​are those determinable in water (i.e., aqueous solutions) at 25°C unless otherwise indicated. Throughout this text, solutions used to cleave protecting groups containing optionally substituted triarylmethyl residues, particularly the DMT protecting group, are also referred to as detritylation cocktails.

[0145] It will be understood that the composition of deprotection mixture M-b' may be the same or different for each iteration of step (b'), unless indicated otherwise in the context of a particular embodiment. Furthermore, when deprotection mixture Mb or M-b' is described as comprising a "protic acid," it will be understood that this encompasses a mixture of protic acids, unless indicated otherwise in the context of a particular embodiment.

[0146] In some embodiments of the method of the present invention, deprotection mixture Mb and each deprotection mixture M-b' comprise a protic acid. In some embodiments of the method of the present invention, deprotection mixture Mb and each deprotection mixture M-b' comprise a protic acid having a pKa of 4 or less, 3.5 or less, 3 or less, 2.5 or less, or 2 or less. In some embodiments of the method of the present invention, deprotection mixture Mb and each deprotection mixture M-b' comprise a protic acid having a pKa in the range of 0 to 4, 0 to 3.5, 0 to 3, 0 to 2.5, or 0 to 2.

[0147] The protonic acid of the deprotection mixture Mb or M-b' is not particularly limited in terms of its chemical structure. For example, the protonic acid is a carboxylic acid, such as dichloroacetic acid (DCA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA), or a sulfonic acid, such as methanesulfonic acid or p-toluenesulfonic acid, or a mineral acid, such as sulfuric acid or hydrochloric acid, or a mixture thereof. Carboxylic acids and sulfonic acids are preferred, and carboxylic acids are more preferred.

[0148] Carboxylic acid for use in the method of the present invention is known to those skilled in the art.Non-limiting examples of such carboxylic acid include monohalogenated acetic acid (e.g., 2-chloroacetic acid), dihalogenated acetic acid (e.g., 2,2-dichloroacetic acid), trihalogenated acetic acid (e.g., 2,2,2-trifluoroacetic acid, 2,2,2-trichloroacetic acid), 2-cyanoacetic acid, dicarboxylic acid (e.g., oxalic acid, malic acid, benzene-1,4-dicarboxylic acid), tricarboxylic acid (e.g., citric acid, benzene-1,3,5-tricarboxylic acid), tetracarboxylic acid (e.g., benzene-1,2,4,5-tetracarboxylic acid), and benzoic acid derivative (e.g., 2-chlorobenzoic acid, 2,4-dichlorobenzoic acid, salicylic acid).Those skilled in the art are also familiar with sulfonic acid for use in the method of the present invention. Non-limiting examples of such sulfonic acids include alkylsulfonic acids of the formula (C1-C6-alkyl)-SO3H (e.g., methanesulfonic acid, ethanesulfonic acid), in which one or more alkyl hydrogen residues are optionally substituted (e.g., 3-hydroxypropane-1-sulfonic acid). Non-limiting examples of sulfonic acids for use in the methods of the present invention further include arylsulfonic acids, in which the aryl moiety is preferably a benzene moiety, optionally substituted (e.g., benzenesulfonic acid, p-toluenesulfonic acid). Structurally more complex sulfonic acids, such as camphorsulfonic acid in any isomeric form, may also be used. Those skilled in the art will also be aware of mineral acids for use in the methods of the present invention. Non-limiting examples of such mineral acids include hydrogen halides (e.g., hydrogen chloride) and sulfuric acid. Descriptions and examples of aliphatic, aromatic, or heteroaromatic amines whose protonated forms have pKa values ​​in the range of 1 to 4 are provided in other sections of this text. In particular, when a mineral acid is used, the process of the present invention employs a preformed salt of said mineral acid with an aliphatic, aromatic or heteroaromatic amine (eg, 4-chloropyridinium hydrochloride).

[0149] According to the method of the present invention, the deprotection mixture Mb is a liquid composition C comprising a solvent, a protonic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups covalently bonded directly to the optionally substituted phenyl moiety. Furthermore, in at least one repetition of step (b'), the deprotection mixture M-b' is a liquid composition C comprising a solvent, a protonic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups covalently bonded directly to the optionally substituted phenyl moiety. It will be understood that two or more liquid compositions C may be the same or different, i.e., contain the same or different components in the same or different amounts or concentrations, so long as all such liquid compositions C meet the general definition of a liquid composition C of the present invention.

[0150] In some embodiments of the method of the present invention, in step (b), a protecting group PG-0 is attached to a hydroxyl moiety of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, particularly adenine. In some embodiments of the method of the present invention, in at least one iteration, preferably each iteration, of step (b') in which a protecting group PG-(x-1) is attached to a hydroxyl moiety of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, particularly adenine, the deprotection mixture M-b' is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety.

[0151] In some embodiments of the methods of the present invention: - the backbone hydroxyl moiety protected by said protecting group PG-0 is part of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine, - in at least one repetition of the coupling cycle comprising steps (b') to (e'), in which the protecting group PG-(x-1) is part of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine, the deprotection mixture M-b' is liquid composition C.

[0152] Steps (b) and (b') of the process of the present invention are typically carried out at temperatures between 15°C and 25°C, and for convenience are simply carried out at ambient temperature. However, it should be understood that either step (b) or (b') can be carried out at any suitable temperature, e.g., between -15°C and 90°C, e.g., temperatures in the ranges of 0-90°C, 10-70°C, 10-60°C, 10-50°C, 10-40°C, 15-30°C, or 15-25°C. Those skilled in the art will appreciate that increasing the reaction temperature typically shortens the reaction time. Means for increasing the reaction temperature are known to those skilled in the art and include, for example, the use of a heating jacket containing a heating medium such as water (e.g., when a double-walled reactor is used), microwave irradiation (particularly when a flow reactor is used), infrared irradiation, and conduction heating.

[0153] The time for carrying out steps (b) and (b') is not particularly limited and varies depending on, for example, the composition of the deprotection mixture Mb or M-b' and the reaction temperature. Those skilled in the art will routinely determine a suitable reaction time, for example, by monitoring the deprotection reaction of step (b) or (b') until complete conversion (i.e., complete removal of the respective protecting groups PG-0 or PG-(x-1)) is indicated. In particular, there is no particular time limit for incubation with the liquid composition C of the present invention, and it is carried out for, for example, at least 5 minutes, 10 minutes, 20 minutes, 30 minutes, or 1 hour.

[0154] Each step (b) and (b') may consist of a single incubation with deprotection mixture Mb or M-b', or optionally include multiple repetitions of such incubation (i.e., multiple incubations), where for each incubation round, a new deprotection mixture Mb or M-b', e.g., liquid composition C, is typically used and drained before the next incubation round (i.e., repeat). For example, a repetition of step (b) or (b') may include 1 to 5, 1 to 4, 1 to 3, or 2 to 3 incubations, each involving incubation with a new deprotection mixture Mb or M-b'. In such cases, the deprotection mixture Mb or M-b', e.g., liquid composition C, used in each incubation round of the same repetition of step (b) or (b') preferably has (essentially) the same composition. In particular, when step (b) or (b') is described as being carried out using a certain specified deprotection mixture Mb or M-b', e.g., liquid composition C, said certain deprotection mixture Mb or M-b', e.g., said liquid composition C, has (essentially) the same composition for all incubation times of each repetition of step (b) or (b'), unless otherwise indicated. This rationale is illustrated by the following three examples:

[0155] As a first example, when step (b) is described as containing a deprotection mixture Mb having a total molar amount of protonic acid in the range of 0.80 to 15.0 equivalents relative to the total molar amount of protecting group PG-0, this condition is met for each of the one or more deprotection mixtures Mb used in one or more incubations in step (b). That is, each of the one or more deprotection mixtures Mb used in one or more incubations in step (b) contains a total molar amount of protonic acid in the range of 0.80 to 15.0 equivalents relative to the total molar amount of protecting group PG-0. As a second example, when step (b) is described as being performed at a temperature in the range of 15 to 25°C, this condition applies to all incubations included in step (b). As a third example, when step (b) is described as being carried out for at least 20 minutes, this refers to the entire time that step (b) is carried out, regardless of whether a single incubation (e.g., 20 minutes) or two incubations (e.g., 10 minutes each) are carried out.

[0156] Step (c) of the method of the present invention is providing a building block B-1 selected from the group consisting of nucleosides and oligonucleotides, the building block B-1 comprising a backbone hydroxyl moiety protected by a protecting group PG-1 comprising an optionally substituted triarylmethyl residue, and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block B-1 via a phosphorus atom.

[0157] Step (c') of the method of the present invention is providing a building block Bx selected from the group consisting of nucleosides and oligonucleotides, where building block Bx comprises a backbone hydroxyl moiety protected by a protecting group PG-x comprising an optionally substituted triarylmethyl residue, and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block Bx via a phosphorus atom.

[0158] The terms "nucleoside," "oligonucleotide," and "backbone hydroxyl moiety" are explained. Any explanations and embodiments relating to the protecting group PG-(x-1) also apply to the protecting group PG-x. Thus, in some embodiments of the methods of the present invention, each protecting group PG-x is a DMT protecting group.

[0159] In some embodiments of the method of the present invention, component B-1 and at least one or each component Bx are selected from the group consisting of nucleosides and oligonucleotides containing 50, 40, 30, 25, 20, 15, 10, or 5 or fewer nucleoside subunits. In some embodiments of the method of the present invention, component B-1 and at least one or each component Bx are nucleosides. In some embodiments of the method of the present invention, component B-1 and at least one or each component Bx are oligonucleotides. In some embodiments of the method of the present invention, component B-1 and at least one or each component Bx are oligonucleotides containing 50, 40, 30, 25, 20, 15, 10, or 5 or fewer nucleoside subunits.

[0160] In some preferred embodiments of the methods of the present invention, each of components B-1 and Bx is: - exactly one backbone hydroxyl moiety protected by a protecting group PG-1 (for B-1) or PG-x (for Bx) comprising an optionally substituted triarylmethyl residue, and - Exactly one phosphorus moiety covalently bonded via a phosphorus atom to an oxygen atom in the backbone of the component Includes.

[0161] In some preferred embodiments of the methods of the present invention, each of components B-1 and Bx is: - exactly one protecting group comprising an optionally substituted triarylmethyl residue, which is said protecting group PG-1 (for B-1) or PG-x (for Bx), and - Exactly one phosphorus moiety covalently bonded via a phosphorus atom to an oxygen atom in the backbone of the component Includes.

[0162] In some embodiments of the methods of the present invention, each of components B-1 and Bx is a compound of formula II: [ka] [In Formula II: Each oxygen atom (O) depicted within each nucleoside subunit y-0 through yq represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; Each nucleoside subunit y-0 to yq is the same or different (i.e., has the same or different chemical structure); PM is the phosphorus moiety; PG is a protecting group PG-1 (for building block B-1) or PG-x (for building block Bx) comprising an optionally substituted triarylmethyl residue; q is an integer greater than or equal to 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 , S.R. z-2 independently selected from the group consisting of: R z-2 is a protecting group which may be the same or different for each repeat unit q].

[0163] In some embodiments of the methods of the present invention, component B-1 or Bx of formula II is a compound of formula II-a: [ka] [In formula II-a: q, PG, Y 2 , Z 2 , R z-2 and PM are defined as in Formula II; B N are nucleobases that are the same or different (i.e., have the same or different chemical structures) in each occurrence; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R XIII is independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, so that each nucleoside subunit has the structure of formula II-a-tc: [ka] In formula II-a-tc: The oxygen atom from which the dashed line originates is the 3'-carbon atom, i.e., R in formula II-a. XII represents an oxygen atom bonded to a carbon atom to which is bonded; The dashed lines represent the connection between each oxygen atom of the nucleoside subunit of formula II-a-tc and the PM or P(Y 2 )(Z 2 ) indicates a covalent chemical bond interconnecting one of the following; The oxygen atom from which the wavy line originates is the 5'-carbon atom, i.e., C(R XIV )(R XV ) represents an oxygen atom bonded to a carbon atom to which it is bonded; The wavy lines represent the connection between each oxygen atom of the nucleoside subunit of formula II-a-tc and the PG or P(Y 2 )(Z 2 ) indicates a covalent chemical bond interconnecting one of the

[0164] In some embodiments of the methods of the present invention, component B-1 or Bx of formula II, particularly formula II-a, is a compound of formula II-b: [ka] [In formula II-b: q, PG, Y 2 , Z 2 , R z-2 , P.M., B. N , R XI , and R XIII is defined as in formula II-a, Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, so that each nucleoside subunit has the structure of formula II-b-tc: [ka] In formula II-b-tc: The oxygen atom from which the dashed line originates is the 3'-carbon atom, i.e., R in formula II-a. XII represents an oxygen atom bonded to a carbon atom to which is bonded; The dashed lines represent the connection between each oxygen atom of the nucleoside subunit of formula II-a-tc and the PM or P(Y 2 )(Z 2 ) indicates a covalent chemical bond interconnecting one of the following; The oxygen atom from which the wavy line originates is the 5'-carbon atom, i.e., C(R XIV )(R XV ) represents an oxygen atom bonded to a carbon atom to which it is bonded; The wavy lines represent the connection between each oxygen atom of the nucleoside subunit of formula II-a-tc and the PG or P(Y 2 )(Z 2 ) indicates a covalent chemical bond interconnecting one of the

[0165] As used herein, the term "phosphorus moiety" is understood in the broadest sense and refers to any group of atoms containing at least one, preferably exactly one, phosphorus atom, where the term "atom group" refers to two or more atoms. In such a group of atoms, each atom does not necessarily have to be covalently bonded to each additional atom in the group of atoms, but each atom must be covalently bonded to at least one additional atom in the group of atoms. From steps (d) and (d'), which will be described in more detail below, it is clear that the "phosphorus moiety covalently bonded to the oxygen atom of the backbone of component B-1 or Bx via a phosphorus atom" (e.g., the phosphorus moiety PM of any one of formulas II, II-a, and II-b) is a phosphorus moiety capable of participating in a condensation reaction with a free hydroxyl group. Those skilled in the art will be aware of phosphorus moieties that satisfy this condition and will be able to select a suitable phosphorus moiety without undue experimentation. Furthermore, those skilled in the art will know that the phosphorus moiety participating in such a condensation reaction typically forms an internucleoside linking group in the oligonucleotide formed by the condensation reaction. The phosphorus moiety, e.g., the phosphorus moiety PM of any one of formulas II, II-a, and II-b, is a phosphorus(III) moiety, also referred to as a P(III) moiety, i.e., a phosphorus moiety comprising a P(III) atom as defined herein. Non-limiting examples of such P(III) moieties are phosphoramidite moieties as disclosed, for example, in X. Wei et al., Tetrahedron 2013, 69, 3615-3637, and H-phosphonate monoester moieties as disclosed, for example, in J. Stawinski and R. Stromberg (2005), Di- and Oligonucleotide Synthesis Using H-Phosphonate Chemistry, in Methods in Molecular Biology, Vol. 288: Oligonucleotide Synthesis: Methods and Applications, edited by P. Herdewijn, Humana Press Inc., Totowa NJ, https: / / doi.org / 10.1385 / 1-59259-823-4:081.Alternatively, the phosphorus moiety, for example, the phosphorus moiety PM of any one of Formulas II, II-a, and II-b, is a phosphorus(V) moiety, also referred to as a P(V) moiety, i.e., a phosphorus moiety containing a P(V) atom as defined herein. Examples of such P(V) moieties include classical aryl phosphate diester moieties and the P(V) moieties disclosed by Baran et al. (Science 2018, 361, 1234-1238 and ACS Central Science 2021, 7, 1473-1485).

[0166] A preferred example of a building block B-1 or Bx in which the phosphorus moiety, eg, the phosphorus moiety PM of formula II, is a phosphoramidite moiety is a compound of formula II-1.

[0167] In some embodiments of the methods of the present invention, each of components B-1 and Bx is a compound of formula II-1: [ka] [In Formula II-1: Each oxygen atom (O) depicted within each nucleoside subunit y-0 through yq represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; Each nucleoside subunit y-0 to yq is the same or different (i.e., has the same or different chemical structure); PG is a protecting group PG-1 (for building block B-1) or PG (for building block Bx) and comprises an optionally substituted triarylmethyl residue; q is an integer greater than or equal to 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 and S.R. z-2 independently selected from the group consisting of: R z-2is a protecting group that is the same or different for each repeat unit q; Z 3 is selected from the group consisting of O and S; R z-3 is a protecting group; R a and R b are each independently a C1-C6 alkyl group, and R a and R b are the same or different, and R a and R b may also be bonded together with the nitrogen atom to which they are attached to form a 5- or 6-membered aliphatic cyclic amine moiety; Step (e) or step (e') is carried out in each coupling cycle.

[0168] In some embodiments of the method of the present invention, component B-1 or Bx of formula II-1 is a compound of formula II-1-a: [ka] [In formula II-1-a: q, PG, Y 2 , Z 2 , R z-2 , Z 3 , R z-3 , R a , and R b is defined as in formula II-1; B N , R XI , R XII , R XIII , R XIV , and R XV is defined as in formula II-a] and step (e) or step (e') is carried out in each coupling cycle.

[0169] In some embodiments of the method of the present invention, component B-1 or Bx of formula II-1, particularly formula II-1-a, is a compound of formula II-1-b: [ka] [In formula II-1-b: q, PG, Y 2 , Z 2 , R z-2 , Z 3 , R z-3 , R a , and R b is defined as in formula II-1, B N , R XI , R XII , R XIII , R XIV , and R XV is defined as in formula II-b] and step (e) or step (e') is carried out in each coupling cycle.

[0170] A preferred example of building block B-1 or Bx, where the phosphorus moiety, e.g., the phosphorus moiety PM of any one of formulas II, II-a, and II-b, is an H-phosphonate monoester moiety, is a compound of formula II-2.

[0171] In some embodiments of the methods of the present invention, each of components B-1 and Bx is a compound of formula II-2: [ka] [In Formula II-2: Each oxygen atom (O) depicted within each nucleoside subunit y-0 through yq represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; Each nucleoside subunit y-0 to yq is the same or different (i.e., has the same or different chemical structure); PG is a protecting group PG-1 (for building block B-1) or PG (for building block Bx) and comprises an optionally substituted triarylmethyl residue; q is an integer greater than or equal to 0; Y 2is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, H, OR z-2 , and S.R. z-2 independently selected from the group consisting of: R z-2 is a protecting group which may be the same or different for each repeat unit q; At least in the final coupling cycle, step (e) or step (e') is carried out.

[0172] In some embodiments of the methods of the present invention, component B-1 or Bx of formula II-2 is a compound of formula II-2-a: [ka] [In formula II-2-a: q, PG, Y 2 , Z 2 , and R z-2 is defined as in formula II-2; B N , R XI , R XII , R XIII , R XIV , and R XV is defined as in formula II-a]; At least in the final coupling cycle, step (e) or step (e') is carried out.

[0173] In some embodiments of the methods of the present invention, component B-1 or Bx of formula II-2, particularly formula II-2-a, is a compound of formula II-2-b: [ka] [In formula II-2-b: q, PG, Y 2 , Z 2 , and R z-2 is defined as in formula II-2; B N , R XI , RXII , R XIII , R XIV , and R XV is defined as in formula II-b]; At least in the final coupling cycle, step (e) or step (e') is carried out.

[0174] In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, the integer q is an integer in the range of 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, 0 to 3, 0 to 2, 0 to 1, or q is 0. It will be understood that a statement that an integer is in the range of, for example, 0 to 150 means that the integer is 0, 1, 2, 3, 4, or 5. In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, the integer q is 0. It will be understood that when the integer q in any one of Formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b is 0, a protecting group PG (i.e., PG-1 for B-1 and PG-x for Bx) is attached to the hydroxyl moiety of each of the remaining nucleoside subunits bearing a phosphorus moiety (e.g., nucleoside subunit y-0 of Formula II, II-1, or II-2).

[0175] In some embodiments, for example, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, the protecting group PG (i.e., PG-1 for B-1 and PG-x for Bx) is selected from the group consisting of a triphenylmethyl group (i.e., a trityl group), a (p-methoxyphenyl)diphenylmethyl group (i.e., an MMT group), and a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group). In some preferred embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, the protecting group PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl group (i.e., a DMT group).

[0176] In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, Y 2 is O for every repeat unit q. In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, Y 2 is S for each repeating unit q.

[0177] In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, R z-2 is a protecting group removable under alkaline conditions, and R z-2 In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, Rz-2 is independently for each repeating unit q a protecting group of the chemical structure CH2-CH2-EWG, where EWG is an electron-withdrawing group, preferably a cyano group. The electron-withdrawing group may be selected from the group consisting of, for example, a cyano group, a halogen atom, such as a chlorine, fluorine, or bromine atom, an aldehyde group, a keto group, a carboxyester group, or a carboxamide group. In some preferred embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, R z-2 is a 2-cyanoethyl group (i.e., CH2-CH2-CN) for every repeat unit q.

[0178] In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, and II-1-b, Z 2 For each repeating unit q, OR z-2 and S.R. z-2 In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, and II-1-b, Z 2 For each repeating unit q, OR z-2 and S.R. z-2 and R is independently selected from the group consisting of z-2 is a 2-cyanoethyl group (i.e., CH2-CH2-CN) for every repeat unit q. In such embodiments, Z 2 is independently selected from the group consisting of O-CH2-CH2-CN and S-CH2-CH2-CN for each repeat unit q. In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, and II-1-b, Z 2 is OR for each repeat unit q z-2 and R z-2 is a 2-cyanoethyl group (i.e., CH2-CH2-CN) for every repeat unit q. In such embodiments, Z 2is O-CH2-CH2-CN for each repeat unit q.

[0179] In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-2, II-2-a, and II-2-b, Z 2 is H for every repeat unit q. In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-2, II-2-a, and II-2-b, Y 2 is O for each repeating unit q, and Z 2 is H for each repeating unit q.

[0180] In some embodiments, in component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, the terminal nucleoside subunit in which the hydroxyl moiety is linked to a PG (i.e., PG-1 for B-1 and PG-x for Bx) protecting group is a nucleoside subunit comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, particularly adenine.

[0181] Those skilled in the art can use any kind of nucleobase B in any one of the components B-1 or Bx of formula II-a, II-b, II-1-a, II-1-b, II-2-a, and II-2-b. N In some embodiments, in component B-1 or Bx of any one of formulas II-a, II-b, II-1-a, II-1-b, II-2-a, and II-2-b, B N is a nucleobase, each occurrence independently selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil. Any nucleobase B of any one of formulas II-a, II-b, II-1-a, II-1-b, II-2-a, and II-2-b NIt will be understood by those skilled in the art that, even if not specifically shown, B is optionally protected, i.e., carries one or more protecting groups. Thus, for example, B N When a reference is made to adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil, this encompasses the aforementioned nucleobases in protected and free forms (i.e., with or without a protecting group). The same rationale applies to nucleobases in general. Nucleobase protecting groups are known to those skilled in the art and are described above, including, for example, the preferred nucleobase protecting groups summarized in Table T-1.

[0182] In some embodiments, each nucleobase of each component B-1 or Bx, particularly each nucleobase B of component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, N teeth, - adenine with a protected exocyclic amino group; - guanine with a protected exocyclic amino group; - cytosine with a protected exocyclic amino group; - 5-methylcytosine with a protected exocyclic amino group; - thymine; and - Uracil are independently selected from the group consisting of:

[0183] In some embodiments, each nucleobase of component B-1 or Bx, particularly each nucleobase B of component B-1 or Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b, N teeth, - adenine whose exocyclic amino group is protected by a benzoyl, isobutyryl or phenoxyacetyl group; - guanine whose exocyclic amino group is protected by an isobutyryl group, a 4-isopropylphenoxyacetyl group or a dimethylformamidino group; - cytosine in which the exocyclic amino group is protected by an acetyl or benzoyl group; - 5-methylcytosine, in which the exocyclic amino group is protected by an acetyl or benzoyl group; - thymine; and - Uracil are independently selected from the group consisting of:

[0184] In some embodiments, in component B-1 or Bx of any one of formulas II-a, II-b, II-1-a, II-1-b, II-2-a, and II-2-b: R XI is, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R XIII is independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of any one of formulas II-a, II-1-a, and II-2-a) or formula II-b-tc (component B-1 or Bx of any one of formulas II-b, II-1-b, and II-2-b) described above.

[0185] In some embodiments, in component B-1 or Bx of any one of formulas II-a, II-b, II-1-a, II-1-b, II-2-a, and II-2-b: R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R XIII is independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of any one of formulas II-a, II-1-a, and II-2-a) or formula II-b-tc (component B-1 or Bx of any one of formulas II-b, II-1-b, and II-2-b) described above.

[0186] In some embodiments, in component B-1 or Bx of any one of formulas II-a, II-b, II-1-a, II-1-b, II-2-a, and II-2-b: R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), and O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy); R XII , R XIII , R XIV , and R XV Each of these is H in its own existence.

[0187] In some embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, Z 3 is O. In some embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, Z 3 is S.

[0188] In some embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, R z-3 is a protecting group removable under alkaline conditions. In some embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, R z-3 is a protecting group of the chemical structure CH2-CH2-EWG, where EWG is an electron-withdrawing group, preferably a cyano group. The electron-withdrawing group may be selected from the group consisting of, for example, a cyano group, a halogen atom, such as a chlorine, fluorine, or bromine atom, an aldehyde group, a keto group, a carboxyester group, or a carboxamide group. In some preferred embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, R z-3 is a 2-cyanoethyl group (i.e., CH2-CH2-CN). In some preferred embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, Z 3 is O and R z-3 is a 2-cyanoethyl group (i.e., CH2-CH2-CN).

[0189] In some embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, R a and R b are each independently a C1-C6 alkyl group, and R a and R b are the same or different, and R a and R b In some embodiments, in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, R a and R b Each of is an isopropyl group (i.e., CH(CH3)2).

[0190] In some embodiments, in component B-1 or Bx of formula II: PM is a phosphorus moiety, preferably selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 , S.R. z-2 independently selected from the group consisting of: R z-2 is a 2-cyanoethyl group in each occurrence.

[0191] In some embodiments, in component B-1 or Bx of formula II: PM is the phosphoramidite moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 and S.R. z-2 independently selected from the group consisting of: R z-2 is a 2-cyanoethyl group in each occurrence.

[0192] In some embodiments, in component B-1 or Bx of formula II: PM is the phosphoramidite moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 is OR for each repeat unit q z-2 and; R z-2 is a 2-cyanoethyl group in each occurrence.

[0193] In some embodiments, in component B-1 or Bx of formula II: PM is an H-phosphonate monoester moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; Y 2 is, for each repeat unit q, independently selected from the group consisting of O and S, preferably O; Z 2 is H for every q repeating units.

[0194] In some embodiments, in component B-1 or Bx of any one of formulas II-a and II-b: PM is a phosphorus moiety, preferably selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0-150, 0-100, 0-75, 0-50, 0-35, 0-30, 0-25, 0-20, 0-15, 0-10, 0-5, 0-3, 0-2, 0-1, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 , S.R. z-2 independently selected from the group consisting of: R z-2 is a 2-cyanoethyl group in each occurrence; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R XIIIis independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of formula II-a) or formula II-b-tc (component B-1 or Bx of formula II-b) described above.

[0195] In some embodiments, in component B-1 or Bx of any one of formulas II-a and II-b: PM is a phosphorus moiety, preferably selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0-150, 0-100, 0-75, 0-50, 0-35, 0-30, 0-25, 0-20, 0-15, 0-10, 0-5, 0-3, 0-2, 0-1, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2, S.R. z-2 independently selected from the group consisting of: R z-2 is a 2-cyanoethyl group in each occurrence; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R XIII is independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of formula II-a) or formula II-b-tc (component B-1 or Bx of formula II-b) described above.

[0196] In some embodiments, in component B-1 or Bx of any one of formulas II-a and II-b: PM is a phosphorus moiety, preferably selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0-50, 0-35, 0-30, 0-25, 0-20, 0-15, 0-10, 0-5, 0-3, 0-2, 0-1, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 , S.R. z-2 independently selected from the group consisting of: R z-2 is a 2-cyanoethyl group in each occurrence; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), and O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy); R XII , R XIII , R XIV , and R XV Each of these is H in its own existence.

[0197] In some embodiments, in component B-1 or Bx of formula II-1: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, ORz-2 and S.R. z-2 and Z are independently selected from the group consisting of 2 is preferably OR for every q repeating units z-2 and; R z-2 is a 2-cyanoethyl group in each occurrence; Z 3 is selected from the group consisting of O and S, preferably O; R z-3 is a 2-cyanoethyl group; R a and R b are each independently a C1-C6 alkyl group, preferably an isopropyl group.

[0198] In some embodiments, in component B-1 or Bx of formula II-1: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 is OR for each repeat unit q z-2 and; R z-2 is a 2-cyanoethyl group in each occurrence; Z 3 is O; R z-3 is a 2-cyanoethyl group; R a and R b Each of is an isopropyl group.

[0199] In some embodiments, in component B-1 or Bx of any one of formulas II-1-a and II-1-b: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 and S.R. z-2 and Z are independently selected from the group consisting of 2 is preferably OR for every q repeating units z-2 and; R z-2 is a 2-cyanoethyl group in each occurrence; Z 3 is selected from the group consisting of O and S, preferably O; R z-3 is a 2-cyanoethyl group; R a and R b each independently represents a C1-C6-alkyl group, preferably an isopropyl group; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—(C1-C5-alkyl), O—(C1-C5-alkyl)-O—(C1-C5-alkyl), O—Si(C1-C5-alkyl)3, and O—CH2—O—Si(C1-C5-alkyl)3; R XIII is independently in each occurrence H or R of the same nucleoside subunit XIII and R XI(i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of formula II-1-a) or formula II-b-tc (component B-1 or Bx of formula II-1-b) described above.

[0200] In some embodiments, in component B-1 or Bx of any one of formulas II-1-a and II-1-b: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 and S.R. z-2 and Z are independently selected from the group consisting of 2 is preferably OR for every q repeating units z-2 and; R z-2 is a 2-cyanoethyl group in each occurrence; Z 3is selected from the group consisting of O and S, preferably O; R z-3 is a 2-cyanoethyl group; R a and R b each independently represents a C1-C6-alkyl group, preferably an isopropyl group; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R XIII is independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of formula II-1-a) or formula II-b-tc (component B-1 or Bx of formula II-1-b) described above.

[0201] In some embodiments, in component B-1 or Bx of any one of formulas II-1-a and II-1-b: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 and S.R. z-2 and Z are independently selected from the group consisting of 2 is preferably OR for every q repeating units z-2 and; R z-2 is a 2-cyanoethyl group in each occurrence; Z 3 is selected from the group consisting of O and S, preferably O; R z-3 is a 2-cyanoethyl group; R a and R b are each independently a C1-C6 alkyl group, preferably an isopropyl group; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), and O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy); R XII , R XIII , R XIV , and R XV Each of these is H in its own existence.

[0202] In some embodiments, in component B-1 or Bx of formula II-2: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer ranging from 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is H; Y 2 is, for each repeat unit q, independently selected from the group consisting of O and S, preferably O; Z 2 is H for every q repeating units.

[0203] In some embodiments, in component B-1 or Bx of any one of formulas II-2-a and II-2-b: PG (i.e., PG-1 for B-1 and PG-x for Bx) is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; q is an integer in the range of 0 to 50, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5, or q is 0; B N are nucleobases which are the same or different in each occurrence; Y 2 is, for each repeat unit q, independently selected from the group consisting of O and S, preferably O; Z 2 is H per repeat unit q; R XI is, at each occurrence, independently selected from the group consisting of H, F, O—CH (i.e., methoxy), O—CH—CH—O—CH (i.e., 2-methoxyethyl-1-oxy), O—Si(CH) (i.e., trimethylsilyloxy), O—Si(CH)(C(CH)) (i.e., tert-butyl(dimethyl)silyloxy), and O—CH—O—Si(C(CH)) (i.e., ((triisopropylsilyl)oxy)-methyloxy); R XIIIis independently in each occurrence H or R of the same nucleoside subunit XIII and R XI (i.e., attached to the 4'- and 2'-C atoms of the same carbohydrate moiety) together form the structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++, where + is the carbon atom at the 4'-carbon atom (i.e., R XIII is the point of attachment to the 2'-carbon (i.e., the carbon atom to which R XI is the point of attachment to the carbon atom to which it is attached; Independently for each nucleoside subunit, R XII , R XIV , and R XV are all H or are linked together, and thus each nucleoside subunit has the structure of formula II-a-tc (component B-1 or Bx of formula II-2-a) or formula II-b-tc (component B-1 or Bx of formula II-2-b) described above.

[0204] In the methods of the present invention, components B-1 or Bx are the same or different (i.e., have the same or different chemical structures) for each repetition of the coupling cycle, unless otherwise indicated in the context of a particular embodiment.

[0205] The term "providing building block B-1 or Bx" in step (c) or (c') is to be understood in the broadest sense. Building block B-1 or Bx for use in the method of the present invention can be obtained commercially, for example, especially when the phosphorus moiety is a phosphoramidite moiety or an H-phosphonate monoester moiety. Alternatively, building block B-1 or Bx for use in the method of the present invention can be obtained by chemical synthesis. Those skilled in the art will know how to synthesize such compounds, and the synthetic route will obviously depend on the chemical structure of the phosphorus moiety. The phosphorus moiety, e.g., the phosphorus moiety PM, is a phosphoramidite moiety, for example, any one of components B-1 or Bx of formula II, II-a, and II-b, particularly any one of components B-1 or Bx of formula II-1, II-1-a, and II-1-b, is synthesized, for example, as disclosed in KV Gothelf et al., Nature Communications 2021 and X. Wei et al., Tetrahedron 2013, 69, 3615-3637. The phosphorus moiety, e.g., the phosphorus moiety PM, is an H-phosphonate monoester moiety, e.g., building block B-1 or Bx of any one of formulas II, II-a, and II-b, particularly building block B-1 or Bx of any one of formulas II-2, II-2-a, and II-2-b, is synthesized as disclosed, for example, in J. Stawinski and R. Stromberg (2005), Di- and Oligonucleotide Synthesis Using H-Phosphonate Chemistry, in Methods in Molecular Biology, Vol. 288: Oligonucleotide Synthesis: Methods and Applications, edited by P. Herdewijn, Humana Press Inc., Totowa NJ, https: / / doi.org / 10.1385 / 1-59259-823-4:081.The phosphorus moiety, e.g., building blocks B-1 or Bx of any one of formulas II, II-a, and II-b, in which the phosphorus moiety PM is a P(V) moiety suitable for chiral phosphorothioate synthesis, are synthesized, for example, as disclosed in P.S. Baran et al., Science 2018, 361, 1234-1238 (see also the supplementary material for this reference).

[0206] Step (d) of the method of the present invention comprises the step of: # and the phosphorus atom of the phosphorus moiety of component B-1. # with component B-1 of step (c), thereby obtaining oligonucleotide O-1 of the first cycle.

[0207] Step (d') of the method of the present invention comprises the step of: (x-1)th cycle oligonucleotide (O-(x-1)) # and the phosphorus atom of the phosphorus moiety of building block Bx, under conditions suitable for forming a covalent bond between the free backbone hydroxyl group of the (x-1)th cycle oligonucleotide (O-(x-1)) obtained in step (b'). # with the building block Bx of step (c'), thereby obtaining the xth cycle oligonucleotide Ox.

[0208] For example, in the fifth coupling cycle (x=5), step (d') is carried out by coupling the fourth cycle oligonucleotide (O-(4)) # and the phosphorus atom of the phosphorus moiety of building block Bx, under conditions suitable for forming a covalent bond between the free backbone hydroxyl group of (5-1) cycle (i.e., fourth cycle) oligonucleotide (O-(5-1)) obtained in step (b'). # (i.e., (O-4) # ) with component B-5 of step (c'), thereby obtaining the fifth cycle oligonucleotide O-5.

[0209] Ingredient C-0 # and the (x-1)th cycle oligonucleotide (O-(x-1)) # are defined. The term "backbone hydroxyl group" refers to a hydroxyl group that is part of the backbone of each nucleoside or oligonucleotide, and will be understood based on the above explanation of the terms "hydroxyl group" and "backbone." Components B-1 and Bx are defined.

[0210] The term "react" in step (d) is used in its broadest sense to mean reacting with the component CO # and component B-1 are present in the same reaction vessel or reactor and are involved in the bond-forming reaction of step (d). The term "reacting" in step (d') is understood in the broadest sense to mean any operation in which the (x-1)th cycle of oligonucleotide (O-(x-1)) # and component Bx are present in the same reaction vessel or reactor and are involved in the bond-forming reaction of step (d'). # or the (x-1)th cycle oligonucleotide (O-(x-1)) # is already placed in a reaction vessel or reactor, and then component B-1 or Bx is added. Alternatively, component B-1 or Bx or a solution thereof is already placed in a reaction vessel or reactor, and then component C-0 is added. # or the (x-1)th cycle oligonucleotide (O-(x-1)) # is added.

[0211] During step (d), component C-0 # A covalent (chemical) bond is formed between the free backbone hydroxyl group of and the phosphorus atom of the phosphorus moiety of component B-1. During step (d'), the (x-1)th cycle oligonucleotide (O-(x-1)) #and the phosphorus atom of the phosphorus moiety of building block Bx. The bond-forming reactions of steps (d) and (d') are also referred to herein as coupling or coupling reactions or condensation or condensation reactions, and steps (d) and (d') are also referred to as coupling steps or condensation steps.

[0212] The product of the bond-forming reaction in step (d) is the first cycle oligonucleotide O-1, which is a first cycle oligonucleotide containing component C-0. # and the nucleoside sequence of component B-1, where the two are interconnected by an internucleoside linking group derived from the phosphorus moiety of component B-1. For example, the product obtained from the bond-forming reaction of step (d') of the fifth coupling cycle (x=5) is the product of the fourth cycle oligonucleotide (O-4). # and the nucleoside sequence of building block B-5, where the two are interconnected by an internucleoside linking group derived from the phosphorus moiety of building block B-5.

[0213] "Suitable conditions" for the bond-forming reactions of steps (d) and (d') form part of the general knowledge of a person skilled in the art. These conditions vary, for example, depending on the chemical structure of the phosphorus moiety to be involved in the bond-forming reaction. Any (reaction) conditions suitable for achieving the desired bond-forming reaction can be used in the method of the present invention.

[0214] The bond-forming reaction of step (d) or (d'), in which the phosphorus moiety involved in the reaction is, for example, a phosphoramidite moiety present in component B-1 or Bx of any one of formulas II-1, II-1-a, and II-1-b, is also referred to herein as a phosphoramidite coupling (reaction). Such phosphoramidite coupling is preferably carried out in the presence of an activator. Any activator used in oligonucleotide synthesis by the so-called phosphoramidite method can be used in the method of the present invention. The activator can be selected, for example, from the group consisting of: tetrazole-type activators, such as 1H-tetrazole, 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), 5-methylthio-1H-tetrazole (MTT), 1-methyl-5-mercaptotetrazole, 1-phenyl-5-mercaptotetrazole, and 5-(4-nitrophenyl)-1H-tetrazole, imidazole-type activators, such as 4,5-dicyanoimidazole (DCI) and 2-bromo-4,5-dicyanoimidacole (2-Br-DCI), 1-hydroxybenzotriazole type activators, such as 1-hydroxybenzotriazole, 1-hydroxy-6-trifluorobenzotriazole, and 1-6-trifluoro-4-nitrobenzotriazole, pyridinium salt type activators, such as pyridinium hydrochloride, pyridinium p-toluenesulfonate, and pyridinium trifluoroacetate, and - Saccharin-type active agents, i.e., salts obtained from the reaction of saccharin with organic bases such as pyridine, collidine, lutidine, picoline, N-methylimidazole, and triethylamine.

[0215] Activators disclosed in X. Wei et al., Tetrahedron 2013, 69, 3615-3637, are used, for example, in phosphoramidite coupling. 1H-tetrazole, 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), and 4,5-dicyanoimidacol (DCI) are preferred. For example, X. Wei et al., Tetrahedron 2013, 69, 3615-3637, also discloses a range of reaction conditions suitable for these activators. N-methylimidazole (NMI) is added together with the activator, which helps adjust the acidity of the solution. The phosphoramidite coupling in step (d) is preferably carried out in a solvent selected from the group consisting of acetonitrile, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), N-methyl-4-piperidone, and mixtures thereof, optionally in combination with a nonpolar solvent such as toluene, xylene, or mesitylene. Acetonitrile, DMF, and mixtures thereof are particularly preferred. The solvent (mixture) used for the phosphoramidite coupling is preferably substantially anhydrous because water reacts with the phosphoramidite. The solvent for phosphoramidite coupling preferably contains 1000 ppm or less, or 750 ppm or less, or 500 ppm or less, or 250 ppm or less, or 100 ppm or less, or 50 ppm or less of water, as determined by standard Karl Fischer titration at approximately 20°C. The phosphoramidite coupling is carried out at temperatures ranging from, for example, 0 to 90°C, 10 to 70°C, 10 to 60°C, 10 to 50°C, 10 to 40°C, 15 to 30°C, or 15 to 25°C. For convenience, the phosphoramidite coupling is simply carried out at room temperature. As the temperature increases, the reaction time decreases. The reaction time also depends on the chemical structure of the reactants and will be routinely selected by one of skill in the art based on reaction monitoring using, for example, thin layer chromatography and / or high performance liquid chromatography (HPLC), optionally coupled with mass spectrometry.

[0216] The bond-forming reaction of step (d) or (d'), in which the phosphorus moiety involved in the reaction is, for example, an H-phosphonate monoester moiety present in component B-1 or Bx of any one of formulas II-2, II-2-a, and II-2-b, is also referred to herein as H-phosphonate coupling (reaction). Such H-phosphonate coupling is typically carried out using a condensing agent. Any condensing agent used in oligonucleotide synthesis by the so-called H-phosphonate method can be used in the method of the present invention. The condensing agent may be selected from the group consisting of, for example, pivaloyl chloride (PvCl), 1-adamantanecarbonyl chloride (AdCl), 2,2-dimethylbutyryl chloride, isobutyryl chloride, diphenyl chlorophosphate, 2,4,6-triisopropylbenzenesulfonyl chloride, bis(pentafluorophenyl)carbonate, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (also called 5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphinane, DMOCP), and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (OXP or BOP-Cl). The building block B-1 or Bx containing the H-phosphonate monoester moiety is pre-activated with the condensing agent (i.e., incubated with the condensing agent), and then reacted with component CO # or the (x-1)th cycle oligonucleotide (O-(x-1)) #and react with them to carry out condensation.Those skilled in the art are familiar with the reaction conditions suitable for such H-phosphonate coupling.For example, J. Stawinski and R. Stromberg (2005), Di- and Oligonucleotide Synthesis Using H-Phosphonate Chemistry, in Methods in Molecular Biology, Vol. 288: Oligonucleotide Synthesis: Methods and Applications, edited by P. Herdewijn, Humana Press Inc., Totowa NJ, https: / / doi.org / 10.1385 / 1-59259-823-4:081, discloses such reaction conditions.The H-phosphonate coupling of step (d) or (d') is preferably carried out in a solvent selected from the group consisting of acetonitrile, pyridine, and mixtures thereof.The solvent (mixture) used for H-phosphonate coupling is preferably substantially anhydrous. The solvent for the phosphoramidite coupling preferably contains no more than 3000 ppm, no more than 2000 ppm, no more than 1500 ppm, no more than 1000 ppm, or no more than 750 ppm, no more than 500 ppm, no more than 250 ppm, no more than 100 ppm, or even no more than 50 ppm of water, as determined by standard Karl Fischer titration. The H-phosphonate coupling is carried out at temperatures ranging from, for example, 0 to 90°C, 10 to 70°C, 10 to 60°C, 10 to 50°C, 10 to 40°C, 15 to 30°C, or 15 to 25°C. For convenience, the H-phosphonate coupling is simply carried out at room temperature. As the temperature increases, the reaction time decreases. The reaction time also varies depending on the chemical structure of the reactants and will be routinely selected by one of skill in the art based on reaction monitoring, for example, using thin-layer chromatography and / or high-performance liquid chromatography (HPLC), optionally coupled with mass spectrometry.

[0217] The bond-forming reaction of step (d) or (d'), in which the phosphorus moiety involved in the reaction is an aryl phosphate diester moiety, is also referred to herein as a phosphotriester coupling reaction. Briefly, the aryl phosphate diester moiety is typically activated with an aryl sulfonyl chloride activator, such as mesitylene-2-sulfonyl chloride (MsCl), usually in the presence of an auxiliary nucleophile, such as 1-methylimidazole. Alternatively, a preformed or in situ generated 1-hydroxybenzotriazole phosphotriester is used as the phosphorus moiety in place of the aryl phosphate diester moiety, for example, in the phosphotriester coupling, also in the presence of an auxiliary nucleophile, such as 1-methylimidazole.

[0218] The bond-forming reaction of step (d) or (d'), in which the phosphorus moiety involved in the reaction is a P(V) moiety that allows for chiral phosphorothioate synthesis, is carried out, for example, as or similarly to that disclosed in Baran et al. (Science 2018, 361, 1234-1238 (see also the supplementary material of said publication) and ACS Central Science 2021, 7, 1473-1485).

[0219] Step (e) of the method of the present invention is optionally a step of incubating the first cycle oligonucleotide O-1 obtained in step (d) with an oxidizing or sulfurizing agent, thereby converting any P(III) atoms in said first cycle oligonucleotide O-1 into P(V) atoms.

[0220] Step (e') of the method of the present invention is optionally a step of incubating the xth cycle oligonucleotide Ox obtained in step (d') with an oxidizing or sulfurizing agent, thereby converting any P(III) atom in the xth cycle oligonucleotide Ox into a P(V) atom.

[0221] The terms first cycle oligonucleotide O-1 and xth cycle oligonucleotide Ox encompass respective oligonucleotides having any type of backbone structure, particularly any type of internucleoside linking group, and therefore it will be understood that the conversion of any P(III) atom to a P(V) atom does not change the name by which said first cycle oligonucleotide O-1 or said xth cycle oligonucleotide Ox is referred to herein. Only the absence of a protecting group PG-0 or PG-(x-1) can be interpreted as a superscripted number sign (i.e., hashtag symbol) " # " is particularly indicated by

[0222] The terms "P(III) atom" and "P(V) atom" are defined above. As used throughout this text, the term "oxidation state" refers to the oxidation states of P(III) atoms, as defined in the IUPAC Recommendations 2016 (P. Karen et al., Pure and Applied Chemistry As stated in

[2016] , 88(8), pp. 831-839, "The charge of an atom after homonuclear bonds are equally divided and heteronuclear bonds are assigned to binding partners according to the Allen electronegativity, except when the electronegative atom is reversibly bound as a Lewis acid ligand, in which case it does not gain electrons from that bond." The Allen electronegativity described in the reference will be used, and the P-H bond electron pair will be assigned to H. As an example, the phosphorus atom in the H phosphonate monoester moiety of any one of formulas II-2, II-2-a, and II-2-b has oxidation state III. As another example, the phosphorus atom in the phosphoramidite moiety of any one of formulas II-1, II-1-a, and II-1-b has oxidation state III. As a third example, the phosphorus atom in the phosphodiester linkage of DNA and RNA has oxidation state V.

[0223] The term "optionally" in steps (e) and (e') indicates that the respective step may or may not be performed in a given iteration of the coupling cycle, unless otherwise indicated in the context of a particular embodiment. As known to those skilled in the art, oligonucleotides containing one or more P(III) atoms, particularly one or more P(III) linking groups, are typically less stable than related oligonucleotides (e.g., those present in DNA and RNA) containing only P(V) atoms, particularly only P(V) linking groups. In some embodiments of the methods of the present invention, target oligonucleotide O T contains only P(V) atoms, in particular only P(V) linking groups. If the oligonucleotide O-1 of the first cycle obtained in step (d) does not contain a P(III) atom, it is not necessary to carry out step (e). If the oligonucleotide Ox of the xth cycle obtained in step (d') does not contain a P(III) atom, it is not necessary to carry out step (e') in the same repetition of the coupling cycle.

[0224] Those skilled in the art will understand that the oxidation state of the phosphorus atom in the internucleoside linking group formed in the bond-forming reaction of step (d) or step (d') typically varies depending on the chemical structure of the phosphorus moiety of the component B-1 or Bx involved in the respective bond-forming reaction. Typically, the oxidation state of the phosphorus atom in such a phosphorus moiety is preserved during the bond-forming reaction of step (d) or (d'). For example, when a phosphotriester coupling as defined herein is performed in step (d) or (d') of the coupling cycle, the phosphorus atom of the resulting phosphotriester internucleoside linking group typically exists as a P(V) atom, i.e., the phosphotriester linking group is a P(V) linking group. The same principle applies to the P(V) chemistry utilized by Baran et al. (Science 2018, 361, pp. 1234-1238 and ACS Central Science 2021, 7, pp. 1473-1485).

[0225] On the other hand, when the phosphorus moiety of component B-1 or Bx involved in the bond-forming reaction of step (d) or (d') contains a P(III) atom, the resulting internucleoside linking group also typically contains a P(III) atom, i.e., is a P(III) linking group. Depending on the stability of the P(III) linking group thus obtained, step (e) or (e') must be performed in the same coupling cycle x, or optionally in a subsequent iteration of the coupling cycle, for example, in the final, i.e., nth, coupling cycle. For example, in the bond-forming reaction of step (d) or (d'), typically, when the phosphorus moiety is covalently bonded via its phosphorus atom to an oxygen atom of the backbone of component B-1 or Bx, for example, when the phosphorus moiety PM in any one of formulas II, II-a, and II-b is, for example, a phosphoramidite moiety as present in any one of formulas II-1, II-1-a, and II-1-b, the phosphite triester product (phosphite triester internucleoside linking group, i.e., P(III) linking group) is a phosphite triester internucleoside linking group. group), whereas if the phosphorus moiety is covalently bonded via its phosphorus atom to an oxygen atom of the backbone of building block B-1 or Bx, e.g., if the phosphorus moiety PM in any one of formulas II, II-a, and II-b is an H-phosphonate monoester moiety, e.g., as present in any one of formulas II-2, II-2-a, and II-2-b, an H-phosphonate diester product (H-phosphonate diester internucleoside linking group, i.e., P(III) linking group) is typically obtained. H-phosphonate diester linking groups are more stable than phosphite triester linking groups, e.g., under the conditions of steps (b) and (b'), and therefore, in each iteration of the coupling cycle in which the phosphorus moiety covalently bonded to the backbone oxygen atom of building block B-1 or Bx via a phosphorus atom, e.g., the phosphorus moiety PM in any one of formulas II, II-a, and II-b, is an H-phosphonate monoester moiety, e.g., as present in any one of formulas II-2, II-2-a, and II-2-b, step (e) or (e') need not be performed.However, at least in the final coupling cycle, step (e) (if the first coupling cycle is the final coupling cycle) or (e') is carried out, thus converting any P(III) atoms into P(V) atoms.

[0226] In some embodiments of the methods of the present invention: - the phosphorus moiety of building block B-1 and each building block Bx is independently selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; - in each coupling cycle in which the phosphorus moiety of building block B-1 or building block Bx is a phosphoramidite moiety, step (e) or (e') is performed; - at least in the final coupling cycle, step (e) or (e') is carried out.

[0227] In some embodiments of the methods of the present invention: - the phosphorus moiety of building block B-1 and each building block Bx, e.g., the phosphorus moiety PM of any one of formulae II, II-a, and II-b, is a phosphoramidite moiety; In each coupling cycle, step (e) or (e') is carried out.

[0228] In some embodiments of the methods of the present invention: - the phosphorus moiety of building block B-1 and each building block Bx, e.g., the phosphorus moiety PM of any one of formulae II, II-a, and II-b, is an H-phosphonate monoester moiety; - at least in the final coupling cycle, step (e) or (e') is carried out.

[0229] In some embodiments of the methods of the present invention: in each coupling cycle, component B-1 or Bx is independently selected from the group consisting of compounds of formula II-1 and formula II-2, preferably from the group consisting of compounds of formula II-1-a and formula II-2-a, in particular from the group consisting of compounds of formula II-1-b and formula II-2-b; - in each coupling cycle in which building block B-1 or Bx is a compound of any one of formulae II-1, II-1-a and II-1-b, step (e) or (e') is carried out; - at least in the last coupling cycle, step (e) or (e') is carried out.

[0230] In some embodiments of the methods of the present invention: in each coupling cycle, component B-1 or Bx is a compound of formula II-1, preferably of formula II-1-a, in particular of formula II-1-b; In each coupling cycle, step (e) or (e') is carried out.

[0231] In some embodiments of the methods of the present invention: in each coupling cycle, building block B-1 or Bx is a compound of formula II-2, preferably of formula II-2-a, in particular of formula II-2-b; - at least in the last coupling cycle, step (e) or (e') is carried out.

[0232] The "oxidizing agent" or "sulfurizing agent" to be used in step (e) or (e') is not particularly limited in terms of its chemical structure, as long as the respective agent can convert any P(III) atom in the respective oligonucleotide O-1 or Ox to a P(V) atom. "Oxidizing agents" and "sulfurizing agents" differ in the means by which they convert P(III) atoms to P(V) atoms. An "oxidizing agent" introduces one or more covalent bonds between a phosphorus atom to be oxidized and an oxygen atom. A "sulfurizing agent" introduces one or more covalent bonds between a phosphorus atom to be sulfurized and a sulfur atom. As used herein, the term "oxidizing agent" preferably refers to any agent capable of converting a phosphite triester linking group to a phosphate triester linking group and an H-phosphonate diester linking group to a phosphate diester (i.e., phosphodiester) linking group. As used herein, the term "sulfurizing agent" preferably refers to any agent capable of converting phosphite triester linkage groups to thiophosphate triester linkage groups and H-phosphonate diester linkage groups to thiophosphate diester (i.e., phosphorothioate) linkage groups.

[0233] Iodine is a preferred oxidizing agent. For example, an aqueous solution of iodine is used, preferably in combination with a base such as pyridine. Optionally, a cosolvent such as tetrahydrofuran (THF) or acetonitrile is added. As an example, a solution of iodine (e.g., 50 mM, i.e., 50 mmol / L) in a mixture of water and pyridine (e.g., 1:9 v / v) is used. Alternatively, peroxides such as tert-butyl hydroperoxide, cumene hydroperoxide, bistrimethylsilyl peroxide, 2-butanone peroxide and hydrogen peroxide, or peroxy acids such as m-chloroperbenzoic acid (mCPBA) are used as oxidizing agents. As another alternative, for example, (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO) is used as an oxidizing agent, for example, as a 0.5 M (i.e., 0.5 mol / L) solution in acetonitrile. The oxidizing agent is applied in the form of a 0.005 to 5.0 M solution, preferably a 0.01 to 1.0 M solution, in a suitable solvent selected from the group consisting of, for example, pyridine, acetonitrile, water, tetrahydrofuran, and mixtures thereof.

[0234] Xanthan hydride (5-amino-3H-1,2,4-dithiazole-3-thione) is a preferred sulfurizing agent. For example, a solution of xanthan hydride in pyridine is used, optionally in combination with a cosolvent such as acetonitrile. In one example, a solution of xanthan hydride in pyridine (e.g., 0.2 M) is used. In another example, a solution of xanthan hydride (e.g., 0.1 M) in a mixture of pyridine and acetonitrile (e.g., 1:1, v / v) is used. Alternatively, 1,4-dithiothreitol (DTT), phenylacetyl disulfide (PADS), 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, 5-ethoxy-3H-1,2,4-dithiazol-3-one (EDITH), or 3-(N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione (CASRN: 1192027-04-5, DDTT) can be used as a sulfurizing agent. The sulfurizing agent is applied in the form of a 0.005 to 5.0 M solution, preferably a 0.01 to 1.0 M solution, in a suitable solvent selected from the group consisting of pyridine, acetonitrile, water, tetrahydrofuran, and mixtures thereof.

[0235] Steps (e) and (e') are preferably carried out at temperatures ranging from 0 to 90°C, 10 to 70°C, 10 to 60°C, 10 to 50°C, 10 to 40°C, 15 to 30°C, or 15 to 25°C. For convenience, steps (e) and (e') are simply carried out at room temperature. As the temperature increases, the reaction time decreases. The reaction time also depends on the chemical structure of the reactants and will be routinely selected by one of skill in the art based on reaction monitoring using, for example, thin layer chromatography and / or high performance liquid chromatography (HPLC), optionally coupled with mass spectrometry.

[0236] The term "incubating" in the context of step (e) refers to any process of combining the first cycle oligonucleotide O-1 obtained in step (d) and an oxidizing or sulfurizing agent defined herein in a reaction vessel or reactor (e.g., a batch reactor or a column reactor). The term "incubating" in the context of step (e') refers to any process of combining the xth cycle oligonucleotide Ox obtained in step (d') of the xth coupling cycle and an oxidizing or sulfurizing agent defined herein in a reaction vessel or reactor (e.g., a batch reactor or a column reactor). Typically, the reaction vessel or reactor already contains the respective oligonucleotide, and then the oxidizing or sulfurizing agent, or a solution thereof, is added.

[0237] In some embodiments of the methods of the present invention: - the first coupling cycle further comprises a step (f) of reacting the free hydroxyl groups with a blocking agent, step (f) being carried out after step (d) or after step (e); and / or - at least one or each of the (n-1) repetitions of the coupling cycle comprising steps (b') to (e') further comprises a step (f') of reacting the free hydroxyl group with a blocking agent, wherein step (f') is performed after step (d') or after step (e').

[0238] In some embodiments of the invention, the first coupling cycle further comprises step (f) of reacting the free hydroxyl group with a blocking agent, where step (f) occurs after step (d) or after step (e'). In some embodiments of the invention, at least one of, or each of, (n-1) iterations of the coupling cycle comprising steps (b') through (e') further comprises step (f') of reacting the free hydroxyl group with a blocking agent, where step (f') occurs after step (d') or after step (e').

[0239] The term "free hydroxyl group" in steps (f) and (f') is understood from the above. The free hydroxyl group formed during step (b) or (b') is intended to participate in the condensation reaction of step (d) or (d'), which consumes the free hydroxyl group by incorporating it into the newly formed internucleoside linking group. However, a portion of the free hydroxyl groups (typically very small, e.g., <1%, <0.5%, or <0.1%) does not participate in the condensation reaction of step (d) or (d'). Such (unreacted) free hydroxyl groups are available to participate in the condensation reaction of step (d) or (d') of the next coupling cycle. However, this is undesirable because it results in an oligonucleotide product lacking one nucleoside subunit. Such an oligonucleotide product is identical only in the absence of a single nucleoside subunit. T and later target oligonucleotide O T Steps (f) and (f') serve to prevent the formation of such difficult-to-remove by-products by blocking the (unreacted) free hydroxyl groups before entering a new iteration of the coupling cycle.

[0240] As used herein, the terms "blocking agent" and "capping agent" are used interchangeably to refer to any chemical reagent capable of acylating, preferably acetylating, free hydroxyl groups. Capping agents for oligonucleotide synthesis are part of the general knowledge of those skilled in the art. Any blocking (i.e., capping) agent known from oligonucleotide synthesis can be used in the method of the present invention. Preferred examples of such blocking agents include carboxylic acid anhydrides, particularly acetic anhydride. For example, acetylation is achieved by treating the growing oligonucleotide chain with pure acetic anhydride or a solution thereof, for example, in acetonitrile. Organic bases, such as N-methylimidazole, pyridine, lutidine (e.g., 2,6-lutidine), collidine, or mixtures thereof, are used as blocking agents. As an example, a 1:1 mixture (v / v) of capping mixture A (Cap A: 20% acetic anhydride in acetonitrile, v / v) and capping mixture B (Cap B: N-methylimidazole, 2,6-lutidine, acetonitrile, 20:30:50 v / v / v) is used as the blocking agent.

[0241] The term "reacting" in steps (f) and (f') is understood in the broadest sense as any operation in which a growing oligonucleotide chain attached to a solid support is contacted with said blocking agent, thus causing blocking / capping (i.e., acylation, preferably acetylation) of (unreacted) free hydroxyl groups.

[0242] It will be understood that because component C-0 is covalently linked to the solid support, the first cycle oligonucleotide O-1 is also covalently linked to said solid support unless cleaved therefrom. It will also be understood that because component B-1 used to prepare first cycle oligonucleotide O-1 contains protecting group PG-1, the first cycle oligonucleotide O-1 will also contain said protecting group PG-1 unless the protecting group PG-1 is cleaved. Such cleavage is carried out in step (b') of the second coupling cycle. If such a second coupling cycle is not carried out, one or more additional protecting groups, including protecting group PG-1 and the solid support, will still be cleaved from the first cycle oligonucleotide O-1.

[0243] In some embodiments of the methods of the present invention: The method comprises incubating the first cycle oligonucleotide O-1 with a deprotection mixture Mg, thereby cleaving the protecting group PG-1 from the first cycle oligonucleotide O-1 to form a first cycle oligonucleotide (O-1) having a free backbone hydroxyl group. # and / or The method comprises the steps of: # from the solid support; When both steps (g) and (h) are performed, they can be performed in either order. When step (e) is performed, step (g) and / or step (h) are preferably performed after step (e). When both steps (g) and (h) are performed, step (h) is preferably performed after step (g). It will be understood that when step (g) and / or step (h) are performed, a second coupling cycle is not performed.

[0244] Since component C-0 is covalently linked to the solid support, it is possible to obtain the oligonucleotide O-1 of the first cycle, as well as any (x-1)th cycle oligonucleotide O-(x-1), (O-(x-1)), if prepared. # It will be understood that any xth cycle oligonucleotide Ox through nth cycle oligonucleotide On are also covalently linked to the solid support unless the respective oligonucleotide is cleaved from the support. It will also be understood that, since the building block Bn used to prepare the nth cycle oligonucleotide On in the last iteration n of the coupling cycle contains a protecting group PG-n, the nth cycle oligonucleotide On will also contain said protecting group PG-n unless the protecting group PG-n is cleaved.

[0245] In some embodiments of the methods of the present invention: The method comprises incubating the nth cycle oligonucleotide On with a deprotection mixture M-g', thereby cleaving the protecting group PG-n from the nth cycle oligonucleotide On to produce an nth cycle oligonucleotide (On) having a free backbone hydroxyl group. # and / or a step (g') of obtaining - The method is to use the oligonucleotide On or (On) in the nth cycle # from the solid support; When both steps (g') and (h') are performed, they can be performed in either order. When step (e') is performed in the final (i.e., nth) coupling cycle, step (g') and / or step (h') are preferably performed after step (e'). When both step (g') and step (h') are performed, step (h') is preferably performed after step (g').

[0246] It will be understood that the descriptions and embodiments relating to deprotection mixture Mb also apply to deprotection mixture Mg, and that the descriptions and embodiments relating to deprotection mixture M-b' also apply to deprotection mixture M-g'.

[0247] In the context of steps (h) and (h'), cleaving an oligonucleotide from a solid support is understood in the broadest sense as any operation, typically a chemical reaction, that results in cleavage of the covalent bond between each oligonucleotide and the solid support. When each oligonucleotide is covalently linked to the solid support via a direct covalent bond, cleavage of the oligonucleotide from the solid support in steps (h) and (h') refers to cleavage of the direct covalent bond. When each oligonucleotide is covalently linked to the solid support via a linker moiety, cleavage of the oligonucleotide from the solid support in steps (h) and (h') refers to cleavage of the covalent bond between the linker moiety and each oligonucleotide. In both cases, each oligonucleotide is no longer covalently linked to the solid support. For example, as shown in Formulas I, Ia, and Ib (when CA is not a covalent chemical bond), if a "capping moiety" is introduced between the terminal nucleoside moiety of component C-0 and the linker moiety or solid support, the capping moiety, in contrast to the linker moiety, is not cleaved from each oligonucleotide during step (h) or step (h'). Thus, as used herein, the term "capping moiety" refers to any moiety conjugated to the terminal nucleoside moiety of an oligonucleotide chain. When present in component C-0, the capping moiety is attached to the final oligonucleotide product, i.e., the target oligonucleotide O. T An example of such a strategy is the insertion of a 3'-GalNAc conjugate, as described for example in WO2009073809.

[0248] Those skilled in the art know how to cleave oligonucleotides from solid supports. Typically, such cleavage is achieved by treating the support-bound oligonucleotide with a base such as an organic amine or an alkali hydroxide, with concentrated aqueous ammonia (i.e., aqueous ammonium hydroxide) being the most common and preferred herein. This base (e.g., ammonia) treatment can be carried out, for example, at room temperature or with heating, for example, to 40-60°C, in an autoclave or sealed container. Under such alkaline conditions, typical nucleobase protecting groups are also cleaved. As non-limiting general examples, any isobutyryl group from the exocyclic amino group of guanine, any benzoyl group from the exocyclic amino group of adenine, and any benzoyl or acetyl group from the exocyclic amino group of cytosine or 5-methylcytosine are typically cleaved (i.e., removed) under such alkaline conditions.

[0249] The protecting group R of component C-0 of any one of formulas I, Ia, and Ib z-1 and a protecting group R of each component B-1 and Bx of any one of formulas II, II-a, II-b, II-1, II-1-a, II-1-b, II-2, II-2-a, and II-2-b. z-2 and a protecting group R of each component B-1 and Bx of any one of formulae II-1, II-1-a, and II-1-b. z-3 It will be understood by those skilled in the art that typically, any such protecting group R is still included in the nth cycle oligonucleotide On that is to be cleaved from the solid support. z-1 , R z-2 , and R z-3 is typically selected to be cleaved (i.e., removed) under alkaline conditions, i.e., during the cleavage step (h'). z-1 , R z-2 , and R z-3 The 2-cyanoethyl protecting group in ##STR1## is a typical example of this strategy. Any 2-cyanoethyl protecting group is removed during base treatment, allowing cleavage from the solid support.

[0250] As known to those skilled in the art, cleavage steps (h) and (h') involve subsequent treatment with different types of bases, for example, first removing the 2-cyanoethyl protecting group using a solution of an organic amine, such as diethylamine (DEA) or triethylamine (TEA), in a suitable solvent, such as acetonitrile, preferably at room temperature, followed by cleavage from the solid support by treatment with concentrated aqueous ammonia, preferably at a temperature in the range of 40-60°C, to remove permanent base-labile protecting groups, such as nucleobase protecting groups.

[0251] In some embodiments of the methods of the invention, the method comprises the step of: # In some embodiments of the methods of the invention, the method further comprises the step (i) of modifying the oligonucleotide On or (On) # The method further comprises a step (i') of modifying

[0252] In the context of steps (i) and (i') of the method of the present invention, the term "modifying" is understood in the broadest sense to encompass "chemically modifying" and / or "biotechnologically modifying" the respective oligonucleotide. In the context of steps (i) and (i') of the method of the present invention, the term "chemically modifying" refers to subjecting the oligonucleotide to be chemically modified to one or more chemical reactions. Such chemical reactions are, for example, conjugation with a carbohydrate moiety, introduction or removal of one or more protecting groups, and intramolecular bond formation to achieve cyclization. In the context of steps (i) and (i') of the method of the present invention, the term "biotechnologically modifying" refers to subjecting the oligonucleotide to be biotechnologically modified to one or more enzymatic reactions. As used herein, the term "enzymatic reaction" refers to any reaction enabled and / or catalyzed by one or more enzymes. For example, one or more enzymatic reactions of steps (i) and (i') are used to link two or more nucleosides or oligonucleotides.

[0253] In some embodiments of the methods of the invention, the method comprises: T The method further comprises a step (k) of isolating the oligonucleotide. Means for isolating oligonucleotides during oligonucleotide synthesis are part of the general knowledge of those skilled in the art. Typically, such a process for isolating an oligonucleotide includes one or more purification steps and one or more steps aimed at obtaining the oligonucleotide in solid form. Chromatographic methods, particularly ion exchange (especially anion exchange) chromatography and reversed-phase (RP) HPLC, e.g., in the form of hydrophobic interaction HPLC, are typically used for purifying oligonucleotides. These techniques are known to those skilled in the art. In addition, the process for isolating an oligonucleotide includes an ultrafiltration and / or desalting step. For example, the solution obtained after cleaving the oligonucleotide from the solid support is subjected to ultrafiltration and / or desalting, ion exchange chromatography, and another round of ultrafiltration and / or desalting. Alternatively, the oligonucleotide cleaved from the support is subjected to reversed-phase (RP) HPLC, e.g., in the form of hydrophobic interaction HPLC. The latter method is preferably carried out when the oligonucleotide still carries a 5'-terminal hydroxyl protecting group, e.g., a DMT group. The 5'-protecting group can also be removed on an RP-HPLC column by passing an acidic solution through the column. If the 5'-terminal protecting group is removed before purification, for example, before cleaving the oligonucleotide from the support, ion exchange chromatography is preferred. Purification is typically followed by one or more steps aimed at obtaining the oligonucleotide in solid form. For example, lyophilization or spray drying is used. In some cases, it is desirable to obtain the oligonucleotide in the form of a salt with a specific counterion. In such cases, salt exchange is typically performed before lyophilization or spray drying.

[0254] In some embodiments of the methods of the present invention, the synthesis is carried out on a scale of at least 100 mmol of target oligonucleotide O. TThis is done in such an embodiment by cleaving the target oligonucleotide O to be obtained from synthesis by the method of the invention. T This means that the maximum theoretical amount of target oligonucleotide O is at least 100 mmol. T The maximum theoretical amount of component C-0 is equal to the total molar amount of component C-0 provided in step (a), assuming all process steps are 100% efficient (i.e., proceed with a quantitative yield of the desired product). For example, if 100 mmol of component C-0 is utilized (e.g., provided in step (a)), the scale of synthesis would be 100 mmol. It will be understood that this scale refers to a single synthesis process (i.e., a single batch), and not the sum of multiple batches handled in parallel or subsequently. Of course, this does not preclude such parallel or subsequently handling of batches according to the methods of the present invention. Because component C-0 is covalently linked to the solid support, the molar mass of component C-0 is not available. In this case, the molar amount of component C-0 is typically assumed to be the same as the molar amount of functional groups (typically hydroxyl moieties) on the solid support used to synthesize the oligonucleotide. As used herein, the term "synthesis" refers to the synthesis of target oligonucleotide O by the methods of the present invention, unless otherwise indicated. T In some embodiments of the methods of the present invention, the synthesis is carried out on a scale of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 mol.

[0255] In one aspect, the present invention provides the use of liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups covalently attached directly to an optionally substituted phenyl moiety, for cleaving protecting groups comprising an optionally substituted triarylmethyl residue, particularly di(p-methoxyphenyl)phenylmethyl protecting groups, from the hydroxyl moiety while suppressing nucleobase cleavage, particularly depurination, during the chemical synthesis of oligonucleotides. In one aspect, the present invention provides the use of liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups covalently attached directly to an optionally substituted phenyl moiety, for cleaving protecting groups comprising an optionally substituted triarylmethyl residue, particularly di(p-methoxyphenyl)phenylmethyl protecting groups, from the hydroxyl moiety while suppressing nucleobase cleavage, particularly depurination, during the solid phase synthesis of oligonucleotides. In one aspect, the present invention provides the use of a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently attached to an optionally substituted phenyl moiety, for cleaving a protecting group comprising an optionally substituted triarylmethyl residue, particularly a di(p-methoxyphenyl)phenylmethyl protecting group, from the hydroxyl moiety while suppressing nucleobase cleavage, particularly depurination, during solid phase synthesis of an oligonucleotide, wherein said hydroxyl moiety is part of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, particularly adenine.

[0256] As used herein, the term "suppressing nucleobase cleavage" is understood in the broadest sense as reducing the degree of (undesired) nucleobase cleavage. Nucleobase cleavage is completely or partially prevented upon cleavage of a triarylmethyl-type protecting group, such as a DMT protecting group. Similarly, the term "suppressing depurination" is understood in the broadest sense as reducing the degree of (undesired) depurination. Depurination is completely or partially prevented upon cleavage of a triarylmethyl-type protecting group, such as a DMT protecting group.

[0257] The degree of nucleobase cleavage / depurination is used to compare various detritylation protocols with respect to undesired nucleobase cleavage / depurination. Briefly, in a chromatogram (preferred detection wavelength: 260 nm) obtained from HPLC-MS analysis of a synthesized oligonucleotide (cleaved from the support if support-assisted synthesis is used), the peak areas (i.e., under the peaks) of the nucleobase cleavage / depurination-derived by-products are summed to obtain the total peak area of ​​all identified nucleobase cleavage / depurination-derived by-products. The total peak area of ​​the nucleobase cleavage / depurination-derived by-products is then divided by the peak area (i.e., under the peak) of the desired product of each oligonucleotide synthesis, and then multiplied by 100% to arrive at a percentage (%) value, thereby determining the percentage (%) of nucleobase cleavage / depurination. Clearly, a low degree of nucleobase cleavage / depurination is desirable. It will be understood that the terms "peak area" and "area under the peak" are used interchangeably herein. It will be appreciated that it is preferable to compare different detritylation protocols for the synthesis of the same target oligonucleotide (leading to the same nucleobase cleavage- / depurination-derived by-products).

[0258] In some embodiments, inhibiting nucleobase cleavage refers to reducing the extent of nucleobase cleavage by at least 5%, 10%, 25%, 50%, 75%, or at least 90% compared to an equivalent detritylation protocol differing only in that a 1.218 M (i.e., 1.218 mol / L) solution of DCA in toluene is used in place of inventive liquid composition C. Similarly, in some embodiments, inhibiting depurination refers to reducing the extent of depurination by at least 5%, 10%, 25%, 50%, 75%, or at least 90% compared to an equivalent detritylation protocol differing only in that a 1.218 M (i.e., 1.218 mol / L) solution of DCA in toluene is used in place of inventive liquid composition C. In this context, the term "equivalent detritylation protocol" refers to a protocol (i.e., procedure or method) for removing protecting groups, including optionally substituted triarylmethyl residues, preferably DMT protecting groups, under similar conditions, e.g., the same substrate, scale, temperature, time, and volume of detritylation cocktail (the only difference being the composition of the detritylation cocktail relative to liquid composition C of the present invention, e.g., 1.218 M DCA in toluene). The meaning of the term "reducing" in the context of the degree of nucleobase cleavage or depurination will be understood by those skilled in the art and is exemplified as follows: if the quotient of the degree of depurination of a first synthesis A, which uses liquid composition C of the present invention for detritylation, divided by the degree of depurination of a second synthesis B, which uses 1.218 M DCA in toluene for detritylation, is 0.95, then the degree of depurination is said to be reduced by 5%.

[0259] The terms "cleaving a protecting group" and "cleaving a protecting group" are understood in the broadest sense and refer to any process that removes a protecting group from an atom or functional group, e.g., a hydroxyl moiety, so that the latter is again available in free form, e.g., a hydroxyl group.

[0260] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: - an oligonucleotide covalently linked to a solid support and comprising a hydroxyl moiety protected by a protecting group comprising an optionally substituted triarylmethyl residue, in particular a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group, and - a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; A composition comprising:

[0261] In preferred embodiments, the composition and / or one or more components are preferably as defined in the context of the methods herein.

[0262] In a preferred embodiment, the protecting group comprising an optionally substituted triarylmethyl residue is as defined in the context of the methods herein, the protic acid is as defined in the context of the methods herein, the at least one alcohol and / or the concentration thereof is as defined in the context of the methods herein, and / or the solvent is as defined in the context of the methods herein.

[0263] In a preferred embodiment, the protecting group comprising an optionally substituted triarylmethyl residue, the protonic acid, the at least one alcohol and / or its concentration, and the solvent are each defined in the context of the method herein.

[0264] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: - an oligonucleotide covalently linked to a solid support and comprising a hydroxyl moiety protected by a protecting group comprising an optionally substituted triarylmethyl residue, in particular a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group, wherein said hydroxyl moiety is part of a nucleoside moiety comprising a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine, and - a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; A composition comprising:

[0265] In some embodiments of the methods, uses, and compositions of the present invention, each protecting group comprising an optionally substituted triarylmethyl residue, in each occurrence, is a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group. In such embodiments of the methods of the present invention, protecting group PG-0, protecting group PG-1, and each protecting group PG-x and each protecting group PG-(x-1) are DMT protecting groups.

[0266] In some embodiments of the methods, uses, and compositions of the present invention, each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: [ka] [In formula D: R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, OH, C1-C6-alkyl, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN. As used herein, according to common practice, an oxygen atom bonded to a carbon is written in parentheses to indicate that it is a carbonyl oxygen atom and does not carry any further substituents. For example, O(C1-C6-alkyl) denotes an alkoxy group in which a C1-C6-alkyl residue is bonded to the oxygen atom, and C(O)(C1-C6-alkyl) denotes an alkanoyl group in which a C1-C6-alkyl residue is bonded to the carbonyl carbon atom.

[0267] In some embodiments, in the alcohol of formula D above, R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 is the residue R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, CH3, OCH3, and OH, with the proviso that at least four of R are H. In some embodiments, in the alcohol of formula D above, R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 is the residue R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, CH3, and OCH3, with the proviso that at least four of

[0268] In some embodiments of the methods, uses, and compositions of the present invention, each of the at least one alcohol having one or more hydroxyl groups covalently bonded directly to its optionally substituted phenyl moiety is independently selected from the group consisting of m-cresol, 4-methoxyphenol, phenol, and resorcinol. In some embodiments of the methods, uses, and compositions of the present invention, each of the at least one alcohol having one or more hydroxyl groups covalently bonded directly to its optionally substituted phenyl moiety is independently selected from the group consisting of m-cresol, 4-methoxyphenol, and phenol. In some embodiments of the methods, uses, and compositions of the present invention, each of the at least one alcohol having one or more hydroxyl groups covalently bonded directly to its optionally substituted phenyl moiety is independently selected from the group consisting of m-cresol and 4-methoxyphenol. In some embodiments of the methods, uses, and compositions of the present invention, the at least one alcohol having one or more hydroxyl groups covalently bonded directly to its optionally substituted phenyl moiety is m-cresol.

[0269] In some embodiments of the methods, uses, and compositions of the present invention, Liquid Composition C comprises the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety in a molar concentration of 0.45-5.6 mol / L, 0.9-5.0 mol / L, 1.0-4.5 mol / L, 1.0-4.0 mol / L, 1.5-4.0 mol / L, 1.5-3.5 mol / L, 2.0-3.5 mol / L, 2.0-3.0 mol / L, or 2.5-3.0 mol / L. As used herein, reference may be made to molar concentrations (e.g., mol / L (i.e., M) or mmol / L (i.e., mM)). The molar concentration of a component of a solution or liquid composition, e.g., Liquid Composition C, is determined herein by dividing the total molar amount of each component added to the solution or liquid composition by the total volume of the solution or liquid composition. The total volume of a solution or liquid composition herein is determined directly by volumetric measurement from the solution or composition after all components have been added. The term "total molar amount of each component" is used to emphasize that when each component is added to the solution or composition in portions, the molar amounts of these separate portions are summed to arrive at the "total molar amount of each component." Similarly, when each component is actually a mixture of two or more components, the "total molar amount of each component" refers to the combined molar amount of these two or more components. For example, if the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is a mixture of 4-methoxyphenol and m-cresol, the total molar amount of the alcohol is the sum of the molar amounts of 4-methoxyphenol and m-cresol. Unless otherwise indicated, volumes herein are determined at 22°C.

[0270] In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety in a molar concentration of 0.45 to 5.60 mol / L; and / or Each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: [ka] [In formula D: R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, OH, C1-C6-alkyl groups, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN.

[0271] In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C comprises a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; - the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic or heteroaromatic amines whose protonated form has a pKa in the range of 1 to 4, and mixtures thereof; each of said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: [ka] [In formula D: R D-1 , R D-2 , R D-3 , R D-4 , and R D-5are independently selected from the group consisting of H, OH, C1-C6-alkyl groups, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN; the sum of the molar concentrations of the alcohols of formula D in the liquid composition C is in the range of 0.45 to 5.6 mol / L, 0.9 to 5.0 mol / L, 1.0 to 4.5 mol / L, 1.0 to 4.0 mol / L, 1.5 to 4.0 mol / L, 1.5 to 3.5 mol / L, 2.0 to 3.5 mol / L, 2.0 to 3.0 mol / L, or 2.5 to 3.0 mol / L.

[0272] In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C does not contain any of trifluoroethanol (i.e., 2,2,2-trifluoroethanol, TFE), hexafluoroisopropanol (i.e., 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP), pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol. In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C does not contain any polyfluorinated alcohol. In this context, the term "polyfluorinated alcohol" refers to an alcohol whose chemical structure has two or more fluorine atoms covalently bonded to the same carbon atom. TFE and HFIP are examples of such polyfluorinated alcohols. In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C does not contain any fluorinated alcohol. In this context, the term "fluorinated alcohol" refers to an alcohol whose chemical structure has one or more fluorine atoms covalently bonded to a carbon atom. Thus, TFE and HFIP are also examples of fluorinated alcohols. However, although 2-fluoroethanol is a fluorinated alcohol as defined herein, it is not a polyfluorinated alcohol as defined herein.

[0273] In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C comprises a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; - the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic or heteroaromatic amines whose protonated form has a pKa in the range of 1 to 4, and mixtures thereof; each of said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: [ka] [In formula D: R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, OH, C1-C6-alkyl groups, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN; Liquid composition C does not contain any of trifluoroethanol (i.e. 2,2,2-trifluoroethanol, TFE), hexafluoroisopropanol (i.e. 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP), pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and preferably does not contain any polyfluorinated alcohols.

[0274] In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C comprises a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; - the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic or heteroaromatic amines whose protonated form has a pKa in the range of 1 to 4, and mixtures thereof; each of said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: [ka] [In formula D: R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, OH, C1-C6-alkyl groups, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN; the sum of the molar concentrations of the alcohols of formula D in the liquid composition C is within the range of 0.45 to 5.6 mol / L, 0.9 to 5.0 mol / L, 1.0 to 4.5 mol / L, 1.0 to 4.0 mol / L, 1.5 to 4.0 mol / L, 1.5 to 3.5 mol / L, 2.0 to 3.5 mol / L, 2.0 to 3.0 mol / L, or 2.5 to 3.0 mol / L; Liquid composition C does not contain any of trifluoroethanol (i.e. 2,2,2-trifluoroethanol, TFE), hexafluoroisopropanol (i.e. 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP), pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and preferably does not contain any polyfluorinated alcohols.

[0275] In some embodiments of the methods, uses, and compositions of the present invention, the total molar amount of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in Liquid Composition C is in the range of 2.0 to 150.0, 2.0 to 120.0, 2.0 to 100.0, 2.0 to 95.0, 2.5 to 95.0, 3.0 to 95.0, 3.0 to 90.0, 3.0 to 85.0, 3.0 to 80.0, 3.0 to 75.0, 3.0 to 70.0, or 3.0 to 65.0 equivalents relative to the total molar amount of nucleobases. The term "total molar amount of nucleobases" refers to the total molar amount of all nucleobases present in the respective mixture, e.g., Liquid Composition C. It is understood that the nucleobases are essentially all, or at least the majority, of the nucleoside subunits of the oligonucleotide from which the protecting group, preferably a DMT group, comprising an optionally substituted triarylmethyl residue is to be cleaved. For example, if Liquid Composition C is used to cleave the DMT protecting group from 1.0 mol of oligonucleotides, each molecule containing 5 nucleobases, then the total molar amount of nucleobases is 5.0 mol.

[0276] In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid contained in Liquid Composition C has a pKa in the range of -10 to 4, -7 to 4, -6 to 4, -5 to 4, -4 to 4, -3 to 4, -2 to 4, -1 to 4, 0 to 4, 0 to 3, or 0 to 2. The term "protonic acid" and the conditions for determining pKa values ​​are described above.

[0277] In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic, or heteroaromatic amines, the protonated forms of which have a pKa in the range of 1 to 4, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, or heteroaromatic amines, the protonated forms of which have a pKa in the range of 1 to 4, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated heteroaromatic amines, the protonated forms of which have a pKa in the range of 1 to 4, and mixtures thereof.

[0278] Examples of carboxylic acids in Liquid Composition C of the present invention are halogenated acetic acids, such as trifluoroacetic acid (TFA), dichloroacetic acid (DCA), and trichloroacetic acid (TCA), with TFA and DCA being preferred. Examples of sulfonic acids in Liquid Composition C of the present invention are methanesulfonic acid and p-toluenesulfonic acid. Examples of mineral acids in Liquid Composition C of the present invention are hydrochloric acid and sulfuric acid. Examples of aliphatic amines in Liquid Composition C of the present invention are triethylamine (TEA) and diisopropylethylamine (DIPEA). Examples of aromatic amines in Liquid Composition C of the present invention are diphenylamine and aniline derivatives having electron-withdrawing substituents. As used herein, the term "electron-withdrawing substituent" refers to a substituent selected from the group consisting of a halogen atom, such as chlorine, fluorine, or bromine, a cyano group, an aldehyde group, a keto group, a carboxyester group, or a carboxamide group, unless otherwise indicated in the context of a particular embodiment. Examples of heteroaromatic amines in the liquid composition C of the present invention include pyrimidine, pyridine, pyrazine, thiazole, pyridazine, pyrazole, or triazole, all of which are optionally substituted with electron-donating or electron-withdrawing substituents. For example, the heteroaromatic amine is pyrimidine, pyridine, thiazole, pyridazine, pyrazole, or 1,2,4-triazole substituted with one or more electron-withdrawing substituents. As another example, the heteroaromatic amine is pyrimidine or pyrazine substituted with one or more electron-donating substituents. The electron-donating substituent is, for example, a methoxy group. Preferably, the heteroaromatic amine is pyridine substituted with one or more electron-withdrawing substituents selected from the group consisting of halogen atoms, cyano groups, aldehyde groups, keto groups, carboxyester groups, and carboxamide groups. Preferably, the heteroaromatic amine is pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of a cyano group and a halogen atom (F, Cl, Br, I). In particular, the heteroaromatic amine is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof, of which 4-cyanopyridine is most preferred.For example, the pKa values ​​of variously substituted pyridinium ions may be obtained from or determined according to the procedure disclosed in A. Fischer et al., Journal of the Chemical Society 1964, pp. 3591-3596.

[0279] It will be understood by those skilled in the art that the protonated form of an aliphatic, aromatic, or heteroaromatic amine is typically obtained by combining the amine with a protonic acid capable of protonating the amine, such as a carboxylic acid, sulfonic acid, or mineral acid. It will be understood that the compound of the acid and the amine is a pre-formed salt of the acid and the amine. Alternatively, the acid and the heteroaromatic amine are added as is (i.e., not as a pre-formed salt). Thus, the amine is combined with the protonic acid, but the protonic acid has a lower pKa value than the protonated form of the amine. The combination of the amine and the protonic acid may occur beforehand to obtain a salt of the protonic acid and the amine. Alternatively, the amine and the protonic acid are combined in liquid composition C.

[0280] In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in liquid composition C is selected from the group consisting of (hetero)alkylsulfonic acids, (hetero)arylsulfonic acids, hydrogen halides, sulfuric acid, protonated heteroaromatic amines whose protonated forms have a pKa in the range of 1 to 4, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in liquid composition C is selected from the group consisting of dihalogenated acetic acids, trihalogenated lactic acids, alkylsulfonic acids, arylsulfonic acids, hydrogen halides, sulfuric acid, protonated heteroaromatic amines whose protonated forms have a pKa in the range of 1 to 4, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in liquid composition C is selected from the group consisting of dihalogenated acetic acids, trihalogenated lactic acids, alkylsulfonic acids, arylsulfonic acids, hydrogen halides, protonated heteroaromatic amines selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, and 3-chloropyridine, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in Liquid Composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, hydrochloric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, and 3-chloropyridine, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid in Liquid Composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, and 3-chloropyridine, and mixtures thereof.In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid contained in Liquid Composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, hydrochloric acid, methanesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, and 3-chloropyridine, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid contained in Liquid Composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, 4-chloropyridinium hydrochloride, and 4-cyanopyridinium trifluoroacetate. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid contained in Liquid Composition C is selected from the group consisting of dichloroacetic acid and 4-cyanopyridinium trifluoroacetic acid. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid contained in Liquid Composition C is dichloroacetic acid. In some embodiments of the methods, uses, and compositions of the present invention, the protonic acid contained in Liquid Composition C is 4-cyanopyridinium trifluoroacetic acid.

[0281] In some embodiments of the methods, uses, and compositions of the present invention, liquid composition C comprises the protonic acid at a concentration of 0.01 to 2.0 mol / L, 0.05 to 1.5 mol / L, 0.05 to 1.25 mol / L, 0.05 to 1.0 mol / L, 0.05 to 0.9 mol / L, 0.05 to 0.8 mol / L, 0.05 to 0.7 mol / L, 0.05 to 0.6 mol / L, 0.05 to 0.5 mol / L, 0.05 to 0.4 mol / L, 0.05 to 0.35 mol / L, 0.06 to 0.35 mol / L, 0.07 to 0.35 mol / L, 0.08 to 0.35 mol / L, 0.09 to 0.35 mol / L, 0.09 to 0.3 mol / L, or 0.09 to 0.25 mol / L. The means for determining the molar concentration of a component of a solution or liquid composition, e.g., Liquid Composition C, are described above and apply to the protonic acid contained in Liquid Composition C. As described above, the protonic acid may also be a protonated aliphatic, aromatic, or heteroaromatic amine, the protonated form of which has a pKa in the range of 1 to 4. As also described above, such a protonated form of an amine is typically obtained by combining the amine with a protonic acid capable of protonating the amine, such as a carboxylic acid, sulfonic acid, or mineral acid. This combination occurs by adding the amine and acid to their respective solutions, or by preforming a salt of the acid and the amine and adding the preformed salt to the solution. When a preformed salt is used, the molar concentration of the protonic acid will be determined based on the total molar amount of the preformed salt used to prepare the liquid composition, e.g., Liquid Composition C, provided that the acid and amine are present in the salt at a 1:1 stoichiometry, as in the case of, for example, 4-chloropyridinium hydrochloride. Otherwise, the stoichiometry of the acid and amine in the salt is routinely considered. However, if the protonic acid and unprotonated amine are added separately and protonation of the amine occurs after addition, the total molar amount of protonic acid initially added with the amine will be used to calculate the concentration of protonic acid in a liquid composition, e.g., Liquid Composition C.As a first example, if 4-cyanopyridine and trifluoroacetic acid (TFA) are added separately during the preparation of liquid composition C, the total molar amount of TFA is used to calculate the molar concentration of the protonic acid in liquid composition C. As a second example, if a preformed salt, such as 4-chloropyridinium hydrochloride, is added during the preparation of liquid composition C, the total molar amount of the salt is used to calculate the molar concentration of the protonic acid in liquid composition C.

[0282] In some embodiments of the methods, uses, and compositions of the present invention, the total molar amount of the protonic acid contained in Liquid Composition C ranges from 0.80 to 15.0, 1.0 to 13.0, or 2.0 to 12.50 equivalents relative to the total molar amount of protecting groups containing optionally substituted triarylmethyl residues, preferably di(p-methoxyphenyl)phenylmethyl (DMT) groups. The term "total molar amount," when referring to protecting groups such as DMT groups, refers to the total molar amount of compounds bearing each protecting group multiplied by the amount of said protecting groups per molecule of those compounds. For example, if a composition contains 1.0 mole of a compound having exactly one DMT group per molecule (and no other compounds in the composition contain any DMT groups), the total molar amount of DMT groups in the composition is equal to the total molar amount of the compounds, which in this example is 1.0 mole.

[0283] In some embodiments of the methods, uses, and compositions of the present invention, the solvent contained in liquid composition C is an aprotic solvent. As used herein, an "aprotic solvent" is a solvent that is not a hydrogen bond donor. Thus, an aprotic solvent is a solvent that does not have either an OH or an NH bond. Thus, alcohols are not aprotic solvents as defined herein. In some embodiments of the methods, uses, and compositions of the present invention, the solvent contained in liquid composition C is a non-halogenated aprotic solvent. As used herein, a "non-halogenated solvent" is a solvent that does not contain halogen atoms (particularly F, Cl, Br, or I) in its chemical structure. In some embodiments of the methods, uses, and compositions of the present invention, the liquid composition C is essentially free of halogenated solvents. Examples of such halogenated solvents are dichloromethane (DCM), chloroform, and 1,2- or 1,1-dichloromethane. As used herein, the term "essentially free of halogenated solvents" preferably means that any halogenated solvents (i.e., solvents containing at least one halogen atom in their chemical structure) collectively account for no more than 3.0%, 2.0%, 1.0%, 0.1%, 0.01%, or 0.001% of the total volume of said liquid composition C. For example, if one or more halogenated solvents are to be added during the preparation of liquid composition C, the total volume of these one or more halogenated solvents added is divided by the total volume determined by volumetric measurement of liquid composition C after all solvents and ingredients have been added, and then multiplied by 100% to arrive at a percentage value representing the volume percentage occupied by said one or more halogenated solvents. The volume of added solvent and the volume of the liquid composition are determined at 22°C. Preferably, no halogenated solvents are added during the preparation of liquid composition C, and any trace amounts of halogenated solvents potentially present in liquid composition C are only impurities of (commercially) obtained solvents or ingredients.

[0284] In some embodiments of the methods, uses, and compositions of the present invention, the solvent contained in liquid composition C is selected from the group consisting of halogenated hydrocarbon solvents, (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, alkyl(hetero)aryl ethers, and mixtures thereof. Non-limiting examples of halogenated hydrocarbon solvents include dichloromethane (DCM), dichloroethane, and chloroform. A non-limiting example of a (hetero)aromatic solvent is benzene. Non-limiting examples of alkyl(hetero)aromatic solvents are toluene, o-xylene, m-xylene, p-xylene, and mesitylene. A non-limiting example of a (hetero)aromatic ether is diphenyl ether. A non-limiting example of an alkyl(hetero)aryl ether is anisole.

[0285] In some embodiments of the methods, uses, and compositions of the present invention, the solvent included in liquid composition C is: benzene, in which one or more hydrogen residues are optionally replaced by C1-C3-alkyl or O(C1-C3-alkyl) groups (for example benzene, toluene, o-xylene, m-xylene, p-xylene, mesitylene or anisole); halogenated C1-C3-alkyl solvents (e.g. dichloromethane, 1,1- or 1,2-dichloroethane, or chloroform); - O(C1-C6-alkyl)2 ethers containing a total of 4 or more carbon atoms (for example, diethyl ether or cyclopentyl methyl ether); aliphatic cyclic ethers containing 4 to 6 ring carbon atoms, preferably 4 or 5 ring carbon atoms, in which one or more hydrogen residues are optionally replaced by C1-C3 alkyl groups (for example, tetrahydrofuran, tetrahydropyran or 1,4-dioxane); - C5-C9 alkyl solvents (for example, pentane, hexane, cyclohexane, heptane, octane or nonane); - C1-C3 alkyl solvents in which exactly one hydrogen residue is replaced by a nitrile group (CN) (e.g., acetonitrile or propionitrile); - ester solvents of the formula (C1-C6-alkyl)-OC(O)-(C1-C6-alkyl) (for example ethyl acetate); and - A mixture of these is selected from the group consisting of:

[0286] In some embodiments of the methods, uses, and compositions of the present invention, the solvent included in liquid composition C is: benzene, in which one or more hydrogen residues are optionally replaced by C1-C3-alkyl or O(C1-C3-alkyl) groups (for example benzene, toluene, o-xylene, m-xylene, p-xylene, mesitylene or anisole); - O(C1-C6-alkyl)2 ethers containing a total of 4 or more carbon atoms (for example, diethyl ether or cyclopentyl methyl ether); aliphatic cyclic ethers containing 4 to 6 ring carbon atoms, preferably 4 or 5 ring carbon atoms, in which one or more hydrogen residues are optionally replaced by C1-C3 alkyl groups (for example, tetrahydrofuran, tetrahydropyran or 1,4-dioxane); - C5-C9 alkyl solvents (for example, pentane, hexane, cyclohexane, heptane, octane or nonane); - C1-C3 alkyl solvents in which exactly one hydrogen residue is replaced by a nitrile group (CN) (e.g., acetonitrile or propionitrile); - ester solvents of the formula (C1-C6-alkyl)-OC(O)-(C1-C6-alkyl) (for example ethyl acetate); and - A mixture of these is selected from the group consisting of:

[0287] In some embodiments of the methods, uses, and compositions of the present invention, the solvent included in liquid composition C is: benzene, in which one or more hydrogen residues are optionally substituted by C1-C3-alkyl or O(C1-C3-alkyl) groups (for example benzene, toluene, o-xylene, m-xylene, p-xylene, mesitylene or anisole); - O(C1-C6-alkyl)2 ethers containing a total of 4 or more carbon atoms (for example, diethyl ether or cyclopentyl methyl ether); aliphatic cyclic ethers containing 4 to 6 ring carbon atoms, preferably 4 or 5 ring carbon atoms, in which one or more hydrogen residues are optionally replaced by C1-C3 alkyl groups (for example, tetrahydrofuran, tetrahydropyran or 1,4-dioxane); - C5-C9 alkyl solvents (e.g., pentane, hexane, cyclohexane, heptane, octane or nonane); and - A mixture of these is selected from the group consisting of:

[0288] In some embodiments of the methods, uses, and compositions of the present invention, the solvent included in liquid composition C is: benzene, in which one or more hydrogen residues are optionally replaced by C1-C3-alkyl or O(C1-C3-alkyl) groups (for example benzene, toluene, o-xylene, m-xylene, p-xylene, mesitylene or anisole); halogenated C1-C3-alkyl solvents (e.g., dichloromethane, 1,1- or 1,2-dichloroethane, or chloroform); and - A mixture of these is selected from the group consisting of:

[0289] In some embodiments of the methods, uses, and compositions of the present invention, the solvent contained in liquid composition C is benzene (e.g., benzene, toluene, o-xylene, m-xylene, p-xylene, mesitylene, or anisole), optionally in which one or more hydrogen residues are replaced by a C1-C3-alkyl or O(C1-C3-alkyl) group, or a mixture of such solvents.

[0290] In some embodiments of the methods, uses, and compositions of the present invention, the solvent in liquid composition C is selected from the group consisting of toluene, anisole, o-xylene, m-xylene, p-xylene, mesitylene, dichloromethane, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the solvent in liquid composition C is selected from the group consisting of toluene, anisole, o-xylene, m-xylene, p-xylene, mesitylene, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the solvent in liquid composition C is selected from the group consisting of toluene, anisole, and mixtures thereof. In some embodiments of the methods, uses, and compositions of the present invention, the solvent in liquid composition C is toluene. In some embodiments of the methods, uses, and compositions of the present invention, the solvent in liquid composition C is anisole. In some embodiments of the methods, uses, and compositions of the present invention, the solvent in liquid composition C is selected from the group consisting of toluene, anisole, dichloromethane, and mixtures thereof.

[0291] In some embodiments of the methods, uses, and compositions of the present invention, the total volume of the solvent included in Liquid Composition C comprises 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, or 60-70% of the total volume of Liquid Composition C. To determine the percentage of the total volume occupied by the solvent, the total volume of the solvent added during preparation of Liquid Composition C is divided by the total volume of Liquid Composition C, and then multiplied by 100% to arrive at a percent (%) value. The total volume of a liquid composition herein is determined directly by volumetric measurement of the composition after all components have been added. The volume of added solvent and the volume of the liquid composition are to be determined at 22°C.

[0292] In some embodiments of the methods, uses, and compositions of the present invention, the total volume of Liquid Composition C is in the range of 5-100 mL, 10-90 mL, 10-80 mL, 20-75 mL, 10-70 mL, 10-60 mL, 20-60 mL, 30-60 mL, or 40-60 mL per millimole (mmol) of protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT group, to be cleaved (i.e., removed). The total volume of Liquid Composition C is routinely adapted by one of skill in the art to ensure efficient mass transport of reagents to the growing oligonucleotide chains and suitable macroscopic properties of the slurry, while minimizing the volume of the slurry containing the solid support.

[0293] In some embodiments of the methods, uses, and compositions of the present invention: the total molar amount of the protonic acids contained in the liquid composition C is in the range of 0.80 to 15.0 equivalents relative to the total molar amount of the protecting groups containing an optionally substituted triarylmethyl residue; the total molar amount of the alcohols having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is in the range of 2.0 to 210.0 equivalents relative to the total molar amount of the nucleobases.

[0294] In some embodiments of the methods, uses, and compositions of the present invention, the liquid composition C further comprises a carbocation scavenger. As used herein, the term "carbocation scavenger" refers to a nucleophilic compound that is used to bind a carbocation or consume the carbocation by formally donating a hydride anion, thereby preventing undesired side reactions of the carbocation. A typical example of such a carbocation is the carbocation formed during cleavage of a protecting group containing an optionally substituted triarylmethyl residue. For example, cleavage of a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group generates a DMT cation (i.e., a di(p-methoxyphenyl)phenylmethyl cation). Examples of carbocation trapping agents are aliphatic alcohols (e.g., methanol and ethanol), water, silanes (e.g., triisopropylsilane (TIS) and triethylsilane (TES)), N-heterocycles (e.g., pyrrole, 3-methylpyrrole, 2,4-dimethylpyrrole indole, 2-methylindole, thiols and thiophenols (e.g., 1,2-ethanedithiol (EDT), 1,4-dithioerythrol (DTE), 1,4-dithiothreitol (DTT), 3,6-dioxa-1,8-octanedithiol (DODT), 1,4-benzenedimethanethiol (BDMT), 1,4-butanedithiol, 2-mercaptoethanol, cysteine, thiophenol, p-thiocresol, and thiomalic acid), and polyalkylbenzenes (e.g., 1,3,5-trimethylbenzene and pentamethylbenzene).

[0295] In some embodiments of the methods, uses, and compositions of the present invention, the contact time between liquid composition C and the oligonucleotide to be cleaved of the protecting group containing an optionally substituted triarylmethyl residue is at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, or even 1 hour or more. Such a relatively long time interval allows the use of a standard pump with a moderate pumping speed for a relatively large reactor (e.g., a column or batch reactor). Such a long contact time can lead to undesired cleavage of the nucleobase, particularly depurination, when used with a detritylation cocktail commonly used in the art, such as a detritylation cocktail containing a large amount of an acid, such as alcohol-free dichloroacetic acid (DCA) as defined in the present invention. A particular advantage of the present invention is that the method of oligonucleotide synthesis can be scaled up without the need to increase the maximum flow rate of the liquid handling system. Furthermore, compared to the standard deprotection protocol using 1.218 M dichloroacetic acid in toluene, the method of the present invention allows for an increase in batch size by at least 5-fold, preferably at least 10, 20, 30, or 40-fold, while using the same liquid handling system to feed and drain liquids from the reaction vessel. Note that for clarity, the reaction vessel is modified to accommodate larger volumes of reagents.

[0296] In some embodiments of the methods, uses, and compositions of the present invention: - each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is an alcohol of formula D above; - the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic or heteroaromatic amines whose protonated form has a pKa in the range of 1 to 4, and mixtures thereof; - the solvent contained in liquid composition C is an aprotic solvent, preferably a non-halogenated aprotic solvent; liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly and covalently bonded to an optionally substituted phenyl moiety, in a molar concentration between 0.45 and 5.6 mol / L;

[0297] In some embodiments of the methods, uses, and compositions of the present invention: - each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is an alcohol of formula D above; - the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated heteroaromatic amines whose protonated form has a pKa in the range of 1 to 4, and mixtures thereof; - the solvent contained in liquid composition C is an aprotic solvent, preferably a non-halogenated aprotic solvent; liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly and covalently bonded to an optionally substituted phenyl moiety, in a molar concentration between 0.45 and 5.6 mol / L;

[0298] In some embodiments of the methods, uses, and compositions of the present invention: - each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is an alcohol of formula D above, wherein R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 is the residue R D-1 , R D-2 , R D-3 , R D-4 , and R D-5are independently selected from the group consisting of H, CH3, and OCH3, with the proviso that at least four of them are H; - the protonic acid contained in liquid composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, hydrochloric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine and 3-chloropyridine, and mixtures thereof; - the solvent contained in composition C is selected from the group consisting of benzene, optionally in which one or more hydrogen residues are replaced by C1-C3-alkyl or O(C1-C3-alkyl) groups, and dichloromethane; liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly and covalently bonded to an optionally substituted phenyl moiety, in a molar concentration between 0.45 and 5.6 mol / L;

[0299] In some embodiments of the methods, uses, and compositions of the present invention: - each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is an alcohol of formula D above, wherein R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 is the residue R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, CH3, and OCH3, with the proviso that at least four of them are H; - the protonic acid contained in liquid composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine and 3-chloropyridine, and mixtures thereof; - the solvent contained in composition C is benzene, optionally in which one or more hydrogen residues are replaced by C1-C3-alkyl groups or O(C1-C3-alkyl); liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly and covalently bonded to an optionally substituted phenyl moiety, in a molar concentration between 0.45 and 5.6 mol / L;

[0300] In some embodiments of the methods, uses, and compositions of the present invention: - each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is selected from the group consisting of m-cresol, 4-methoxyphenol, and phenol; - the protonic acid contained in liquid composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine and 3-chloropyridine, and mixtures thereof; - the solvent contained in composition C is selected from the group consisting of toluene, anisole, o-xylene, m-xylene, p-xylene, mesitylene, and mixtures thereof; liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly and covalently bonded to an optionally substituted phenyl moiety, in a molar concentration between 0.45 and 5.6 mol / L;

[0301] In some embodiments of the methods, uses, and compositions of the present invention: - each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety contained in liquid composition C is selected from the group consisting of m-cresol, 4-methoxyphenol, and phenol; - the protonic acid contained in liquid composition C is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, hydrochloric acid, methanesulfonic acid, a combination of any of these acids with a heteroaromatic amine selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine and 3-chloropyridine, and mixtures thereof; - the solvent contained in composition C is selected from the group consisting of toluene, anisole, o-xylene, m-xylene, p-xylene, mesitylene, and mixtures thereof; - liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety in a molar concentration of 0.45 to 5.6 mol / L; Liquid composition C contains the protonic acid in a molar concentration of 0.01 to 2.0 mol / L.

[0302] The present invention also refers to the use of a liquid composition C (deprotection mixture Mb) comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety, for cleaving one or more protecting groups PG-0 from a nucleoside or oligonucleotide.

[0303] Preferably, the liquid composition C (deprotection mixture Mb) and / or one or more of its components are as described in the context of the method or composition.

[0304] The present invention relates, for example, to the following phrases:

[0305] 1. Targeted Oligonucleotides T 1. A method for solid phase synthesis of a compound represented by the formula (I), comprising step (b) of incubating a nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from the nucleoside or oligonucleotide, wherein the deprotection mixture Mb is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety.

[0306] 2. Targeted Oligonucleotides T comprises a first cycle oligonucleotide O-1, and the method comprises the following steps (a) and the following steps (b) to (e): (a) providing a component C-0 selected from the group consisting of nucleosides and oligonucleotides, component C-0 being covalently linked to a solid support and comprising backbone hydroxyl moieties protected by a protecting group PG-0 comprising an optionally substituted triarylmethyl residue; (b) incubating component C-0 of step (a) with a deprotection mixture Mb, thereby cleaving the protecting group PG-0 from component C-0 to produce component C-0 having a free backbone hydroxyl group; # obtaining the (c) providing a building block B-1 selected from the group consisting of nucleosides and oligonucleotides, building block B-1 comprising a backbone hydroxyl moiety protected by a protecting group PG-1 comprising an optionally substituted triarylmethyl residue, and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block B-1 via a phosphorus atom; (d) Component C-0 # and the phosphorus atom of the phosphorus moiety of component B-1. #with component B-1 of step (c), thereby obtaining oligonucleotide O-1 of the first cycle; (e) optionally incubating the first cycle oligonucleotide O-1 obtained in step (d) with an oxidizing or sulfurizing agent, thereby converting any P(III) atoms in said first cycle oligonucleotide O-1 to P(V) atoms. a first coupling cycle comprising: In step (b), the deprotection mixture Mb is a liquid composition C comprising a solvent, a protonic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; How to follow clause 1.

[0307] 3. Targeted Oligonucleotides T comprises an n-th cycle of oligonucleotide On, and the method further comprises performing (n-1) repetitions of a coupling cycle comprising the following steps (b') to (e'), where n is an integer ranging from 2 to 99 and indicates the total number of coupling cycles performed to obtain the n-th cycle of oligonucleotide On, and each individual coupling cycle comprising the following steps (b') to (e') is identified by a serial number x, which is performed one by one from 2 to n: (b') The (x-1)th cycle oligonucleotide O-(x-1) obtained in the previous coupling cycle is incubated with the deprotection mixture M-b', thereby cleaving the protecting group PG-(x-1) from the (x-1)th cycle oligonucleotide O-(x-1) to form the (x-1)th cycle oligonucleotide (O-(x-1)) having a free backbone hydroxyl group. # obtaining the (c') providing a building block Bx selected from the group consisting of nucleosides and oligonucleotides, wherein building block Bx comprises a backbone hydroxyl moiety protected by a protecting group PG-x comprising an optionally substituted triarylmethyl residue, and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block Bx via a phosphorus atom; (d') (x-1)th cycle oligonucleotide (O-(x-1)) # and the phosphorus atom of the phosphorus moiety of building block Bx, under conditions suitable for forming a covalent bond between the free backbone hydroxyl group of the (x-1)th cycle oligonucleotide (O-(x-1)) obtained in step (b'). # with the building block Bx of step (c'), thereby obtaining the xth cycle oligonucleotide Ox; (e') optionally incubating the xth cycle oligonucleotide Ox obtained in step (d') with an oxidizing or sulfurizing agent, thereby converting any P(III) atom in said xth cycle oligonucleotide Ox into a P(V) atom; wherein in at least one repetition of step (b'), the deprotection mixture M-b' is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety; How to follow clause 2.

[0308] 4.- The phosphorus moiety of building block B-1 and each building block Bx is independently selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; - in each coupling cycle in which the phosphorus moiety of building block B-1 or building block Bx is a phosphoramidite moiety, step (e) or (e') is performed; - in at least the final coupling cycle, step (e) or (e') is carried out, Follow either clause 2 or 3.

[0309] 5. The nucleoside or oligonucleotide of phrase 1 and component C-0 of phrase 2 covalently linked to a solid support is a compound of formula I: [ka] [In Formula I: Each oxygen atom (O) depicted within each nucleoside subunit x-0 through xm represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; each of the nucleoside subunits x-0 through xm is the same or different; PG-0 is a protecting group comprising an optionally substituted triarylmethyl residue; m is an integer greater than or equal to 0; Y 1 is independently selected from the group consisting of O and S for each repeat unit m; Z 1 For each repeat unit m, OR z-1 and S.R. z-1 independently selected from the group consisting of: R z-1 is a protecting group that is the same or different for each repeat unit m; CA is a capping moiety or covalent chemical bond; L is a linker moiety or a covalent chemical bond; SM is solid support] This method follows one of clauses 1 to 4.

[0310] 6. Each of components B-1 and Bx is a compound of formula II-1: [ka] [In Formula II-1: Each oxygen atom (O) depicted within each nucleoside subunit y-0 through yq represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; each nucleoside subunit y-0 to yq is the same or different; PG is a protecting group PG-1 or PG-x, comprising an optionally substituted triarylmethyl residue; q is an integer greater than or equal to 0; Y 2 is independently selected from the group consisting of O and S for each repeat unit q; Z 2 For each repeating unit q, OR z-2 , S.R. z-2 independently selected from the group consisting of: R z-2 is a protecting group that is the same or different for each repeat unit q; Z 3 is selected from the group consisting of O and S; R z-3 is a protecting group; R a and R b are each independently a C1-C6 alkyl group, and R a and R b are the same or different, and R a and R b may also be bonded together with the nitrogen atom to which they are attached to form a 5- or 6-membered aliphatic cyclic amine moiety; Step (e) or step (e') is performed in each coupling cycle; Follow one of clauses 2-5.

[0311] 7.- The first coupling cycle further comprises a step (f) of reacting the free hydroxyl groups with a blocking agent, step (f) being carried out after step (d) or after step (e); and / or - at least one of the (n-1) repetitions of the coupling cycle comprising steps (b') to (e') further comprises a step (f') of reacting a free hydroxyl group with a blocking agent, and step (f') is performed after step (d') or after step (e'); Follow one of clauses 2-6.

[0312] 8.—The method comprises incubating the first cycle oligonucleotide O-1 with a deprotection mixture Mg, thereby cleaving the protecting group PG-1 from the first cycle oligonucleotide O-1 to produce a first cycle oligonucleotide (O-1) having a free backbone hydroxyl group. # and / or - the method comprises the step of: - preparing the oligonucleotide O-1 or (O-1) in the first cycle; # from the solid support; When steps (g) and (h) are both performed, they can be performed in either order; Follow clauses 2 and one of 4-7.

[0313] 9.- The method comprises incubating the nth cycle oligonucleotide On with a deprotection mixture M-g', thereby cleaving the protecting group PG-n from the nth cycle oligonucleotide On to produce the nth cycle oligonucleotide (On) having a free backbone hydroxyl group. # and / or a step (g') of obtaining - the method is the nth cycle of oligonucleotide On or (On) # from the solid support; When steps (g') and (h') are both performed, they can be performed in either order. Follow one of clauses 3-7.

[0314] 10. The method according to any one of clauses 1-9, wherein at least steps (b) and (b') are carried out in a batch reactor, or at least steps (b) and (b') are carried out in a column reactor, and the flow rate of liquid composition C through the column reactor is less than 300 cm / hr.

[0315] 11.- The backbone hydroxyl moiety protected by the protecting group PG-0 is part of a nucleoside moiety containing a purine-type nucleobase; - in at least one repetition of the coupling cycle comprising steps (b') to (e'), wherein the protecting group PG-(x-1) is part of a nucleoside moiety comprising a purine-type nucleobase, the deprotection mixture M-b' is liquid composition C; Follow one of clauses 1-10.

[0316] 12. The method according to any one of clauses 1-11, wherein each protecting group comprising an optionally substituted triarylmethyl residue is a di(p-methoxyphenyl)phenylmethyl protecting group.

[0317] 13. Liquid composition C comprises said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety in a molar concentration of 0.45 to 5.60 mol / L, and / or Each of the at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: [ka] [In formula D: R D-1 , R D-2 , R D-3 , R D-4 , and R D-5 are independently selected from the group consisting of H, OH, C1-C6-alkyl groups, O(C1-C6-alkyl), C(O)(C1-C6-alkyl), C(O)O(C1-C6-alkyl), F, Cl, Br, I, and CN. A method according to any one of clau...

Claims

1. Target Oligonucleotide O Ｔ 1. A method for the solid phase synthesis of (I), comprising step (b) of incubating a nucleoside or oligonucleotide covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group PG-0 with a deprotection mixture M-b, thereby cleaving the protecting group PG-0 from the nucleoside or oligonucleotide, wherein said deprotection mixture M-b is a liquid composition C comprising a solvent, a protic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups covalently attached directly to an optionally substituted phenyl moiety; the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic or heteroaromatic amines, the protonated forms of which have a pKa in the range of 1 to 4, and mixtures thereof; each of said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: 【Chemical 1】 [In formula D: R Ｄ－１ , R Ｄ－２ , R Ｄ－３ , R Ｄ－４ , and R Ｄ－５ is H, OH, C １ ~C ６ - alkyl group, O(C １ ~C ６ -alkyl), C(O)(C １ ~C ６ -alkyl), C(O)O(C １ ~C ６ -alkyl), F, Cl, Br, I, and CN. That's the method.

2. Target Oligonucleotide O Ｔ comprises a first cycle oligonucleotide O-1, and the method comprises the following steps (a) and the following steps (b) to (e): (a) providing a component C-0 selected from the group consisting of nucleosides and oligonucleotides, component C-0 being covalently linked to a solid support and comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group PG-0; (b) incubating component C-0 of step (a) with deprotection mixture Mb, thereby cleaving the protecting group PG-0 from component C-0 to produce component C-0 having a free backbone hydroxyl group; ＃ obtaining (c) providing a building block B-1 selected from the group consisting of nucleosides and oligonucleotides, building block B-1 comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group PG-1 and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block B-1 through a phosphorus atom; (d) Component C-0 ＃ and the phosphorus atom of the phosphorus moiety of component B-1 under conditions suitable for forming a covalent bond between the free backbone hydroxyl group of component C-0 of step (b). ＃ with component B-1 of step (c), thereby obtaining oligonucleotide O-1 of the first cycle; (e) optionally incubating the first cycle oligonucleotide O-1 obtained in step (d) with an oxidizing or sulfurizing agent, thereby converting any P(III) atoms in said first cycle oligonucleotide O-1 to P(V) atoms. a first coupling cycle comprising:

2. The method of claim 1, wherein in step (b), the deprotection mixture Mb is the liquid composition C defined in claim 1.

3. Target Oligonucleotide O Ｔ comprises an nth cycle oligonucleotide O-n, and the method further comprises performing (n-1) repetitions of a coupling cycle comprising the following steps (b') to (e'), where n is an integer ranging from 2 to 99 and indicates the total number of coupling cycles performed to obtain the nth cycle oligonucleotide O-n, and each individual coupling cycle comprising the following steps (b') to (e') is identified by a sequential number x, which is performed one by one from 2 to n: (b') The (x-1)th cycle oligonucleotide O-(x-1) obtained in the previous coupling cycle is incubated with the deprotection mixture M-b', thereby cleaving the di(p-methoxyphenyl)phenylmethyl protecting group PG-(x-1) from the (x-1)th cycle oligonucleotide O-(x-1) to give the (x-1)th cycle oligonucleotide (O-(x-1)) having a free backbone hydroxyl group. ＃ obtaining (c') providing a building block B-x selected from the group consisting of nucleosides and oligonucleotides, wherein building block B-x comprises a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group PG-x and a phosphorus moiety covalently bonded to a backbone oxygen atom of building block B-x through a phosphorus atom; (d') (x-1)th cycle oligonucleotide (O-(x-1)) ＃ and the phosphorus atom of the phosphorus moiety of building block B-x, under conditions suitable for forming a covalent bond between the free backbone hydroxyl group of the (x-1)th cycle oligonucleotide (O-(x-1)) obtained in step (b'). ＃ with component B-x of step (c'), thereby obtaining oligonucleotide O-x of the xth cycle; (e') optionally incubating the x-th cycle oligonucleotide O-x obtained in step (d') with an oxidizing or sulfurizing agent, thereby converting any P(III) atom in said x-th cycle oligonucleotide O-x to a P(V) atom; 3. The method of claim 2, wherein in at least one repetition of step (b'), the deprotection mixture Mb' is liquid composition C as defined in claim 1.

4. the phosphorus moiety of building block B-1 and each building block B-x is independently selected from the group consisting of a phosphoramidite moiety and an H-phosphonate monoester moiety; - in each coupling cycle in which the phosphorus moiety of building block B-1 or building block B-x is a phosphoramidite moiety, step (e) or (e') is performed; The method according to any one of claims 2 or 3, wherein step (e) or (e') is carried out at least in the final coupling cycle.

5. The nucleoside or oligonucleotide of claim 1 and component C-0 of claim 2 that is covalently linked to a solid support is a compound of formula I: 【Chemistry 2】 [In Formula I: Each oxygen atom (O) depicted within each nucleoside subunit x-0 through x-m represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; each of the nucleoside subunits x-0 through x-m is the same or different; PG-0 is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer equal to or greater than 0; Y １ is independently selected from the group consisting of O and S for each repeat unit m; Z １ is O-R for each repeating unit m. ｚ－１ and S.-R. ｚ－１ are independently selected from the group consisting of: R ｚ－１ is a protecting group that is the same or different for each repeat unit m; CA is a capping moiety or covalent chemical bond; L is a linker moiety or a covalent chemical bond; SM is a solid support. The method according to any one of claims 1 to 4, wherein

6. Each of components B-1 and B-x is a compound of formula II-1: 【Chemistry 3】 [In formula II-1: Each oxygen atom (O) depicted within each nucleoside subunit y-0 through y-q represents the oxygen atom of a hydroxyl moiety of the respective nucleoside subunit; each nucleoside subunit y-0 to y-q is the same or different; PG is a protecting group PG-1 or PG-x, which is a di(p-methoxyphenyl)phenylmethyl protecting group; q is an integer equal to or greater than 0; Y ２ is independently selected from the group consisting of O and S for each repeat unit q; Z ２ is O-R for each repeating unit q. ｚ－２ , S.R. ｚ－２ are independently selected from the group consisting of: R ｚ－２ is a protecting group that is the same or different for each repeat unit q; Z ３ is selected from the group consisting of O and S; R ｚ－３ is a protecting group; R ａ and R ｂ Each of the is independently C １ ~C ６ - is an alkyl group, and R ａ and R ｂ are the same or different, and R ａ and R ｂ may also be bonded together with the nitrogen atom to which they are attached to form a 5- or 6-membered aliphatic cyclic amine moiety; The method according to any one of claims 2 to 5, wherein step (e) or step (e') is carried out in each coupling cycle.

7. the first coupling cycle further comprises a step (f) of reacting the free hydroxyl groups with a blocking agent, step (f) being carried out after step (d) or after step (e); and / or 7. The method according to any one of claims 2 to 6, wherein at least one of the (n-1) repetitions of the coupling cycle comprising steps (b') to (e') further comprises a step (f') of reacting a free hydroxyl group with a blocking agent, wherein step (f') is performed after step (d') or after step (e').

8. The method comprises incubating the first cycle oligonucleotide O-1 with a deprotection mixture Mg, thereby cleaving the protecting group PG-1 from the first cycle oligonucleotide O-1 to produce a first cycle oligonucleotide (O-1) having a free backbone hydroxyl group. ＃ and / or further comprising step (g) of obtaining - the method comprises the step of: - the first cycle of oligonucleotide O-1 or (O-1) ＃ from the solid support; The method of any one of claims 2 or 4-7, wherein steps (g) and (h), if both, are performed, can be performed in either order.

9. the method comprises incubating the nth cycle oligonucleotide O-n with a deprotection mixture M-g′, thereby cleaving the protecting group PG-n from the nth cycle oligonucleotide O-n to produce an nth cycle oligonucleotide (O-n) having a free backbone hydroxyl group. ＃ and / or a step (g') of obtaining - the method comprises the step of: - the nth cycle of oligonucleotides O-n or (O-n) ＃ from the solid support; The method of any one of claims 3 to 7, wherein steps (g') and (h'), if both, are performed, can be performed in either order.

10. 10. The method according to any one of claims 1 to 9, wherein at least steps (b) and (b') are carried out in a batch reactor, or at least steps (b) and (b') are carried out in a column reactor, and the flow rate of liquid composition C passing through the column reactor is less than 300 cm / hour.

11. - the backbone hydroxyl moiety protected by said protecting group PG-0 is part of a nucleoside moiety containing a purine-type nucleobase; 11. The method according to any one of claims 1 to 10, wherein in at least one repetition of the coupling cycle comprising steps (b') to (e'), in which the protecting group PG-(x-1) is part of a nucleoside moiety comprising a purine-type nucleobase, the deprotection mixture M-b' is liquid composition C.

12. The synthesis is carried out on a scale of at least 100 mmol of target oligonucleotide O Ｔ The method according to any one of claims 1 to 11, wherein the method is carried out by

13. 13. The method according to any one of claims 1 to 12, wherein liquid composition C comprises said at least one alcohol according to formula D in a molar concentration of 0.45 to 5.60 mol / L.

14. The method according to any one of claims 1 to 13, wherein the solvent contained in liquid composition C is a non-halogenated aprotic solvent.

15. - an oligonucleotide covalently linked to a solid support and comprising a hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, and a composition comprising a liquid composition C comprising a solvent, a protonic acid having a pKa of 4 or less, and at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety, the protonic acid contained in liquid composition C is selected from the group consisting of carboxylic acids, sulfonic acids, mineral acids, protonated aliphatic, aromatic or heteroaromatic amines, the protonated forms of which have a pKa in the range of 1 to 4, and mixtures thereof; each of said at least one alcohol having one or more hydroxyl groups directly covalently bonded to an optionally substituted phenyl moiety is independently an alcohol of formula D: 【Chemistry 4】 [In formula D: R Ｄ－１ , R Ｄ－２ , R Ｄ－３ , R Ｄ－４ , and R Ｄ－５ is H, OH, C １ ~C ６ - alkyl group, O(C １ ~C ６ -alkyl), C(O)(C １ ~C ６ -alkyl), C(O)O(C １ ~C ６ -alkyl), F, Cl, Br, I, and CN; Preferably, the composition and / or one or more components are as defined in any one or more of claims 1 to 14, In particular, a composition in which the concentration of said at least one alcohol of formula D is as defined in claim 13 and / or said solvent is as defined in claim 14.

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