Improved Oligonucleotide Synthesis

Abstract

Improved protocols for the synthesis of oligonucleotides are provided that reduce the level of depurination. A liquid composition for the cleavage of di(p-methoxyphenyl)phenylmethyl protecting groups from hydroxyl groups and its use in the chemical synthesis of oligonucleotides is disclosed.

Description

[Technical field]

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

[0002] Generally speaking, the iterative method of chemical oligonucleotide synthesis usually relies on the use of a first type of protecting group for those functional groups located in the base / base analogue and ribose / ribose analogue moieties that are not involved in chain elongation, and a second type of protecting group to control backbone elongation. The latter type is a temporary protecting group included in the building block to be added. Its function is to avoid double insertion of building blocks and / or multimerization of building block moieties. The first and second types of protecting groups are typically orthogonal to each other, which means that one type can be removed under conditions that do not affect the other type of protecting group.

[0003] The first type of protecting group is usually cleavable under alkaline conditions and is "permanent" in that they are only removed once the chain construction is completed; the second type of temporary protecting group is typically cleavable under acidic conditions and is removed once per synthesis cycle. Triarylmethyl protecting groups are usually used for temporary protection. Thus, a coupling cycle involves coupling a protected building block to the unprotected end of the oligonucleotide backbone, followed by deprotection of the oligonucleotide thus extended to prepare it for the subsequent coupling cycle. To facilitate separation from the reagents after each step, the growing oligonucleotide chain is usually attached to a supporting moiety, which is a solid support or a soluble modified tag.

[0004] For phosphoramidite oligonucleotide synthesis, the synthesis cycle usually begins with selective deprotection of the hydroxyl groups of the ribose moiety, e.g., the 5'-hydroxyl group. The growing oligonucleotide chain is then incubated with appropriately protected nucleoside phosphoramidites and an activating reagent. The DMT (dimethoxytrityl, also known as di(p-methoxyphenyl)phenylmethyl) group is often used as a temporary protecting group. The tricoordinate phosphite triester resulting from the coupling reaction is then oxidized or sulfurized, allowing the routine synthesis of phosphate triesters, phosphorothioates, phosphorodithioates, or mixed backbone structures. An optional capping step is used to block any unreacted hydroxyl groups, thereby avoiding the appearance of deletion sequences that differ from the target oligonucleotide by the absence of only a single nucleoside subunit in subsequent coupling rounds.

[0005] For phosphonate oligonucleotide synthesis, nucleoside H-phosphonate monoesters with acid-sensitive temporary protecting groups such as DMT are used. The H-phosphonate diester linkage between nucleosides is typically oxidized or sulfurized at the end of chain construction. Depending on the reaction conditions used, phosphodiester linkages, phosphorothioate linkages, phosphoroselenoate or phosphoroamidate analogs, and other derivatives are produced in this process.

[0006] The process of removing temporary triarylmethyl-type protecting groups has been found to be surprisingly difficult for the synthesis of high-quality oligonucleotides.Triarylmethyl-type protecting groups, especially DMT groups, are well known and commonly used in organic synthesis.Various cleavage protocols exist, and one example for the cleavage of tritylamine is described in US Pat. No. 5,399,633.

[0007] However, in the context of oligonucleotide synthesis, the DMT cleavage step poses the risk of acid-induced base cleavage, since both reactions are acid-catalyzed. This side reaction is particularly prominent for guanine and adenine-type bases and is called depurination. To avoid depurination, various strategies have been explored. Alternative temporary protecting groups, nucleobase protecting groups that reduce basicity, and coupling schemes using di- and trinucleotides have been tested, but these approaches have not proven their value for routine oligonucleotide synthesis. In addition, the cleavage reaction itself and the reaction conditions have been studied. Non-Patent Document 1 demonstrated that the cleavage of the DMT group from nucleotides in non-aqueous solutions can occur by a concerted general acid-catalyzed mechanism, in contrast to the deamination of 4,4'-dimethoxytritylamine, which appears to be involved in the initial protonation step.

[0008] Habus and Agarwal (Patent Document 2) teach that a detritylation agent containing 2% dichloroacetic acid (DCA) and 0.1% lower alcohol, e.g., ethanol or methanol, and / or 0.1-1% 1H-pyrrole suppresses depurination. Both Non-Patent Document 2 and Non-Patent Document 3 teach increasing the concentration of dichloroacetic acid (DCA) in the detritylation reagent from 3% to 10% and keeping the contact time between the detritylation agent and the oligonucleotide to a minimum to favor detritylation over depurination. This approach is still used for industrial-scale production of oligonucleotides, such as those highly useful as active pharmaceutical ingredients (APIs). However, limiting the contact time between the detritylation reagent and the oligonucleotide severely limits the scalability of the process, as it limits the maximum pumping capacity of the liquid handling system. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 8,299,206 [Patent Document 2] WO96 / 03417 issue [Non-patent literature]

[0010] [Non-Patent Document 1] Russell et al. (Org. Biomol. Chem. 2009, 7, 52-57) [Non-Patent Document 2] Septak (Nucleic Acids Research, 1996, Vol. 24, No. 15, pp. 3053-3058) [Non-Patent Document 3] Cheruvallath(Organic Process Research & Development 2003, 7, pp. 917-920) Summary of the Invention [Problem to be solved by the invention]

[0011] There is a long felt and unmet need for a method for depurination, i.e., deprotection of the (backbone-associated) hydroxyl groups of a growing oligonucleotide while suppressing undesired base cleavage, that is scalable and suitable for large-scale production of oligonucleotides. [Means for solving the problem]

[0012] The present invention provides a solution to this problem.

[0013] In one aspect, the present invention provides a use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting 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 a base and a salt of a strong acid, the cation of the salt having a pka in the range of 1 to 4, for the cleavage of a protecting group comprising an optionally substituted triarylmethyl residue from a hydroxyl group during the chemical synthesis of an oligonucleotide. The liquid composition C is particularly used to suppress acid-induced nucleobase cleavage, in particular depurination, while causing the cleavage of a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from a hydroxyl group during the chemical synthesis of an oligonucleotide.

[0014] As used herein, the indefinite articles "a" and "an" mean "one or more," unless otherwise indicated.

[0015] Thus, one aspect of the present invention relates to the use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of which has a pKa in the range of 1 to 4, for suppressing nucleobase cleavage, in particular depurination, while effecting cleavage of a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from a hydroxyl group during chemical synthesis of an oligonucleotide.

[0016] As used herein, an "aprotic solvent" is a solvent that is not a hydrogen bond donor. Thus, an aprotic solvent is a solvent without OH or NH bonds.

[0017] One aspect of the present invention relates to the use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4 and the strong acid has a pKa less than 1, for suppressing nucleobase cleavage, in particular depurination, while effecting cleavage of di(p-methoxyphenyl)phenylmethyl (DMT) protecting groups from hydroxyl groups during chemical synthesis of oligonucleotides.

[0018] As known to those of skill in the art, "nucleobase cleavage" typically occurs at purine nucleobases such as adenine and guanine, which is referred to as "depurination." Adenine is particularly prone to depurination.

[0019] The term "salt of a base and a strong acid" will be understood by those skilled in the art to refer to a group of molecules of the base and the strong acid, in which a part of the base molecules and a part of the acid molecules are associated with each other, for example, by electrostatic interaction. Typically, the strong acid donates a proton to the base. However, this should not be interpreted as meaning that all the molecules of the base and all the molecules of the strong acid in the liquid composition C are always associated with each other. In fact, as known to those skilled in the art, a solution of a salt contains ions in solvated form. In addition, the protonation of the base by the strong acid is followed by deprotonation of the protonated base (i.e., the cation of the salt), for example by the transfer of a proton to an oxygen atom from which a protective group, including an optionally substituted triarylmethyl residue (e.g., DMT group), is removed. The strong acid also protonates species other than the base in the first place, and participates in hydrogen bonding with such species, for example, with the nucleobase of an oligonucleotide. Thus, as used throughout the text, the phrase "a liquid composition comprising an aprotic solvent and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4" is synonymous with the phrase "a liquid composition comprising an aprotic solvent, a base whose protonated form has a pKa in the range of 1 to 4, and a strong acid." Both phrases are used interchangeably throughout the text.

[0020] Thus, one aspect of the present invention relates to the use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, a base whose protonated form has a pKa in the range of 1 to 4, and a strong acid having a pKa less than 1, to suppress nucleobase cleavage, in particular depurination, while effecting cleavage of the di(p-methoxyphenyl)phenylmethyl (DMT) protecting group from the hydroxyl group during chemical synthesis of an oligonucleotide.

[0021] Furthermore, the present invention also relates to the use of a liquid composition C comprising a non-halogenated and / or (hetero)aromatic aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol (also known as 1,1,1,3,3,3-hexafluoro-2-propanol), pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4 and the strong acid has a pKa less than 1, for suppressing nucleobase cleavage, in particular depurination, while effecting cleavage of a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from a hydroxyl group during chemical synthesis of an oligonucleotide.

[0022] Furthermore, the present invention also relates to the use of a liquid composition C comprising a non-halogenated and / or (hetero)aromatic aprotic solvent, trifluoroethanol and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4 and the strong acid having a pKa below 1, for suppressing nucleobase cleavage, in particular depurination, while effecting cleavage of a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from a hydroxyl group during chemical synthesis of an oligonucleotide.

[0023] Furthermore, the present invention relates to the use of a liquid composition C comprising a non-halogenated and / or (hetero)aromatic aprotic solvent, hexafluoroisopropanol and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4, for suppressing nucleobase cleavage while effecting cleavage of a protecting group, comprising an optionally substituted triarylmethyl residue, from a hydroxyl group during chemical synthesis of an oligonucleotide.

[0024] DMT (dimethoxytrityl) is a preferred triarylmethyl protecting group (or triarylmethyl residue), and preferred salts or strong acid and base combinations for DMT cleavage are detailed herein below. Unless otherwise indicated, the terms dimethoxytrityl group, dimethoxytrityl protecting group, dimethoxytrityl residue, DMT group, and DMT residue are used interchangeably to refer to the di(p-methoxyphenyl)phenylmethyl protecting group.

[0025] As used in this context, the term "inhibit" is understood in a broad sense as reducing the rate of (undesirable) nucleobase cleavage (e.g., depurination), which is completely or partially prevented upon cleavage of a triarylmethyl-type protecting group, e.g., a DMT protecting group.

[0026] The degree of nucleobase cleavage / depurination is used to compare different detritylation protocols with respect to undesired nucleobase cleavage. Briefly, in the chromatogram obtained from the HPLC-MS analysis of the synthesized oligonucleotide (the oligonucleotide cleaved from the support when the support-assisted synthesis is used), the peak areas of the nucleobase cleavage / depurination by-products are summed to obtain the total peak area of ​​all the identified nucleobase cleavage / depurination by-products. The degree of nucleobase cleavage / depurination in percentage (%) is then determined by dividing the total peak area of ​​the nucleobase cleavage / depurination by-products by the area of ​​the product of each oligonucleotide synthesis (i.e., the target oligonucleotide), followed by multiplying by 100% to arrive at a value in percentage (%).

[0027] In some embodiments, inhibiting nucleobase cleavage refers to reducing the extent of nucleobase cleavage by at least 5%, or at least 10%, or at least 25%, or at least 50%, or at least 75%, or at least 90%, in comparison to an equivalent detritylation step differing only in that 10% DCA (v / v) in toluene is used instead of liquid composition C of the present invention. Similarly, in some embodiments, inhibiting depurination refers to reducing the extent of depurination by at least 5%, or at least 10%, or at least 25%, or at least 50%, or at least 75%, or at least 90%, in comparison to an equivalent detritylation step differing only in that 10% DCA (v / v) in toluene is used instead of liquid composition C of the present invention.

[0028] In this context, the terms "equivalent cleavage step" and "equivalent detritylation step" refer to a step of removing a protecting group, including an optionally substituted triarylmethyl residue, preferably a DMT protecting group, under similar conditions, e.g., with the same substrate, scale, temperature, time and volume of detritylation cocktail, differing only with respect to the composition of the detritylation cocktail, e.g., 10% DCA (v / v) in toluene versus liquid composition C of the present invention.

[0029] The meaning of the term "reducing" in this context of the degree of nucleobase cleavage or the degree of depurination will be understood by the skilled artisan and is exemplified as follows: if the degree of depurination of a first synthesis A, which uses liquid composition C of the present invention for detritylation, is divided by the degree of depurination of a second synthesis B, which uses 10% DCA (v / v) in toluene for detritylation, provides a quotient of 0.95, then the degree of depurination is said to be reduced by 5%.

[0030] One aspect of the present invention relates to the use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, a base, and a salt of a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4 and the strong acid has a pKa less than 1, for suppressing cleavage of adenine or guanine, particularly adenine, from hydroxyl groups while effecting cleavage of the di(p-methoxyphenyl)phenylmethyl (DMT) protecting group during chemical synthesis of oligonucleotides.

[0031] In another aspect, the present invention provides a liquid composition C for use in cleaving a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from a hydroxyl group, the composition comprising a non-halogenated and / or (hetero)aromatic aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4.

[0032] One aspect of the invention relates to a liquid composition C for use in cleaving a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group from a hydroxyl group, the composition comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, a base and a salt of a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4, and the strong acid has a pKa less than 1, and the aprotic solvent is non-halogenated and selected from the group consisting of (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers.

[0033] Thus, one aspect of the invention relates to a liquid composition C for use in cleaving a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group from a hydroxyl group, the composition comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, a base whose protonated form has a pKa in the range of 1 to 4, and a strong acid having a pKa less than 1, wherein the aprotic solvent is non-halogenated and selected from the group consisting of (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers.

[0034] In some embodiments of liquid composition C of the present invention, the liquid composition is for use in cleaving a di(p-(methoxyphenyl)phenylmethyl (DMT) protecting group from a hydroxyl group, wherein the nucleoside moiety bearing the DMT group comprises a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0035] In another embodiment, the present invention provides a method for the synthesis of oligonucleotides, comprising steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group; b) cleaving the protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating free backbone hydroxyl moieties; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) and c); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4. A method is provided.

[0036] One aspect of the present invention is a method for the synthesis of oligonucleotides, comprising the following steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; b) cleaving the di(p-methoxyphenyl)phenylmethyl (DMT) protecting group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating free backbone hydroxyl moieties; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group, thereby generating a covalent linkage between the oxygen atom of the free hydroxyl group and the phosphorus atom of the building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4, and the strong acid has a pKa of less than 1. It concerns the method.

[0037] Therefore, one aspect of the present invention is a method for the synthesis of oligonucleotides, comprising the following steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; b) cleaving the di(p-methoxyphenyl)phenylmethyl (DMT) protecting group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating free backbone hydroxyl moieties; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group, thereby generating a covalent linkage between the oxygen atom of the free hydroxyl group and the phosphorus atom of the building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, a base whose protonated form has a pKa in the range of 1 to 4, and a strong acid having a pKa of less than 1; It concerns the method.

[0038] In some embodiments, the present invention provides a method for the synthesis of an oligonucleotide, comprising steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group; b) cleaving a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus(III) moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group, thereby generating a covalent linkage between the oxygen atom of the free hydroxyl group and the phosphorus(III) atom of the building block; d) oxidizing or sulfurizing the phosphorus(III) atom to a phosphorus(V) atom, thereby creating the desired type of internucleoside linkage; e) optionally repeating the sequence of steps b) through d) or repeating the sequence of steps b) and c) before performing step d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4. A method is provided.

[0039] Protecting groups that contain optionally substituted triarylmethyl residues are also designated triarylmethyl-based residues, triarylmethyl-type groups, or trityl-type residues, triarylmethyl groups, or trityl groups, and the like.

[0040] Some embodiments of the present invention provide a method for the synthesis of oligonucleotides, comprising the steps of: a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group; b) cleaving the di(p-methoxyphenyl)phenylmethyl (DMT) protecting group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating free backbone hydroxyl moieties; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus(III) moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a di(p-methoxyphenyl)phenylmethyl (DMT) protecting group, thereby generating a covalent linkage between the oxygen atom of the free hydroxyl group and the phosphorus(III) atom of the building block; d) oxidizing or sulfurizing the phosphorus(III) atom to a phosphorus(V) atom, thereby creating the desired type of internucleoside linkage; e) optionally repeating the sequence of steps b) through d) or repeating the sequence of steps b) and c) before performing step d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4, and the strong acid has a pKa of less than 1. It concerns the method.

[0041] In some embodiments of the method of the invention comprising steps a) to f) above, in at least one iteration of step a), the backbone hydroxyl group protected by a DMT protecting group is part of a nucleoside moiety that comprises a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0042] In some embodiments of the method of the invention comprising steps a) through f) above, step e) is performed and includes repeating the order of steps b) through d). In some embodiments of the method of the invention comprising steps a) through f) above, step e) is performed and includes repeating the order of steps b) through d) 4-99 times, 9-99 times, 14-99 times, 19-99 times, 19-49 times, 19-29 times, 4-22 times, or 4-19 times.

[0043] In some embodiments of the methods of the invention comprising steps a) through f) above, step e) is performed and comprises repeating the sequence of steps b) through d) 4 to 99 times, with the proviso that in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 iterations of step b), the backbone hydroxyl group from which the DMT protecting group is cleaved is part of a nucleoside moiety that comprises a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine. In some embodiments of the methods of the invention comprising steps a) through f) above, step e) is performed and comprises repeating the sequence of steps b) through d) 9 to 99 times, with the proviso that in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 iterations of step b), the backbone hydroxyl group from which the DMT protecting group is cleaved is part of a nucleoside moiety that comprises a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine. In some embodiments of the methods of the invention comprising steps a) through f) above, step e) is performed and comprises repeating the sequence of steps b) through d) 19 to 99 times, with the proviso that in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 iterations of step b), the backbone hydroxyl group from which the DMT protecting group is cleaved is part of a nucleoside moiety that comprises a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0044] In some embodiments of the method comprising steps a) through f) above, step a) comprises reacting a compound according to formula I: [ka] [In formula: R 1is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y, independently for each repeat unit, n, is selected from the group consisting of S and O; Z, independently for each repeat unit n, is selected from the group consisting of O and S; R 2 are the same or different protecting groups for each repeat unit n; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting part. preparing a - step c) comprises reacting the free hydroxyl group resulting from step b) with a nucleoside or oligonucleotide phosphoramidite building block, the building block comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and a phosphorus(III) atom of said building block; - step e) optionally comprises repeating the sequence of steps b) to d).

[0045] In some embodiments, the compound according to formula I is a compound according to formula Ia: [ka] [In the formula, R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y, independently for each repeat unit, n, is selected from the group consisting of S and O; Z, independently for each repeat unit n, is selected from the group consisting of O and S; R2 are the same or different protecting groups for each repeat unit n; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting part; B N are the same or different nucleobases in each occurrence; R I is independently selected at each occurrence 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 II is, independently in each occurrence, H or R II and R I represents the chemical structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++ (where + is the 4' carbon atom (i.e., R II is the point of attachment to the 2' carbon (i.e., the carbon atom to which R I (a) forms a methylene or ethylene bridge with the carbon atom to which it is attached. It is.

[0046] In some embodiments, Z in formula I or Ia is O for each repeat unit n.

[0047] In some embodiments, CA in formula I and / or Ia is a single bond. In some embodiments, L in formula I and / or Ia is a single bond. In some embodiments, CA and L in formula I and / or Ia are single bonds. It will be understood that when both CA and L in formula I and / or Ia are single bonds, they together represent the same single bond linking the supporting moiety to the nucleoside x0.

[0048] In some embodiments, the integer n in Formula I and / or Ia is in the range of 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 35, 0 to 30, 0 to 20, 0 to 10, 4 to 30, 9 to 30, 9 to 25, or 9 to 20. When the integer n in Formula I and / or Ia is 0, the protecting group R 1 is still included in the compounds described in formula I and / or Ia, and will be understood to be attached to the oxygen atom of nucleoside x0 that would be involved in a covalent bond to the phosphorus atom of the first repeat unit n if n was not 0.

[0049] In some embodiments, where step a) comprises providing a compound according to formula I and / or Ia, -OR 1 The terminal nucleoside xn carrying the group is a nucleoside containing a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0050] For example, in one embodiment, the present invention provides a method for synthesizing an oligonucleotide, comprising the steps of: * ) to f * ): a * ) a compound according to formula I [ka] [In formula: R 1 is a protecting group comprising an optionally substituted triarylmethyl moiety; n is an integer equal to or greater than 0; Y, independently for each repeat unit, n, is selected from the group consisting of S and O; Z, independently for each repeat unit n, is selected from the group consisting of O and S; R 2 are the same or different protecting groups for each repeat unit n; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond (i.e., CA is absent); L is a linker moiety or a single bond (i.e., L is absent); SM is the supporting part. preparing a b * ) cleaving the protecting group, including the optionally substituted triarylmethyl residue R1, from the compound according to formula I by incubating said compound with a liquid composition C; c * ) reacting the free hydroxyl group resulting from step b) with a nucleoside or oligonucleotide phosphoramidite building block comprising a backbone hydroxyl moiety protected by a protecting group comprising an optionally substituted triarylmethyl residue, thereby producing a covalent linkage between the oxygen atom of said free hydroxyl group and a phosphorus(III) atom of said building block; d * ) oxidizing or sulfurizing said phosphorus(III) moieties to phosphorus(V) moieties; e * ) optionally repeating any of steps b) to d) in any order; and f * ) cleaving the oligonucleotide from the linker portion. Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4. It concerns the method.

[0051] It should be noted that the order of steps presented herein may be varied and that steps marked as optional may be omitted.

[0052] In some embodiments of the method comprising steps a) through f) above, step a) comprises reacting a compound according to formula III [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; Y is O; Z is H; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting part. preparing a - step c) comprises reacting the free hydroxyl group resulting from step b) with an H-phosphonate building block, the building block comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent link between the oxygen atom of said free hydroxyl group and the phosphorus(III) atom of said building block; - step e) optionally comprises constructing the desired oligonucleotide sequence by repeating the order of steps b) and c) before carrying out step d).

[0053] In some embodiments, the compound according to formula III may be a compound according to formula III-a: [ka] [In the formula, R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y is O; Z is H; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting part; B N are the same or different nucleobases in each occurrence; R I is independently selected at each occurrence 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 II is, independently in each occurrence, H or R II and R I represents the chemical structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++ (where + is the 4' carbon atom (i.e., R II is the point of attachment to the 2' carbon (i.e., the carbon atom to which R I (a) forms a methylene or ethylene bridge with the carbon atom to which it is attached. It is.

[0054] In some embodiments, CA in formula III and / or III-a is a single bond. In some embodiments, L in formula III and / or III-a is a single bond. In some embodiments, CA and L in formula III and / or III-a are single bonds. It will be understood that when both CA and L in formula III and / or III-a are single bonds, they together represent the same single bond that connects the supporting moiety to the nucleoside x0.

[0055] In some embodiments, the integer n in Formula III and / or IIIa is in the range of 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 35, 0 to 30, 0 to 20, 0 to 10, 4 to 30, 9 to 30, 9 to 25, or 9 to 20. When the integer n in Formula III and / or IIIa is 0, the protecting group R 1It will be understood that is still included in the compounds described in formula III and / or IIIa and is attached to the oxygen atom of nucleoside x0 that would be involved in a covalent bond to the phosphorus atom of the first repeat unit n if n was not 0.

[0056] In some embodiments, where step a) comprises providing a compound according to formula III and / or IIIa, -OR 1 The terminal nucleoside xn carrying the group is a nucleoside containing a purine-type nucleobase, preferably a nucleobase selected from the group consisting of adenine and guanine, in particular adenine.

[0057] Each nucleobase B in any of formulas Ia and III-a N It will be understood that each occurrence of is the same or different and is optionally protected, i.e., has one or more protecting groups. Those skilled in the art are familiar with nucleobase protecting groups and know how to select them for a particular synthesis. In particular, any exocyclic amino group, such as that present in adenine, guanine, and cytosine, is protected. Table T-1 shows a non-limiting overview of commonly used protecting groups.

[0058] In some embodiments, in Formula Ia and / or Formula III-a: B N is independently at each occurrence selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R I is independently at each occurrence selected from the group consisting of H, F, -O-CH3 (i.e., methoxy), -O-CH2-CH2-O-CH3 (i.e., 2-methoxyethyl-1-oxy), -O-Si(CH3)3 (i.e., trimethylsilyloxy), -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy), and -O-CH2-O-Si(C(CH3)3)3 (i.e., ((triisopropylsilyl)oxy)-methyloxy); R IIis H in each occurrence.

[0059] In some embodiments, in Formula Ia and / or III-a: B N is independently at each occurrence selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R I is independently at each occurrence selected from the group consisting of H, F, -O-CH3 (i.e., methoxy); R II is H in each occurrence.

[0060] In some embodiments, in Formula Ia and / or III-a: B N is independently selected at each occurrence from the group consisting of adenine whose exocyclic amino group is benzoyl protected, guanine whose exocyclic amino group is isobutyryl protected, cytosine, thymine, and uracil whose exocyclic amino group is benzoyl or acetyl protected; R I is independently at each occurrence selected from the group consisting of H, F, -O-CH3 (i.e., methoxy); R II is H in each occurrence.

[0061] In another embodiment, the present invention provides a method for synthesizing an oligonucleotide, comprising the steps of: * ) to f * ): a * ) a compound according to formula III [ka] [In formula: R 1 is a protecting group comprising an optionally substituted triarylmethyl moiety; n is an integer equal to or greater than 0; Y is O and Z is H; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond (i.e., CA is absent); L is a linker moiety or a single bond (i.e., L is absent); SM is the supporting part. preparing a b * ) cleaving the protecting group comprising the optionally substituted triarylmethyl residue from the compound according to formula III by incubating said compound with a liquid composition C; c * )Process b * reacting a free hydroxyl group resulting from the reaction of 1,2-dihydro-1,3-triphenylphosphine with an H-phosphonate building block, the building block comprising a backbone hydroxyl moiety protected by a protecting group comprising an optionally substituted triarylmethyl residue, thereby producing a covalent linkage between the oxygen atom of the free hydroxyl group and the phosphorus(III) atom of the building block; d * ) oxidizing or sulfurizing said phosphorus(III) moieties to phosphorus(V) moieties; e * ) optionally, prior to step d'), repeating steps b) and c) for subsequent coupling of additional building blocks; and f * ) cleaving the oligonucleotide from the linker portion. Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4. A method is provided.

[0062] As used herein, the expression oligonucleotide is used in the most general manner to refer to any oligomer comprising at least two nucleosides linked by a phosphodiester bond or by an analog structure as shown in formula A below, and any isomeric forms and stereoisomers thereof. In a preferred embodiment, an oligonucleotide in the sense of the present invention has the structure according to formula A or a salt thereof: [ka] [wherein, in formula A: * and ** each independently of the other represents a point of attachment to each of the nucleoside moieties (i.e., the preceding and following nucleoside moieties, respectively) or H (i.e., in the case of the termini of the oligonucleotide); X1 is selected from S and O; X2 is any possible stable atom or functional group, preferably -OH, -OR, -NHR, C3-C 20 -(hetero)aromatic moiety, -CH3, -F, -Cl, -SH, -SeH, and -SR; where R is any residue, preferably a residue having no more than 30 carbon atoms, preferably a branched, unbranched or cyclic C1-C 20 (Hetero)alkyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkenyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkynyl residues, C6-C 20 Aryl residues, C3-C 19 heteroaryl residues, said residues being deuterium, halogen, branched, unbranched or cyclic C1-C 20 (Hetero)alkyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkenyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkynyl residues, C1-C 20 (Hetero)cycloalkyl residues, C6-C20 Aryl residues and / or C3-C 19 optionally substituted with a heteroaryl residue] The nucleoside moiety comprises two or more nucleoside moieties conjugated to each other via a linker comprising or consisting of:

[0063] As used throughout the present invention, the terms "alkyl" and "heteroalkyl" are understood in the broadest sense. They are, for example, methyl or ethyl or isopropyl. Heteroalkyl contains at least one heteroatom, such as, for example, N, O, S or P. They also include alkoxy (e.g., methoxy (-O-CH3, -OMe)) and similar residues such as -NHCH3 or -N(CH3)2, or salts thereof. Preferably, alkyl is a deuterium, halogen, branched, unbranched or cyclic C1-C 20 (Hetero)alkyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkenyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkynyl residues, C1-C 20 (Hetero)cycloalkyl residues, C6-C 20 Aryl residues and / or C3-C 19 Branched, unbranched or cyclic C1-C, optionally substituted with heteroaryl residues 20 It is (hetero)alkyl.

[0064] As used throughout the present invention, the terms "alkenyl" and "heteroalkenyl" are understood in the broadest sense and have one, two or more double bonds. Heteroalkenyl contains at least one heteroatom, such as, for example, N, O, S or P. Preferably, alkenyl is a deuterium, halogen, branched, unbranched or cyclic C1-C 20 (Hetero)alkyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkenyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkynyl residues, C1-C 20(Hetero)cycloalkyl residues, C6-C 20 Aryl residues and / or C3-C 19 Branched, unbranched or cyclic C1-C, optionally substituted with heteroaryl residues 20 (hetero)alkenyl.

[0065] As used throughout the present invention, the terms "alkynyl" and "heteroalkynyl" are understood in the broadest sense and have one, two or more triple bonds. Heteroalkynyl contains at least one heteroatom, such as, for example, N, O, S or P. Preferably, alkynyl is a deuterium, halogen, branched, unbranched or cyclic C1-C 20 (Hetero)alkyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkenyl residues, branched, unbranched or cyclic C2-C 20 (Hetero)alkynyl residues, C1-C 20 (Hetero)cycloalkyl residues, C6-C 20 Aryl residues and / or C3-C 19 Branched, unbranched or cyclic C1-C, optionally substituted with heteroaryl residues 20 (hetero)alkynyl.

[0066] As used throughout the present invention, the terms "aryl" and "heteroaryl" are understood in the broadest sense. Heteroaryl preferably contains at least one heteroatom, such as, for example, N, O, S and / or P, in the ring structure.

[0067] The type of internucleoside linkage is not necessarily the same at all positions in an oligonucleotide, for example the middle of the oligonucleotide chain may contain phosphodiester linkages and phosphorothioate linkages may be present at the ends of the chain.

[0068] It is clear from formula A that each nucleoside contains two hydroxyl moieties (i.e., hydroxyl groups or moieties derived from hydroxyl groups, typically participating in an ester bond linking the residue of formula A with the nucleotide moiety), which participate in an internucleoside linkage, i.e., a phosphoester type bond with a phosphorus (V) moiety located between adjacent nucleosides. One of the hydroxyl moieties participates in the internucleoside linkage to the preceding nucleoside, and the other hydroxyl moiety participates in the internucleoside linkage to the subsequent nucleoside. These hydroxyl moieties are therefore referred to throughout the text as "main chain hydroxyl moieties" or "main chain hydroxyl groups". Obviously, the first and last nucleoside units participate in only one internucleoside linkage.

[0069] Thus, they either contain free backbone hydroxyl groups (e.g., the so-called 5' and 3' OH groups in the case of a phosphoribose backbone as found in DNA and RNA), or the hydroxyl moiety is linked to another moiety, e.g., a capping structure.

[0070] As used herein, the terms "internucleoside linkage" and "internucleoside linkage" are used interchangeably.

[0071] Oligonucleotides are conjugated to additional moieties for various purposes.Conjugation is through terminal backbone hydroxyl groups or through other functional groups of nucleoside moieties, which are located at the end of oligonucleotide chain or in the chain.For example, the 5'OH group of the antisense strand of siRNA is modified by vinylphosphonate to inhibit cellular degradation and improve efficacy.Another example is conjugation to N-acetylgalactosamine (GalNAc), which is of interest for targeting oligonucleotide delivery to hepatocytes. Often, a GalNAc structure branched to accommodate three or four GalNAc moieties is conjugated, for example, to the 5' OH group of an oligonucleotide (see, e.g., WO2016055601), to the 3' OH group (see, e.g., WO2009073809), 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).

[0072] Nucleoside unit is a glycosylamine that contains a sugar moiety, usually a ribose moiety, and a nucleic acid base.As used herein, the term nucleoside or nucleoside unit encompasses naturally occurring nucleosides and non-natural compounds, in which the ribose moiety and / or the nucleic acid base are modified or replaced by functional equivalents or abasic sites.Examples of natural nucleosides include, but are not limited to, adenosine, guanosine, cytidine, ribothymidine, uridine, desoxyuridine, desoxythymidine, inosine, desoxyadenosine, desoxyguanosine, desoxycytidine, and methylated derivatives thereof.Further examples are queosine, archaeosine, wybutosine, lysidine, and N. 6-threonylcarbamoyl adenosine. Unnatural nucleosides include modified ribose moieties, which are, for example, "locked" (i.e., contain a methylene bridge connecting the 2' oxygen and the 4' carbon), exhibit ethylene bridges, or have -F, -OMe (-O-CH3), and / or 2-methoxyethyl-1-oxy (-O-CH2-CH2-O-CH3, also known as MOE, -O-methoxyethyl) substituents. In addition, the ribose moiety is replaced by a functional equivalent, for example, another pentose, such as arabinose, a hexose, such as mannose, or a glycerol moiety. As used herein, the term nucleobase encompasses unnatural nucleobases and naturally occurring nucleobases, such as adenine, guanine, uracil, cytosine, and thymine.

[0073] Unnatural nucleobases are functional equivalents of natural nucleobases in that they can specifically interact with complementary nucleobases.The interaction between complementary nucleobases is mediated by hydrogen bonds, known as Watson-Crick base pairing.Such unnatural nucleobases are derivatives of purine or pyrimidine, which can specifically interact with another nucleobase.In this specification, the nucleobase derived from purine is also called purine nucleobase, and the nucleobase derived from pyrimidine is also called pyrimidine nucleobase.Adenine and guanine are naturally occurring purine nucleobases.Cytosine, thymine and uracil are naturally occurring pyrimidine nucleobases.

[0074] As used herein, the terms "oligonucleotide" and "polynucleotide" are understood interchangeably.From the above description, it can be seen that non-limiting examples of oligonucleotide are deoxynucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), constrained ethyl nucleic acid analog (cEt), bridged nucleic acid (BNA), tricyclo DNA, unlocked nucleic acid (UNA), small interfering RNA (siRNA), micro RNA, antisense oligonucleotide (ASO), gapmer, glycerin nucleic acid, oligonucleotide phosphorothioate, phosphorodithioate, diastereomerically pure phosphorothioate, and its derivatives and analogs.The expression "oligonucleotide" further includes the conjugate of oligonucleotide moiety with other moieties such as peptide, carbohydrate, etc. Particularly interesting examples of oligonucleotides are nucleosides that contain modified ribose moieties, such as nucleosides with 2'-substituents selected from -F, -OMe (-O-CH3), or 2-methoxyethyl-1-oxy (-O-CH2-CH2-O-CH3, also known as MOE, -O-methoxyethyl substituent), and phosphorothioate build-up from nucleobases that can form Watson-Crick base pairs with natural target sequences. Those skilled in the art are well aware of the fact that the above moieties have a number of stereoisomers, all of which are encompassed by the above definition. "Oligonucleotide", as used herein, refers to a collection of oligonucleotide molecules that essentially have the same sequence, which is a mixture of a number of distinct stereoisomers, or is enriched in or consists essentially of one particular stereoisomeric form.

[0075] It will be understood by those skilled in the art that oligonucleotides, as used herein, may optionally have any counterion known in the art, such as chloride, acetate, carbonate, hydrocarbon, sodium, potassium, magnesium, trifluoroacetic acid (i.e., trifluoroacetate TFA), bromide, perchlorate, ammonium, and / or the cation or anion of a residue of a protecting group. In addition, the oligonucleotide may optionally be covalently or non-covalently associated with one or more trace scavengers, such as, for example, triisopropylsilane (TIS), dithiothreitol (DTT), anisole, thioanisole, or 1,2-ethanedithiol.

[0076] As used herein, the expression "nucleoside building block" refers to a monomer or oligomer building block that allows the construction of an oligonucleotide chain. Those skilled in the art will readily understand that essentially any coupling chemistry compatible with the use of a temporary protecting group, preferably a DMT protecting group, including an optionally substituted triarylmethyl residue, can be used in the method of the present invention, and the chemical nature of the phosphorus moiety will vary accordingly. In some embodiments, the building block comprises an H-phosphonate moiety. In other embodiments, the building block is a phosphorus V reagent, for example, as taught in WO2019200273. In yet other embodiments, the building block is a phosphoramidite building block of the general structure shown in formula II: [ka] [In formula: R 1 is a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group; m is an integer equal to or greater than 0; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z is independently selected for each position (i.e., for each terminal position and for each repeat unit m) from the group consisting of O and S; R 2 are the same or different protecting groups for each position (i.e., the terminal positions and for each repeat unit m); R 3 and R 4 are the same or different protecting groups; Each of the nucleosides xm0 to xm may be the same or different. It is.

[0077] In some embodiments of the method of the invention, the method comprises the above steps a) to f), wherein the nucleoside or oligonucleotide building block (in step c)) is a compound according to formula II: [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer equal to or greater than 0; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z is independently selected for each position (i.e., for each terminal position and for each repeat unit m) from the group consisting of O and S; R 2 are the same or different protecting groups for each position; R 3 and R 4 are the same or different protecting groups; Each of the nucleosides xm0 to xm may be the same or different. It is.

[0078] Specific non-limiting examples of mononucleoside phosphoramidite building blocks are shown in Figures 1 and 2. It should be noted that nucleosides xm0-xm and x0-xn in Formulas I, II and III differ from each other with respect to the identity and substitution pattern of the ribose or ribose analog moiety (also known as ribose surrogate), with respect to the nucleobase, and with respect to the presence and identity of any protecting groups.

[0079] In some embodiments, the compound according to formula II may be a compound according to formula II-a: [ka] [In the formula, R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer equal to or greater than 0; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z is independently, for each occurrence, selected from the group consisting of O and S; R 2 are the same or different protecting groups for each position; R 3 and R 4 are the same or different protecting groups; B N are the same or different nucleobases in each occurrence; R III is independently selected at each occurrence 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 IV is, independently in each occurrence, H or R III and R IV represents the chemical structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++ (where + is the 4' carbon atom (i.e., RIV is the point of attachment to the 2' carbon (i.e., the carbon atom to which R III (a) forms a methylene or ethylene bridge with the carbon atom to which it is attached. It is.

[0080] In some embodiments, Z in formula II and / or II-a is O at each position (ie, at the terminal positions and for each repeat unit m).

[0081] In some embodiments, different types of building blocks with different coupling chemistries are used in different coupling cycles, for example, phosphorothioate-linked nucleosides are introduced using P(V) chemistry and phosphodiester-linked nucleosides are introduced using P(III) chemistry.

[0082] In some embodiments, in Formula II and / or II-a, m is an integer from 0 to 2, i.e., the building block is a mononucleoside, dinucleotide, or trinucleotide phosphoramidite building block. When the integer m in Formula II and / or II-a is 0, the protecting group R 1 It will be understood that is still included in the compounds described in formula II and / or II-a and is attached to the oxygen atom of the nucleoside xm0 that would be involved in a covalent bond to the phosphorus atom of the first repeat unit m if m was not 0.

[0083] Each nucleobase B in formula III-a BN It will be understood that each occurrence of is the same or different and is optionally protected, i.e., has one or more protecting groups. Those skilled in the art are familiar with nucleobase protecting groups and know how to select them for a particular synthesis. In particular, any exocyclic amino group, such as that present in adenine, guanine, and cytosine, is protected. Table T-1 shows a non-limiting overview of commonly used protecting groups.

[0084] In some embodiments, in Formula II-a: B N is independently at each occurrence selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R III is independently at each occurrence selected from the group consisting of H, F, -O-CH3 (i.e., methoxy), -O-CH2-CH2-O-CH3 (i.e., 2-methoxyethyl-1-oxy), and -O-Si(CH3)3 (i.e., trimethylsilyloxy), -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy), and -O-CH2-O-Si(C(CH3)3)3 (i.e., ((triisopropylsilyl)oxy)-methyloxy); R IV is H in each occurrence.

[0085] In some embodiments, in Formula II-a: B N is independently at each occurrence selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R III is independently at each occurrence selected from the group consisting of H, F, -O-CH3 (i.e., methoxy); R IV is H in each occurrence.

[0086] In some embodiments, in Formula II-a: B N is independently selected at each occurrence from the group consisting of adenine whose exocyclic amino group is benzoyl protected, guanine whose exocyclic amino group is isobutyryl protected, cytosine, thymine, and uracil whose exocyclic amino group is benzoyl or acetyl protected; R III is independently at each occurrence selected from the group consisting of H, F, -O-CH3 (i.e., methoxy); RIV is H in each occurrence.

[0087] The term "protecting group" as used herein is understood in the broadest sense as a group introduced into a molecule by chemical modification of a functional group in order to block the group from reacting in a subsequent process step, e.g., to prevent side reactions with the backbone or nucleobase of an oligonucleotide. Suitable protecting groups R of formulae I, Ia, II, and II-a are 2 A typical example for is the 2-cyanoethyl group; R 3 and R 4 A typical example for both is the isopropyl group. Therefore, in some preferred embodiments, R 2 is a 2-cyanoethyl group at each position (i.e., for each repeat unit n). In some preferred embodiments, R 2 is a 2-cyanoethyl group at each position (i.e., at each occurrence). In some preferred embodiments, R 3 and R 4 are each independently a C1 to C6 alkyl group, and more preferably, R 3 and R 4 and R are both isopropyl groups. Alternatively, in the compounds according to formula II and / or II-a, R 2 and R 3 forms an ethylene bridge and the phosphoramidite takes the form of a chiral group of formula B, where ** represents the point of attachment to the backbone hydroxyl group).

[0088] [ka]

[0089] The building blocks used in the method of the present invention show additional protective groups, for example to block the exocyclic amino group of nucleobase from side reactions.Suitable nucleoside derivatives are commercially available.Table T-1 shows a non-limiting overview of the protective groups that are commonly used for nucleoside building blocks.

[0090] [Table 1]

[0091] Of particular interest for the present invention are the protecting groups each containing an optionally substituted triarylmethyl residue, which are used as temporary hydroxyl protecting groups to avoid multiple insertions of the same nucleoside or oligonucleotide building block.Those skilled in the art are well aware of the fact that the properties of such protecting groups each containing an optionally substituted triarylmethyl residue vary according to their substitution pattern.Substituents that stabilize the carbocation leaving group facilitate cleavage, so that the corresponding protecting group is cleaved under less acidic conditions.The present invention is preferably used with protecting groups each containing an optionally substituted triarylmethyl residue, which contain at least one substituent, which, for example, in at least one substituent, exhibits the effect of stabilizing the carbocation, the inductive effect of stabilizing the carbocation and / or the mesomeric effect of stabilizing the carbocation.For example, the triarylmethyl protecting group comprises at least one substituent selected from the group consisting of a methoxy group and a halogen atom.

[0092] Preferred protecting groups each comprising an optionally substituted triarylmethyl residue include the p-methoxyphenyldiphenylmethyl (MMT) group, the di(p-methoxyphenyl)phenylmethyl (DMT) group, the tri(p-methoxyphenyl)methyl ether (TMT) group, the 4,4'-dimethoxy-3''-[N-(imidazolylmethyl)]trityl (IDT) group, the 4,4'-dimethoxy-3''-[N-(imidazolylethyl)carbamoyl]trityl ether (IET) group, the bis(4-methoxyphenyl)-1'-pyrenylmethyl (Bmpm) group, and the 4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4',4''-dimethoxytrityl (Tbf-DMT) group. The DMT group is particularly preferred. The protecting groups each comprising an optionally substituted triarylmethyl residue are the same or different for the different building blocks used in the method according to the invention.

[0093] Herein, the process of cleaving protecting groups containing optionally substituted triarylmethyl moieties is referred to as "detritylation."

[0094] Step a) of the method described herein includes preparing a nucleoside or oligonucleotide bound to a support moiety. This is understood in the broadest possible sense. For example, nucleosides loaded onto solid supports for many standard nucleosides are easily obtained from commercial suppliers such as Merck and Cytiva, among others. Alternatively, a suitably protected nucleoside is loaded onto a given support moiety. Those skilled in the art will be aware of and routinely select a large number of possible immobilization strategies for this purpose. Generally speaking, unless otherwise indicated in the context of a particular embodiment, the first nucleoside (referred to as nucleoside x0 in formulas I and III) is immobilized to the support moiety in any conceivable manner. For example, the first nucleoside is covalently linked via one of the backbone hydroxyl groups or via another functional group located on the ribose / ribose analog moiety or nucleobase. Unless otherwise indicated in the context of a particular embodiment, it should be noted that when step a) includes preparing an oligonucleotide bound to a support moiety, this is not limited to embodiments in which the oligonucleotide is bound to a support moiety via one of its terminal nucleosides. However, immobilization via the 3' end is particularly common, as are many methods of oligonucleotide synthesis that involve the addition of the next building block to the 5' end of a phosphoribose backbone.

[0095] The link between the support moiety and the nucleoside is established using a linker moiety. As used herein, the expression "linker moiety" refers to a moiety that exhibits at least two functional groups, where one of said groups can be bound to a functional group on the support moiety and the other can be bound to a functional group on the nucleoside. As an example, a succinic acid linker or a hydroquinone diacetate linker mediates the linking of a hydroxyl group of a nucleoside (e.g., the 3' hydroxyl group of a ribose moiety) to an amino group present on the support moiety (e.g., the aminomethyl group of a polymer resin obtained from the copolymerization of poly(styrene) with divinylbenzene). Other commonly used linkers are so-called universal linkers, for example of the "Unylinker" type described in US Pat. No. 7,202,264. When a support moiety modified with a linker such as the universal linker is used, step a) of the method comprises loading a first nucleoside or oligonucleotide onto the support moiety via a universal linker.

[0096] At the end of the chain construction, the newly synthesized oligonucleotide is cleaved from the support moiety, i.e., the bond between the oligonucleotide and the support moiety, or, if applicable, between the oligonucleotide and the linker, is broken to release the oligonucleotide (step f)). However, in some cases, it is desirable to attach a capping moiety to the first nucleoside. This is achieved by including a capping moiety between the support moiety, or, if applicable, between the linker and the first nucleoside. In other words: the capping moiety differs from the linker moiety in that it is not removed from the oligonucleotide during the final cleavage of the oligonucleotide from the support moiety. Thus, as used herein, the expression "capping moiety" refers to any moiety that is conjugated to the final nucleoside of the oligonucleotide chain. The capping moiety is included in the final oligonucleotide product. In the case where a capping moiety is present between the first nucleoside and the linker moiety or between the first nucleoside and the support moiety, it is attached to the 3' end of the oligonucleotide. An example for such a strategy is the insertion of a 3'GalNac conjugate as described, for example, in WO2009073809.

[0097] Any support moiety known in the art can be used in the present invention. As used herein, the term "support moiety" refers to any macroscopic or molecular structure to which the oligonucleotide to be synthesized is anchored and allows separation of the synthesized oligonucleotide from the liquid phase. Therefore, this term encompasses the terms "resin" and "resin", which are understood in the broadest sense as a specific structure useful for solid-phase oligonucleotide synthesis. The terms "resin", "solid support" and "solid phase" are used interchangeably herein. For solid-phase oligonucleotide synthesis, the support moiety is an insoluble solid or gel-like substrate. 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 complexes, such as Tentagel™ resin. Step a) of the present method includes conditioning the solid support by washing steps with various solvents. In some embodiments of the method of the present invention, including the above steps a) to f), the support moiety is a solid support (and therefore the method is a method for solid-phase synthesis of oligonucleotides).

[0098] Those skilled in the art know how to cleave an oligonucleotide (i.e., a fully assembled oligonucleotide chain) from a support (i.e., a support moiety) after support-assisted synthesis. The terms support and support moiety are used interchangeably herein. Typically, such cleavage (as in step f) of the method of the present invention) is accomplished by treating the support-bound oligonucleotide with a base such as an organic amine or an alkali hydroxide, where concentrated aqueous ammonia (i.e., aqueous ammonium hydroxide) is the most common and most preferred herein. This base (e.g., ammonia) treatment is carried out at room temperature or under heating, e.g., at 40-60°C, e.g., in an autoclave or sealed container. Under such alkaline conditions, typical nucleobase protecting groups are similarly cleaved. As non-limiting examples, but not typical, 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 are typically cleaved (i.e., removed) under such alkaline conditions. The protecting group R of formula I, Ia, II, and II-a 2 are also typically selected such that they are cleaved (i.e., removed) under alkaline conditions, and thus during the cleavage step f). 2 is a prime example for this strategy. The optional 2-cyanoethyl protecting group R 2 is removed in the course of base treatment, resulting in cleavage from the support. However, as known to those skilled in the art, cleavage step f) can include subsequent treatment with different types of base. First, a solution of an organic amine, such as diethylamine or trimethylamine, in a suitable solvent, such as acetonitrile, is used, preferably at room temperature, to remove the 2-cyanoethyl protecting group, followed by treatment with concentrated aqueous ammonia, resulting in cleavage from the support and removal of base-labile permanent protecting groups, such as nucleobase protecting groups.

[0099] Furthermore, the term "support moiety" encompasses any molecular tag that allows the separation of the tag with the synthesized oligonucleotide to which it is tethered from the liquid phase. For liquid phase synthesis, the support moiety is a tag that modifies solubility, as disclosed, for example, in WO2012157723. Such support moieties are also referred to as pseudo-solid-phase protecting groups. They essentially function to modify the solubility of the growing oligonucleotide chain by attaching to a hydrophobic moiety. This allows the separation of the growing oligonucleotide chain from other reagents by a simple precipitation or extraction step. In some embodiments of the method of the present invention, including the above steps a) to f), the support moiety is a pseudo-solid-phase protecting group (and thus the method is for liquid phase synthesis of oligonucleotides).

[0100] Based on the foregoing, it will be apparent to one of skill in the art that the term "support moiety," as used herein, refers to a population of entities, e.g., bead-like particles or molecular tags, and is used in the singular for readability purposes only.

[0101] Steps c) of the method, i.e., coupling of the building blocks to the growing oligonucleotide chain, and d), i.e., oxidation or sulfurization of the phosphorus(III) moieties, depend on the coupling chemistry selected and are routinely performed by those skilled in the art. In order to avoid the formation of deletion sequences that are difficult to remove, the method of the present invention comprises a further step of blocking (also referred to as capping) the unreacted free hydroxyl groups after step c) or step d). In some embodiments, the method of the present invention comprising the above steps a) to f) further comprises step g) of blocking the unreacted free hydroxyl groups after step c) or after step d). In these embodiments, step g) is performed after at least one repetition of step c) or step d), but typically not after both steps c) and step d) of the same extension cycle (i.e., coupling cycle). Thus, in some embodiments, the further step g) is performed after at least one repetition of step c) or step d). In some embodiments, the further step g) is performed at least once, preferably exactly once, in each repetition of steps b)-c) or b)-d). In some embodiments, a further step g) is performed after each iteration of step c). In some embodiments, a further step g) is performed after each iteration of step d). It should be noted that the order of steps shown in the claims and the description of the method is not necessarily binding unless otherwise indicated. The method may also include further steps. For example, the constructed oligonucleotide moiety is conjugated to another moiety before carrying out the cleavage step f) of the method. Furthermore, the skilled artisan will appreciate that an intervening washing step is used, for example, when changing the composition of the solvent between process steps or to remove traces of reagents from previous method steps. As another example, step f) of the method of the invention may proceed to the last (and isolated, i.e., not part of an extension cycle) step b) to remove the 5'-terminal hydroxyl protecting group R from the nucleoside or oligonucleotide building block of the last iteration of step c). 1 Remove this last R 1 The removal may also be carried out after step f).

[0102] In step b) of the method disclosed herein, cleavage of the temporary protecting group from the backbone hydroxyl group is carried out by incubating the nucleoside or oligonucleotide compound with liquid composition C. As used herein, the expression "composition C" refers to a cleavage composition comprising the specified components. The reference sign "C" is added to improve readability and should not be interpreted as implying any limitation.

[0103] Cleavage of the positional protecting group, including the optionally substituted triarylmethyl residue, is usually carried out at ambient temperature, for example, at a temperature of 15 to 35°C. However, it should be understood that the reaction may be carried out at any suitable temperature, for example, at a temperature of 0 to 90°C. The incubation with liquid composition C is carried out for at least 5 minutes, 10 minutes, 20 minutes, or 30 minutes, without any particular time constraint. The incubation includes mixing the liquid composition C with the compound for deprotection in a stirred bed reactor, a sparged bed reactor (wherein agitation is achieved by passing nitrogen gas through the reaction solution), a batch reactor, or circulating the liquid composition C through a column reactor for the required time. The cleavage step is repeated several times with fresh composition C.

[0104] It is a particular advantage of the present invention 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 10% dichloroacetic acid (DCA), the method of the present invention allows the batch size to be increased by at least 5 times, preferably at least 10, 20, 30 or 40 times, while using the same liquid handling system for supplying liquid and draining liquid from the reaction vessel.For the sake of clarity, it is noted that the reaction vessel may be modified to accommodate a larger amount of reagent.

[0105] The methods of the present application are particularly used on a scale of greater than 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, meaning that the maximum theoretical amount of expected final product is 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 per single synthesis process. The theoretical amount of expected final product is calculated based on the molar amount of starting nucleoside or oligonucleotide attached to the support moiety provided in step a), under the assumption that all subsequent process steps are 100% efficient (i.e., proceed with quantitative yield of the desired product). In some embodiments of the method of the invention comprising steps a) through f) above, the synthesis is carried out on a scale of 100 mmol or more of oligonucleotide product.

[0106] Solid-phase oligonucleotide synthesis is usually carried out using a column reactor packed with a solid support to which the growing oligonucleotide chain is tethered. The column reactor typically includes a bottom frit and a top frit, between which the solid support is packed. The size and diameter of the reaction vessel are selected according to the intended scale of synthesis. For example, a column reactor 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 is used. In certain embodiments, a column reactor with a maximum internal volume of 1-500 liters, 5-450 liters, 10-400 liters, 50-300 liters, or 100-300 liters is used. The inventors have surprisingly found that the flow rate of liquid composition C through the column reactor is sometimes rather low, for example, lower than 300 cm / h, lower than 290 cm / h, lower than 280 cm / h, lower than 270 cm / h, lower than 260 cm / h, lower than 250 cm / h, lower than 240 cm / h, lower than 230 cm / h, lower than 220 cm / h, lower than 210 cm / h, lower than 200 cm / h, lower than 190 cm / h, lower than 180 cm / h, or lower than 160 cm / h. This dramatically increases the scalability of the process. Therefore, in some embodiments of the method of the present invention comprising the above steps a) to f), the method is carried out in a column reactor, and the flow rate of liquid composition C through the column reactor is lower than 300 cm / h in at least one repetition of step b). In some embodiments of the process of the invention comprising the above steps a) to f), the process is carried out in a column reactor and the flow rate of liquid composition C through the column reactor is less than 300 cm / hr in each repetition of step b).

[0107] Alternatively, the inventors have found that batch reactors are used in the process of the present invention. In one embodiment, the batch reactor is a stirred bed reactor. Such reactors include a mixing device and a bottom frit or filter cloth to hold the support inside the reactor, for example, when discharging the solvent and dissolved components. As a further example, a batch reactor having a maximum internal volume of 1-1000 liters, 5-650 liters, 5-300 liters, 10-600 liters, 50-500 liters, or more than 1000 liters is used. As a further example, a batch reactor 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 is used. In some embodiments of the method of the present invention comprising steps a) through f) above, at least one repetition of step b) is carried out in a batch reactor. In some embodiments of the method of the present invention comprising steps a) through f) above, at least step b) is carried out in a batch reactor. In some embodiments of the method of the present invention comprising steps a) through f) above, each repetition of step b) is carried out in a batch reactor.

[0108] In a preferred embodiment, the contact time between composition C (which can be used as a composition for cleaving a protecting group comprising an optionally substituted triarylmethyl residue, preferably a DMT protecting group) and an oligonucleotide bound to a support moiety (e.g., a solid phase in a reaction vessel) 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 more than 1 hour.

[0109] Such a comparable long time interval allows the use of standard pumps with moderate pump flow rates for comparable large size reactors (e.g., column reactors or batch reactors).Such a long contact time, when used for cleavage solutions commonly used in the art, such as cleavage solutions containing a higher content of acid, such as dichloroacetic acid (DCA) without base and alcohol as defined in the present invention, can result in undesired cleavage of nucleic acid bases, in particular depurination.

[0110] As is known to those of skill in the art, oligonucleotide synthesis (including cleavage from the support, if a support is used) is typically followed by a step of isolating the oligonucleotide.

[0111] Therefore, in some embodiments, the method of the present invention comprising the above steps a) to f) further comprises the following step h): h) isolating the oligonucleotide cleaved from the support Further includes:

[0112] The means of isolating oligonucleotides during chemical oligonucleotide synthesis form part of the general knowledge of the skilled artisan. Typically, such a step of isolating an oligonucleotide comprises one or more purification steps and one or more steps that aid in obtaining the oligonucleotide in solid form. For oligonucleotide purification, typically, chromatographic methods are used, in particular ion exchange (particularly anion exchange) chromatography, and reversed phase (RP) HPLC, for example in the form of hydrophobic interaction HPLC. These techniques are known to those skilled in the art. In addition, the step of isolating an oligonucleotide comprises an ultrafiltration and / or desalting step. For example, the solution obtained after cleaving an oligonucleotide from a support in a support-assisted oligonucleotide synthesis is subjected to another round of ultrafiltration and / or desalting, ion exchange chromatography, and ultrafiltration and / or desalting. Alternatively, the oligonucleotide cleaved from the support is subjected to a reversed phase (RP) HPLC, for example in the form of hydrophobic interaction HPLC. The latter method is preferably carried out when the oligonucleotide still bears a 5'-terminal hydroxyl protecting group, for example a DMT group. The 5'-protecting group is further removed in a RP-HPLC column by passing an acidic solution through the column. When the 5'-terminal protecting group is removed before purification, for example before the oligonucleotide is cleaved from the support, ion exchange chromatography is preferred. Purification is typically followed by one or more steps that help obtain the oligonucleotide in solid form. For example, freeze-drying and spray-drying are used. In some cases, it is desirable to obtain the oligonucleotide in the form of a salt with a certain counterion. In such cases, salt exchange is typically performed before freeze-drying or spray-drying.

[0113] Preferably, the salt contained in liquid composition C is not very hygroscopic and is soluble in the mixture of organic solvents present in cleavage composition C (i.e., liquid composition C). Such salts are obtained from the reaction of a weak base with a strong acid. Those skilled in the art are aware of how acids and bases form salts, and that salt formation typically occurs with a certain stoichiometry depending on the charge of the ions involved. An acid that has a single acidic functional group and therefore donates a single proton is called a monobasic acid, and its conjugate base (i.e., in its deprotonated form) has a single negative charge. When a molecule of such a monobasic acid, such as trifluoroacetic acid (TFA), hydrochloric acid (HCl) or methanesulfonic acid (MSA), donates a proton to a base that has a single basic functional group (resulting in a single positive charge upon protonation), such as a pyridine derivative, salt formation typically occurs with a 1:1 stoichiometry of said acid and said base. It is understood that said salts are soluble in liquid composition C of the present invention, as shown above.

[0114] The base contained in liquid composition C functions to deprotonate the strong acid, resulting in a protonated form of the base (i.e., the cation of the salt) that is less acidic than the strong acid (see below). It is preferred that the total molar amount of the base contained in liquid composition C is equal to or greater than the total molar amount of the strong acid.

[0115] In some preferred embodiments, the total molar amount of the base in the liquid composition C is equal to or greater than the total molar amount of the strong acid. In some embodiments, the total molar amount of the base in the liquid composition C is within the range of 1.0 to 3.0, 1.0 to 2.0, 1.00 to 1.50, 1.00 to 1.40, 1.00 to 1.30, 1.00 to 1.20, 1.00 to 1.10, or even 1.000 to 1.050 equivalents relative to the total molar amount of the strong acid. It will be understood that the total molar amount of the base refers to the total molar amount of the base added to the liquid composition C and does not distinguish between protonated and unprotonated molecules of the base. Similarly, it will be understood that the molar amount of the strong acid refers to the total molar amount of the strong acid added to the liquid composition C and does not distinguish between protonated and unprotonated molecules of the strong acid. The term "total molar amount" of a particular species, such as a base, a strong acid, or at least one alcohol, of composition C refers to the sum of the molar amounts of two or more bases or acids or alcohols. For example, if the base contained in composition C is a mixture of 4-cyanopyridine and 3-cyanopyridine, the total molar amount of said base is the sum of the molar amounts of 4-cyanopyridine and 3-cyanopyridine. The same theory applies, for example, to said strong acid and said alcohol.

[0116] In the context of this application, a weak base is a base whose conjugated acid has a pKa of 1-4. Similarly, in the context of this application, a strong acid is an acid whose pKa is less than 1. As used herein, unless otherwise indicated, pKa is so determined in water (i.e., aqueous solution) at 25°C. Means for determining the pKa value of an acid or base are known to those skilled in the art, and for the most common acids and bases, pKa values ​​are readily available from the literature. For example, the pKa values ​​of the bases of the liquid composition C referred to herein, in particular the aromatic cyclic amine bases (e.g., pyridine derivatives), are taken from A.Fischer, WJGalloway, J.Vaughan, Journal of the Chemical Society 1964, pp. 3591-3596, or are determined according to the procedures disclosed therein.

[0117] Preferred acids include trifluoroacetic acid, hydrochloric acid, and sulfonic acid. In some embodiments of the liquid composition C, its use, and method of the present invention, the strong acid contained in the composition C is selected from the group consisting of carboxylic acid, mineral acid, sulfonic acid, and mixtures thereof. In some embodiments of the liquid composition C, its use, and method of the present invention, the strong acid contained in the composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof, preferably, the strong acid is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof. In some embodiments of the liquid composition C, its use, and method of the present invention, the strong acid contained in the composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, and mixtures thereof. In some embodiments of the liquid composition C, its use, and method of the present invention, the strong acid contained in the composition C is trifluoroacetic acid.

[0118] Preferred bases include cyclic amines and heterocyclic amines.The base is selected from the group consisting of pyrimidine, pyridine, pyrazine, thiazole, pyridazine, pyrazole or triazole, which is optionally substituted with electron donating or electron withdrawing groups.For example, the base is pyrimidine, pyridine, thiazole, pyridazine, pyrazole and 1,2,4-triazole, which is substituted with one or more electron withdrawing substituents.In some embodiments of the liquid composition C of the present invention, its use and method, the base contained in composition C is a cyclic amine, and preferably, the cyclic amine is selected from the group consisting of pyridine, pyrimidine, pyrazine, thiazole, pyridazine, pyrazole and triazole. In some embodiments of the liquid composition C of the present invention, its use and method, the base contained in the composition C is a cyclic amine, preferably the cyclic amine is selected from the group consisting of pyridine, pyrimidine, pyrazine, thiazole, pyridazine, pyrazole, and triazole, which is optionally substituted with an electron-donating or electron-withdrawing substituent. In this specification, the terms "group", "residue", "moiety", and "substituent" are used interchangeably, so that, for example, the terms "electron-withdrawing group" and "electron-withdrawing substituent" are used interchangeably. The electron-withdrawing substituent is selected from the group consisting of a halogen atom such as a chlorine, fluorine, or bromine atom, a cyano group, an aldehyde group, a keto group, a carboxy ester group, or a carboxamide group. As another example, the base is a pyrimidine or pyrazine substituted with one or more electron-donating substituents. The electron-donating substituent is a methoxy group. As used herein, residues (i.e., substituents or groups) are interchangeably represented by their full names, e.g., a chlorine atom or a cyano group, or in the form, e.g., Cl (for chlorine) or -CN (for cyano). In such cases, particularly when more than one atom is included in the residue, a hyphen is used to indicate the atom at which the residue is attached to the core structure, e.g., the carbon atom of a cyano group.

[0119] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is a pyridine substituted with one or more electron-withdrawing substituents selected from the group consisting of halogen atoms, cyano groups, aldehyde groups, keto groups, carboxy ester groups, and carboxamide groups.

[0120] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C 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.

[0121] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof. In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, and 3-chloropyridine.

[0122] In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of -CN, Cl, Br, F, and I. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of -CN, Cl, Br, and F. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of -CN, Cl, and Br. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of -CN and Cl. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of -CN and Cl.

[0123] In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is 4-cyanopyridine. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is 3-cyanopyridine. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is 4-chloropyridine. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is 3-chloropyridine. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is 4-bromopyridine. In some embodiments of the liquid composition C, its use and method of the present invention, the base contained in the composition C is 3-bromopyridine.

[0124] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is a cyclic amine of formula S-1: [ka] [wherein, in formula S-1: Q 1 is N or CR 7 and; Q 2 is N or CR 8 and; Q 3 is N or CR 9 and; R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, a C1-C5 alkyl group, and an electron-withdrawing substituent; However, Q 1 , Q 2 , and Q 3 If not N, then R 5 , R 6 , R 7 , R 8 , and R 9 At least one substituent selected from the group consisting of is an electron-withdrawing substituent; and further, Q 1 , Q 2 , and Q 3 Only one of them is N. It is.

[0125] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is a cyclic amine of formula S-1, where: Q 1 is N or CR 7 and; Q 2 is N or CR 8 and; Q 3 is N or CR 9 and; R 5 , R6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, a C1-C5 alkyl group, and an electron-withdrawing substituent selected from the group consisting of -CN, Cl, Br, and F; However, Q 1 , Q 2 , and Q 3 If not N, then R 5 , R 6 , R 7 , R 8 , and R 9 At least one substituent selected from the group consisting of is an electron-withdrawing substituent; and further, Q 1 , Q 2 , and Q 3 Only one of them is N.

[0126] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is a cyclic amine of formula S-1, where: Q 1 CR 7 and; Q 2 CR 8 and; Q 3 CR 9 and; R 5 , R 6 , R 7 , R 8 , and R 9 are independently selected from the group consisting of H and electron-withdrawing substituents selected from the group consisting of -CN, Cl, Br, and F; However, R 5 , R 6 , R 7 , R 8 , and R 9 At least one, and preferably exactly one, substituent selected from the group consisting of is an electron-withdrawing substituent.

[0127] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is a cyclic amine of formula S-1, where: Q 1 CR 7 and; Q 2 CR 8 and; Q 3 CR 9 and; R 5 , R 6 , R 7 , R 8 , and R 9 are independently selected from the group consisting of H and electron-withdrawing substituents selected from the group consisting of -CN, Cl, and Br; However, R 5 , R 6 , R 7 , R 8 , and R 9 At least one, and preferably exactly one, substituent selected from the group consisting of is an electron-withdrawing substituent.

[0128] In some embodiments of the liquid composition C, its uses and methods of the present invention, the base contained in composition C is a cyclic amine of formula S-1, where: Q 1 CR 7 and; Q 2 CR 8 and; Q 3 CR 9 and; R 5 , R 6 , R 7 , R 8 , and R 9 are independently selected from the group consisting of H and electron-withdrawing substituents selected from the group consisting of -CN and Cl; However, R 5 , R 6 , R 7 , R 8 , and R 9At least one, and preferably exactly one, substituent selected from the group consisting of is an electron-withdrawing substituent.

[0129] One aspect of the present invention is the use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, for suppressing nucleobase cleavage while effecting cleavage of di(p-methoxyphenyl)phenylmethyl protecting groups from hydroxyl groups during chemical synthesis of oligonucleotides; the base is a pyridine substituted with one or more electron-withdrawing substituents selected from the group consisting of halogen atoms, cyano groups, aldehyde groups, keto groups, carboxy ester groups, and carboxamide groups; the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof; Regarding use.

[0130] One aspect of the present invention is the use of a liquid composition C comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, for suppressing nucleobase cleavage while effecting cleavage of di(p-methoxyphenyl)phenylmethyl protecting groups from hydroxyl groups during chemical synthesis of oligonucleotides; the base is a 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, more preferably the base is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof; the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; Regarding use.

[0131] A further embodiment of the invention is a liquid composition C for use in cleaving a di(p-methoxyphenyl)phenylmethyl protecting group from a hydroxyl group, the composition comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein: - the aprotic solvent is not halogenated and is selected from the group consisting of (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, or alkyl(hetero)aryl ethers; the base is a pyridine substituted with one or more electron-withdrawing substituents selected from the group consisting of halogen atoms, cyano groups, aldehyde groups, keto groups, carboxy ester groups, and carboxamide groups; the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid and sulfonic acid; Concerning liquid composition C.

[0132] A further embodiment of the invention is a liquid composition C for use in cleaving a di(p-methoxyphenyl)phenylmethyl protecting group from a hydroxyl group, the composition comprising a non-halogenated and / or (hetero)aromatic aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein: - the aprotic solvent is non-halogenated and is selected from the group consisting of (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers; the base is a 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, more preferably the base is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof; the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; Concerning liquid composition C.

[0133] A further aspect of the present invention relates to a method for the synthesis of oligonucleotides, comprising the steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group; b) cleaving the di(p-methoxyphenyl)phenylmethyl protecting group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby producing a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C comprises an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein: the base is a pyridine substituted with one or more electron-withdrawing substituents selected from the group consisting of halogen atoms, cyano groups, aldehyde groups, keto groups, carboxy ester groups, and carboxamide groups; the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof; It concerns the method.

[0134] A further aspect of the present invention is a method for the synthesis of oligonucleotides comprising the following steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group; b) cleaving the di(p-methoxyphenyl)phenylmethyl protecting group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby producing a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C comprises an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, wherein: the base is a 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, more preferably the base is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof; the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; It concerns the method.

[0135] The salt contained in the cleavage composition acts as a proton donor in the deprotection reaction. Thus, the skilled person will select the salt so that the pKa of its protonated cation corresponds to the nature of the triarylmethyl group to be cleaved. For example, for the cleavage of a protecting group containing an optionally substituted triarylmethyl residue with two methoxy substituents, for example, the salt used for the cleavage of a dimethoxytrityl (DMT, preferably di(p-methoxyphenyl)phenylmethyl) group, preferably contains a protonated cation having a pKa of 1 to 2.5, preferably a pKa of 1 to 2.5. In some embodiments, the protonated form of the base contained in the liquid composition C (i.e., the cation of the salt) has a pKa in the range of 1 to 4, 1 to 3.5, 1 to 3, 1 to 2.5, or 1 to 2. Examples are the conjugated acids of 4-cyanopyridine, 3-cyanopyridine, pyrimidine, 2-methylpyrazine, 2,5-dimethylpyrazine, and 3-bromopyridine. Preferably, the cleavage composition of the present invention is 4-cyanopyridinium trifluoroacetate, 4-cyanopyridinium chloride, 4-cyanopyridinium sulfonate, e.g., 4-cyanopyridinium alkylsulfonate or 4-cyanopyridinium p-toluenesulfonate, 3-cyanopyridinium trifluoroacetate, 3-cyanopyridinium chloride, 3-cyanopyridinium sulfonate, e.g., 3-cyanopyridinium alkylsulfonate or 3-cyanopyridinium p-toluenesulfonate, pyrimidinium trifluoroacetate, pyrimidinium chloride, pyrimidinium sulfonate, e.g., pyrimidinium pyrimidinium alkylsulfonate or pyrimidinium p-toluenesulfonate, 2-methylpyrazinium trifluoroacetate, 2-methylpyrazinium chloride, 2-methylpyrazinium sulfonate, such as 2-methylpyrazinium alkylsulfonate or 2-methylpyrazinium p-toluenesulfonate, 2,5-dimethylpyrazinium trifluoroacetate, 2,5-dimethylpyrazinium chloride, and 2,5-dimethylpyrazinium sulfonate, such as 2,5-dimethylpyrazinium alkylsulfonate or 2,5-dimethylpyrazinium p-toluenesulfonate.4-Cyanopyridinium trifluoroacetate is particularly preferred.

[0136] In some embodiments of the liquid composition C of the present invention, its use and method, the salt of the base and the strong acid is selected from the group consisting of 4-cyanopyridinium trifluoroacetate, 3-cyanopyridinium trifluoroacetate, 3-chloropyridinium trifluoroacetate, and 4-chloropyridinium hydrochloride. In some embodiments of the liquid composition C of the present invention, its use and method, the salt of the base and the strong acid is selected from the group consisting of 4-cyanopyridinium trifluoroacetate, 3-cyanopyridinium trifluoroacetate, and 3-chloropyridinium trifluoroacetate. In some embodiments of the liquid composition C of the present invention, its use and method, the salt of the base and the strong acid is 4-cyanopyridinium trifluoroacetate.

[0137] Composition C comprises at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol and nonafluoro-tertiary butyl alcohol, or a mixture thereof. The use of trifluoroethanol, hexafluoroisopropanol, or a mixture thereof is particularly preferred.

[0138] In some embodiments of the liquid composition C, its use and method of the present invention, the at least one alcohol contained in the liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof. In some embodiments of the liquid composition C, its use and method of the present invention, the at least one alcohol contained in the liquid composition C is trifluoroethanol. In some embodiments of the liquid composition C, its use and method of the present invention, the at least one alcohol contained in the liquid composition C is hexafluoroisopropanol.

[0139] The total molar amount of said alcohols in the cleavage composition is at least 2, 5, 10, 20, 40, 60, 70, 80, or 100 times higher than the total amount of nucleobases.

[0140] In some embodiments, the molar concentration of the alcohol in composition C is at least 2 times higher than the molar concentration of the cation of the salt contained in composition C. In embodiments, it should be understood that composition C includes two or more of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and the molar concentration of the alcohol in composition C is calculated as the sum of the molar amounts of each of the alcohols divided by the liquid volume of composition C. In some embodiments, the molar concentration of the alcohol in composition C is at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, or 50 times higher than the molar concentration of the cation of the salt contained in composition C.

[0141] In some embodiments of the liquid composition C, its use and method of the present invention, the molar concentration of the alcohol in the composition C is at least 2 times higher than the molar concentration of the base contained in the composition C. In some embodiments of the liquid composition C, its use and method of the present invention, the molar concentration of the alcohol in the liquid composition C is at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, or 50 times higher than the molar concentration of the base contained in the composition C. The term "said alcohol" includes mixtures of alcohols, such as a mixture of TFE and HFIP, when such mixtures are present in the liquid composition C. Similarly, the term "said base" includes mixtures of bases, such as a mixture of 4-cyanopyridine and 3-cyanopyridine, when such mixtures are present in the liquid composition C.

[0142] In some embodiments, composition C further comprises a carbocation scavenger. As used herein, the expression "carbocation scavenger" refers to a nucleophilic compound that is used to bind to the formed carbocation and thereby prevent unwanted side reactions of the carbocation, or a compound that consumes the carbocation by formal donation of a hydride anion. Examples of carbocation scavengers include alcohols and phenols [e.g., methanol, ethanol, phenol, cresol, water], phenol ethers [e.g., anisole, 1,3-dimethoxybenzene (dimethylanisole), 1,3,5-trimethoxybenzene]; thioethers [e.g., dimethylsulfide, methylethylsulfide, thioanisole-, silanes-e.g., triisopropylsilane (TIS), triethylsilane (TES)], N-heterocycles [e.g., pyrrole, 3-methylpyrrole, 2,4-dimethylpyrrole, indole, 2 -methylindole, succinimide, phthalimide], 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 polyalkylbenzenes [e.g., 1,3,5-trimethylbenzene, pentamethylbenzene].

[0143] The aprotic solvent contained in composition C is preferably non-halogenated.

[0144] Suitable solvents are selected from the group consisting of (hetero)aromatic solvents, alkyl (hetero)aromatic solvents or (hetero)aromatic ethers, preferably selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene and diphenyl ether. Composition C comprises an aprotic non-halogenated and / or (hetero)aromatic solvent and an alcohol selected from the group of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol and nonafluoro-tertiary butyl alcohol plus a further halogenated solvent.

[0145] In some embodiments of the uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of halogenated hydrocarbon solvents, (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers.

[0146] 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.

[0147] In some embodiments of the uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, dichloromethane, and mixtures thereof. In some embodiments of the uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, dichloromethane, and mixtures thereof. In some embodiments of the uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of toluene, anisole, dichloromethane, and mixtures thereof.

[0148] In some embodiments of the uses and methods of the present invention, the aprotic solvent contained in composition C is non-halogenated (i.e., does not contain any halogen atoms in its chemical structure). In some embodiments of the uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof.

[0149] In some embodiments of the liquid composition C, its uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof. In some embodiments of the liquid composition C, its uses and methods of the present invention, the aprotic solvent contained in composition C is selected from the group consisting of toluene, anisole, and mixtures thereof.

[0150] In some embodiments of the liquid composition C of the present invention, the aprotic solvent is selected from the group consisting of toluene, anisole, and mixtures thereof. In some embodiments of the liquid composition C of the present invention, the aprotic solvent is toluene. In some embodiments of the liquid composition C of the present invention, the aprotic solvent is anisole.

[0151] In some embodiments of liquid composition C, its use, and its method of the present invention, the total volume of the aprotic solvent contained in liquid composition C accounts for 60 to 99%, 70 to 98%, 80 to 98%, 81 to 98%, or 81 to 95% of the total volume of liquid composition C. In some embodiments of liquid composition C, its use, and its method of the present invention, the total volume of the alcohol contained in liquid composition C accounts for 1 to 40%, 2 to 30%, 2 to 20%, 2 to 19%, or 5 to 19% of the total volume of liquid composition C.

[0152] The term "total volume of liquid composition C" refers to the combination of the volumes of alcohol, aprotic solvent, and any other liquid components that are combined to form liquid composition C. The change in the total volume due to dissolving a solid component, such as a base, is not considered for said "total volume of liquid composition C" if it is solid at room temperature. Similarly, the volume of the oligonucleotide and the support is not considered as part of said "total volume of liquid composition C". For example, if liquid composition C consists of TFE (as alcohol), toluene (as aprotic solvent), TFA (as strong acid), and 4-cyanopyridine (as base), the "total volume of liquid composition C" is the combination of the volumes of TFE, toluene, and TFA. The term "total volume" is used to indicate that, for example, if the alcohol is actually a mixture of alcohols, such as a mixture of TFE and HFIP, said "total volume" refers to the sum of the volumes of the alcohols, such as TFE and HFIP. The same logic applies to the "total volume" of the protic solvent.

[0153] In some embodiments of the liquid composition C, its uses and methods of the present invention: - the total volume of the aprotic solvent contained in the liquid composition C is 60 to 99% of the total volume of the liquid composition C; The total volume of the alcohol contained in the liquid composition C accounts for 1 to 40% of the entire volume of the liquid composition C.

[0154] In some embodiments of the liquid composition C, its uses and methods of the present invention: - the total volume of the aprotic solvent contained in the liquid composition C is 70 to 98% of the total volume of the liquid composition C; The total volume of the alcohol contained in the liquid composition C is 2 to 30% of the total volume of the liquid composition C.

[0155] In some embodiments of the liquid composition C, its uses and methods of the present invention: - the total volume of the aprotic solvent contained in the liquid composition C is 81 to 98% of the total volume of the liquid composition C; The total volume of the alcohol contained in the liquid composition C is 2 to 19% of the total volume of the liquid composition C.

[0156] In some embodiments of the liquid composition C, its uses and methods of the present invention: - the total volume of the aprotic solvent contained in liquid composition C accounts for 81 to 95% of the total volume of liquid composition C; The total volume of the alcohol contained in the liquid composition C is 5 to 19% of the total volume of the liquid composition C.

[0157] In some embodiments of the liquid composition C, its uses and methods of the present invention, the total volume of the liquid composition C is within the range of 5 to 100 mL, 10 to 90 mL, 10 to 80 mL, 20 to 75 mL, 10 to 70 mL, 10 to 60 mL, 20 to 50 mL, 20 to 40 mL, or 25 to 37.5 mL per millimole (mmol) of hydroxyl protecting group, preferably DMT group, containing an optionally substituted triarylmethyl residue to be cleaved (i.e., removed).

[0158] In some embodiments of the liquid composition C, its use and method of the present invention, the total molar amount of strong acid in the liquid composition C is within the range of 0.80 to 15.0, 1.0 to 13.0, 2.0 to 12.50, or 2.30 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 the respective protecting groups multiplied by the amount of said protecting groups per molecule of these compounds. For example, if composition C contains 1 mole of compounds having exactly one DMT group per molecule, the total molar amount of DMT groups in composition C would be equal to the total molar amount of said compounds, which in this example would also be 1 mole.

[0159] In some embodiments of the liquid composition C, its use and method of the present invention, the total molar amount of the alcohol in the liquid composition C is within 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 the nucleobases. The term "total molar amount of the nucleobases" refers to the total molar amount of all the nucleobases contained in the liquid composition C. It is understood that essentially all, or at least most, of the nucleobases that are part of the nucleoside units of the oligonucleotide (i.e., oligonucleotide molecule) are cleaved from the protecting group, preferably the DMT group, which includes an optionally substituted triarylmethyl residue. For example, if composition C comprises 1 mole of a compound whose molecules each contain 5 nucleobases, then the total molar amount of nucleobases in said composition C is 5 moles.

[0160] In some embodiments of the liquid composition C, its uses and methods of the present invention, in the liquid composition C: - the total molar amount of the strong acid is in the range of 0.80 to 15.0 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl (DMT) groups; The total molar amount of the alcohol is within the range of 2.0 to 150.0 equivalents relative to the total molar amount of the nucleic acid base.

[0161] In some embodiments of the liquid composition C, its uses and methods of the present invention, in the liquid composition C: - the total molar amount of the strong acid is in the range of 1.0 to 13.0 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl (DMT) groups; The total molar amount of the alcohol is within the range of 2.0 to 100.0 equivalents relative to the total molar amount of the nucleic acid base.

[0162] In some embodiments of the liquid composition C, its uses and methods of the present invention, in the liquid composition C: - the total molar amount of the strong acid is in the range of 2.0 to 12.5 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl (DMT) groups; The total molar amount of the alcohol is within the range of 3.0 to 95.0 equivalents relative to the total molar amount of the nucleic acid base.

[0163] In some preferred embodiments, the present invention provides a method for the synthesis of oligonucleotides, comprising steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) group; b) cleaving the DMT group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a DMT group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C includes an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4. A method is provided.

[0164] In some preferred embodiments, the present invention provides a method for the synthesis of oligonucleotides, comprising steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) group; b) cleaving the DMT group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a DMT group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C comprises a non-halogenated and / or (hetero)aromatic aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4; A method is provided.

[0165] In some preferred embodiments, the present invention provides a method for the synthesis of oligonucleotides, comprising steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) group; b) cleaving the DMT group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a DMT group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C comprises a non-halogenated and / or (hetero)aromatic aprotic solvent, trifluoroethanol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4; A method is provided.

[0166] In some preferred embodiments, the present invention provides a method for the synthesis of oligonucleotides, comprising steps a) to f): a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl (DMT) group; b) cleaving the DMT group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a DMT group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C comprises a non-halogenated and / or (hetero)aromatic aprotic solvent, hexafluoroisopropanol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4; A method is provided.

[0167] The salts in the above embodiments are, for example, 4-cyanopyridinium trifluoroacetate, 4-cyanopyridinium chloride, 4-cyanopyridinium sulfonate, such as 4-cyanopyridinium alkylsulfonate or 4-cyanopyridinium p-toluenesulfonate, 3-cyanopyridinium trifluoroacetate, 3-cyanopyridinium chloride, 3-cyanopyridinium sulfonate, such as 3-cyanopyridinium alkylsulfonate or 3-cyanopyridinium p-toluenesulfonate, pyrimidinium trifluoroacetate, pyrimidinium chloride, pyrimidinium sulfonate, such as pyrimidinium alkylsulfonate or pyrimidinium p-toluenesulfonate, 2-methyl-pyrazinium trifluoroacetate, 2-methyl-pyrazinium chloride, a The pyrazinium sulfonate is selected from the group consisting of 2-methyl-pyrazinium sulfonate, such as 2-methyl-pyrazinium alkyl sulfonate or 2-methyl-pyrazinium p-toluene sulfonate, 2,5-dimethyl-pyrazinium trifluoroacetate, 2,5-dimethyl-pyrazinium chloride, and 2,5-dimethyl-pyrazinium sulfonate, such as 2,5-dimethyl-pyrazinium alkyl sulfonate or 2,5-dimethyl-pyrazinium p-toluene sulfonate. 4-cyanopyridinium trifluoroacetate is particularly preferred. In some embodiments of the liquid composition C, its uses and methods of the present invention, the salt of a base and a strong acid is selected from the group consisting of 4-cyanopyridinium trifluoroacetate, 4-cyanopyridinium methanesulfonate, 4-cyanopyridinium hydrochloride, 3-cyanopyridinium trifluoroacetate, 3-cyanopyridinium methanesulfonate, 3-cyanopyridinium hydrochloride, 4-chloropyridinium trifluoroacetate, 4-chloropyridinium methanesulfonate, 4-chloropyridinium hydrochloride, 3-chloropyridinium trifluoroacetate, 3-chloropyridinium methanesulfonate, and 3-chloropyridinium hydrochloride.In some embodiments of the liquid composition C, its uses and methods of the present invention, the salt of a base and a strong acid is selected from the group consisting of 4-cyanopyridinium trifluoroacetate, 4-cyanopyridinium methanesulfonate, 3-cyanopyridinium trifluoroacetate, 4-chloropyridinium hydrochloride, and 3-chloropyridinium trifluoroacetate. In some embodiments of the liquid composition C, its uses and methods of the present invention, the salt of a base and a strong acid is 4-cyanopyridinium trifluoroacetate.

[0168] The explanations and details given herein with respect to the method of the invention are equally applicable to the use of the liquid composition C according to the invention and to the liquid composition itself, unless otherwise indicated.

[0169] In some embodiments of the uses and / or methods of the invention: the aprotic solvent contained in composition C is selected from the group consisting of halogenated hydrocarbon solvents, (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers; - the at least one alcohol contained in composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; - the base contained in composition C is a pyridine substituted with one or more electron-withdrawing substituents selected from the group consisting of halogen atoms, cyano groups, aldehyde groups, keto groups, carboxy ester groups, and carboxamide groups; The strong acid contained in composition C is selected from the group consisting of carboxylic acids, mineral acids, sulfonic acids, and mixtures thereof.

[0170] In some embodiments of the uses and / or methods of the invention: the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, dichloromethane, and mixtures thereof; - the at least one alcohol contained in composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; - the base contained in composition C is pyridine in which exactly one hydrogen radical is replaced by an electron-withdrawing substituent selected from the group consisting of cyano groups and halogen atoms; The strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof.

[0171] In some embodiments of the uses and / or methods of the invention: the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, dichloromethane, and mixtures thereof; - the at least one alcohol contained in composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; The base contained in composition C is a pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of cyano groups and halogen atoms: - the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof; - the total volume of the aprotic solvent contained in the liquid composition C is 60 to 99% of the total volume of the liquid composition C; - the total volume of the alcohol contained in the liquid composition C is 1-40% of the total volume of the liquid composition C; the total molar amount of the strong acids is in the range of 0.80 to 15.0 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; - the total molar amount of the alcohol is in the range of 2.0 to 150.0 equivalents relative to the total molar amount of the nucleobase; The total molar amount of the base is in the range of 1.0 to 3.0 equivalents relative to the total molar amount of the strong acid.

[0172] In some embodiments of the uses and / or methods and / or liquid composition C of the present invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; The base contained in composition C is a pyridine in which exactly one hydrogen residue is replaced by an electron-withdrawing substituent selected from the group consisting of cyano groups and halogen atoms: - the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof; - the total volume of the aprotic solvent contained in the liquid composition C is 60 to 99% of the total volume of the liquid composition C; - the total volume of the alcohol contained in the liquid composition C is 1-40% of the total volume of the liquid composition C; the total molar amount of the strong acids is in the range of 0.80 to 15.0 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; - the total molar amount of the alcohol is in the range of 2.0 to 150.0 equivalents relative to the total molar amount of the nucleobase; The total molar amount of the base is in the range of 1.0 to 3.0 equivalents relative to the total molar amount of the strong acid.

[0173] In some embodiments of the uses and / or methods and / or liquid composition C of the present invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; - the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof; The strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof.

[0174] In some embodiments of the uses and / or methods and / or liquid composition C of the present invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof: - the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; - the total volume of the aprotic solvent contained in the liquid composition C is 70 to 98% of the total volume of the liquid composition C; - the total volume of the alcohol contained in the liquid composition C is 2-30% of the total volume of the liquid composition C; the total molar amount of the strong acid is in the range of 1.0 to 13.0 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; - the total molar amount of the alcohol is in the range of 2.0 to 100.0 equivalents relative to the total molar amount of the nucleobase; The total molar amount of the base is in the range of 1.0 to 2.0 equivalents relative to the total molar amount of the strong acid.

[0175] In some embodiments of the uses and / or methods and / or liquid composition C of the present invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof: - the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; - the total volume of the aprotic solvent contained in the liquid composition C is 70 to 98% of the total volume of the liquid composition C; - the total volume of the alcohol contained in the liquid composition C is 2-30% of the total volume of the liquid composition C; the total molar amount of the strong acids is in the range of 2.0 to 12.5 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; - the total molar amount of the alcohol is in the range of 3.0 to 95.0 equivalents relative to the total molar amount of the nucleobase; The total molar amount of the base is within the range of 1.00 to 1.50 equivalents relative to the total molar amount of the strong acid.

[0176] In some embodiments of the uses and / or methods of the invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, anisole, dichloromethane, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; - the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof; The strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof.

[0177] In some embodiments of the uses and / or methods of the invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, anisole, dichloromethane, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof: - the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; - the total volume of the aprotic solvent contained in the liquid composition C is 81 to 98% of the total volume of the liquid composition C; - the total volume of alcohol contained in liquid composition C is 2-19% of the total volume of liquid composition C; the total molar amount of the strong acids is in the range of 2.0 to 12.5 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; - the total molar amount of the alcohol is in the range of 3.0 to 95.0 equivalents relative to the total molar amount of the nucleobase; The total molar amount of the base is in the range of 1.000 to 1.050 equivalents relative to the total molar amount of the strong acid.

[0178] In some embodiments of the uses and / or methods and / or liquid composition C of the present invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, anisole, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; - the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof; The strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof.

[0179] In some embodiments of the uses and / or methods and / or liquid composition C of the present invention: - the aprotic solvent contained in composition C is selected from the group consisting of toluene, anisole, and mixtures thereof; - the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof; the base contained in composition C is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof: - the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, and mixtures thereof; - the total volume of the aprotic solvent contained in the liquid composition C is 81 to 98% of the total volume of the liquid composition C; - the total volume of alcohol contained in liquid composition C is 2-19% of the total volume of liquid composition C; the total molar amount of the strong acids is in the range of 2.0 to 12.5 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; - the total molar amount of the alcohol is in the range of 3.0 to 95.0 equivalents relative to the total molar amount of the nucleobase; The total molar amount of the base is in the range of 1.000 to 1.050 equivalents relative to the total molar amount of the strong acid.

[0180] In some embodiments of the methods of the present invention, step a) comprises reacting a compound according to formula I [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y, independently for each repeat unit, n, is selected from the group consisting of S and O; Z, independently for each repeat unit n, is selected from the group consisting of O and S; R 2 are the same or different protecting groups for each repeat unit n; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting part. preparing a - step c) comprises reacting the free hydroxyl group resulting from step b) with a nucleoside or oligonucleotide phosphoramidite building block, the building block comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and a phosphorus(III) atom of said building block, wherein; The nucleoside or oligonucleotide phosphoramidite building blocks are compounds according to formula II: [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer equal to or greater than 0; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z is independently, for each occurrence, selected from the group consisting of O and S; R 2are the same or different protecting groups for each position; R 3 and R 4 are the same or different protecting groups; Each of the nucleosides xm0 to xm may be the same or different. and; - step e) optionally comprises repeating the sequence of steps b) to d).

[0181] In some embodiments of the methods of the present invention, step a) comprises reacting a compound according to formula Ia [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y, independently for each repeat unit, n, is selected from the group consisting of S and O; Z, independently for each repeat unit n, is selected from the group consisting of O and S; R 2 are the same or different protecting groups for each repeat unit n; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting part; B N are the same or different nucleobases in each occurrence; R I is independently selected at each occurrence 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 II is, independently in each occurrence, H or R II and R Irepresents the chemical structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++ (where + is the 4' carbon atom (i.e., R II is the point of attachment to the 2' carbon (i.e., the carbon atom to which R I (a) forms a methylene or ethylene bridge with the carbon atom to which it is attached. preparing a - step c) comprises reacting the free hydroxyl group resulting from step b) with a nucleoside or oligonucleotide phosphoramidite building block, the building block comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and a phosphorus(III) atom of said building block, wherein; The nucleoside or oligonucleotide phosphoramidite building blocks are compounds according to formula II-a: [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer equal to or greater than 0; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z is independently, for each occurrence, selected from the group consisting of O and S; R 2 are the same or different protecting groups for each position; R 3 and R 4 are the same or different protecting groups; B N are the same or different nucleobases in each occurrence; R IIIis independently selected at each occurrence 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 IV is, independently in each occurrence, H or R III and R IV represents the chemical structure +-CH2-O-++, +-CH(CH3)-O-++, or +-CH2-CH2-O-++ (where + is the 4' carbon atom (i.e., R IV is the point of attachment to the 2' carbon (i.e., the carbon atom to which R III (a) forms a methylene or ethylene bridge with the carbon atom to which it is attached. and; - step e) optionally comprises repeating the sequence of steps b) to d).

[0182] In some embodiments of the methods of the present invention, step a) comprises reacting a compound according to formula Ia [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer ranging from 0 to 150; Y, independently for each repeat unit, n, is selected from the group consisting of S and O; Z, for each repeat unit n, is O; R 2 is a 2-cyanoethyl group in each occurrence; CA is a single bond; L is a linker moiety or a single bond; SM is the supporting part; B Nis independently at each occurrence selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R I is independently at each occurrence selected from the group consisting of H, F, and -O-CH3 (i.e., methoxy); R II is H in each occurrence] preparing a - step c) comprises reacting the free hydroxyl group resulting from step b) with a nucleoside or oligonucleotide phosphoramidite building block, the building block comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and a phosphorus(III) atom of said building block, wherein; The nucleoside or oligonucleotide phosphoramidite building blocks are compounds according to formula II-a: [ka] [In formula: R 1 is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer ranging from 0 to 2; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z, for each occurrence, is O; R 2 is, in each occurrence, a 2-cyanoethyl group; R 3 and R 4 are both isopropyl groups; B N is independently at each occurrence selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R IIIis independently at each occurrence selected from the group consisting of H, F, and -O-CH3 (i.e., methoxy); R IV is H in each occurrence] and; - step e) optionally comprises repeating the sequence of steps b) to d).

[0183] The following figures and examples, including the experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the claims. [Brief description of the drawings]

[0184] [Figure 1] Non-limiting examples of building blocks suitable for the synthesis of oligonucleotides with a phosphoribose backbone: (a) building blocks for the synthesis of DNA by the phosphoramidite approach; (b) building blocks for the synthesis of RNA by the phosphoramidite approach (the terms "Dmt" and "DMT" are used interchangeably herein); (c) phosphoramidite building blocks with 2'-O-methyl ribose; (d) phosphoramidite building blocks with 2'-O-(2-methoxyethyl) ribose; (e) phosphoramidite building blocks with 2'-fluoro ribose. [Diagram 2]Figure 1 shows phosphoramidite building blocks for the synthesis of oligonucleotide analogues, including ribose surrogates (a) protected building blocks for obtaining arabinose nucleic acids (ANA), (b) building blocks for obtaining 2'-fluoroarabinose nucleic acids (FANA), (c) building blocks for obtaining threose nucleic acids (TNA). The following types of building blocks are obtained as stereoisomers, and the structures shown represent one of these compounds: (d) building blocks for obtaining 1',3'-anhydrothreitol nucleic acids (HNA), (e) building blocks for obtaining locked nucleic acids (LNA), (f) building blocks for obtaining ethylene-bridged nucleic acids (ENA), (g) building blocks for obtaining tricyclic nucleic acids (tc-DNA), (h) building blocks for obtaining cyclohexane nucleic acids (CeNA), and (i) shows a (GNA) building block in which the ribose is replaced by glycerol. The protected building block (j) for unlocked nucleic acids (UNA) is obtained by oxidative ring cleavage of ribose. [Diagram 3] Analytical HPLC trace of crude 5'-TTT TAT TTT T-3' (SEQ ID NO: 1). Dashed line - EOP4149994 / 02: DMT deprotection using 10% DCA in toluene (2x10 min); Purity (HPLC): 89%. Solid line - EOP4149994 / 01: DMT deprotection using cleavage cocktail (2x10 min); Purity (HPLC): 93%. Capping was not performed in each of the coupling cycles. [Figure 4] Analytical HPLC trace of crude 5'-ATA CCG ATT AAG CGA AGT TT-3' (SEQ ID NO:2). Dashed line - EOP4149467 / 22: DMT deprotection using 10% DCA in toluene (2x10 min); Purity (HPLC): 50%. Solid line - EOP4149467 / 24: DMT deprotection using cleavage cocktail (2x10 min); Purity (HPLC): 64%. Capping was not performed in each of the coupling cycles. [Diagram 5]Analytical HPLC traces of crude 5'-ATA CCG ATT AAG CGA AGT TT-3' (SEQ ID NO:2). Dashed line - EOP4149467 / 01: DMT deprotection using 10% DCA in toluene (1 x 1 min); Purity (HPLC): 68%. Solid line - EOP4149467 / 02: DMT deprotection using cleavage cocktail (2 x 10 min); Purity (HPLC): 79%. Figure 5b is an enlarged view of Figure 5a with overlay of the traces corresponding to both samples. [Figure 6] Analytical HPLC traces of crude 5'-AAA AT-3'. Solid line - EOP4153156 / 03-5: DMT deprotection using 2.0% DCA, 0.1% ethanol (v / v) in DCM (5+20 min); Purity (HPLC-MS UV trace): 91%. Dashed line - EOP4153156 / 04-5: DMT deprotection using the cleavage cocktail of the invention (5+20 min); Purity (HPLC-MS UV trace): 94%. Figure 6b is an expanded view of Figure 6a, where the peaks have been assigned peak IDs as listed in Tables B.1 and B.2. [Figure 7] Analytical HPLC traces of crude 5'-[mAs][fCs][mA][fA][fA][fA][mG][fC][mA][fA][mA][mA][mC][fA][mG][fG][mU][fC][mU][mA][mGs][mAs][mA]-3'. Solid line - EOP4150430 / 28-1: DMT deprotection using the cleavage cocktail of the invention in the last cycle (5+5+5+20 min); Purity (HPLC-MS UV trace): 78%. Dashed line - EOP4150430 / 36-1: DMT deprotection using 10% DCA in toluene in the last cycle (5+5+5+20 min); Purity (HPLC-MS UV trace): 50%. Figure 7b is an expanded view of Figure 7a, marking the target products and peak areas containing the depurination products described in Tables C.1 and C.2.

[0185] The invention is illustrated by the examples, figures and claims. EXAMPLES

[0186] Common methods 1. Determination of Weight Gain and Yield To determine the theoretical weight gain, the mass of the dimethoxytrityl group was subtracted from the amount of starting resin according to the scale of synthesis. The sum of the β-cyanoethyl phosphate triesters of base-protected nucleobases was added according to the coupling cycle applied for the sequence. The experimental weight gain was calculated as the difference between the determined mass of the dry resin minus the mass of the starting resin, where the dimethoxytrityl group was already subtracted. The yield is given in percent as the ratio between the experimental weight gain and the theoretical weight gain.

[0187] More specifically, the experimental weight gain was determined by subtracting the mass of the solid support from the mass of the oligonucleotide on the dried resin after the end of all the extension cycles and the final dimethoxytrityl removal step. To determine the mass of the solid support, the mass of the dimethoxytrityl group was subtracted from the mass of the starting resin (consisting of the solid support with the dimethoxytrityl group) according to the scale of synthesis and the molecular weight of the dimethoxytrityl group. The theoretical maximum weight gain was determined as the sum of the β-cyanoethyl protected phosphate triesters of the nucleobase-protected nucleosides according to the coupling cycles applied to the sequence, assuming quantitative yields in all steps. Unless otherwise indicated, the yields are given in percent (%) and were obtained by dividing the experimental weight gain by the theoretical maximum weight gain, followed by multiplying by 100% to obtain the percentage value.

[0188] 2. Purity Determination Unless otherwise indicated, the purity of oligonucleotides was determined by analytical reversed-phase HPLC using a C18 stationary phase, with elution provided by a gradient (Eluent A: HFIP / TEA / H2O; Eluent B: Methanol). The detection wavelength was 260 nm. Unless otherwise indicated, the reported oligonucleotide purity in percent (%) was obtained by dividing the area under the peak of the target oligonucleotide by the sum of the areas of all meaningful peaks, followed by multiplication by 100% to obtain a percent (%) value. Unless otherwise indicated, the UV trace was integrated applying a minimum relative area threshold of 0.05%. Unless otherwise indicated, purity determinations were performed using support-cleaved oligonucleotides. Unless otherwise indicated, such support-cleaved oligonucleotides were obtained as described in section "3. Synthesis of Oligonucleotides".

[0189] Depurination assay The effect of extended contact times on the depurination of A and G was assessed by comparing a detritylation cocktail with 10% dichloroacetic acid (DCA) in toluene and a cleavage time of 2 x 10 min. Adenosine was chosen because it is the most susceptible to depurination.

[0190] The capping step was omitted in all synthesis cycles, therefore any observed strand breaks are expected to be the result of different detritylation protocols.

[0191] The superiority of the deprotection cocktail is shown by comparison with standard conditions of 10% DCA in toluene and 2 x 10 min cleavage time. For the sequence 5'-TTT TAT TTT T-3' (SEQ ID NO: 1), a 4% increase in purity was observed (see FIG. 3). In the case of the 10% DCA protocol, 1537 Da [MH] - A species of 5'-phosphorylated-TTT TT-3' was detected by LC-MS. This species was assigned to the sequence 5'-phosphorylated-TTT TT-3'. This species is believed to be the result of depurination.

[0192] For the more challenging 20 mere 5'-ATA CCG ATT AAG CGA AGT TT-3' (SEQ ID NO:2), a 14% increase in purity was observed (see FIG. 4).

[0193] 3. Oligonucleotide Synthesis General Protocol (GP) A: Preparation of phosphoramidite solutions Benzoyl-protected adenosine (A Bz ), benzoyl-protected cytidine (C Bz ) [or, where specifically indicated, acetyl-protected cytidine (C Ac )], isobutyryl-protected guanosine (G iBu 5'-DMT protected nucleoside phosphoramidite derivatives of riboflavin (R), thymidine (T), and uridine (U) bearing any 2'-modification (2'-deoxy, 2'-fluoro, or 2'-O-methyl, i.e., 2'-methoxy) were typically dissolved in dry acetonitrile or a combination of dry acetonitrile and N,N-dimethylformamide at a concentration of 0.2 M. Molecular sieves (3 Å) were added and the headspace was flushed with nitrogen. The solutions were then dried for a minimum of 12 h.

[0194] General Protocol (GP) B: Detritylation using amine salts Detritylation cocktails were typically prepared by dissolving a preformed salt composed of a base and a strong acid, or a base and a strong acid, in a mixture of the indicated aprotic solvent, a fluorinated alcohol, and, where appropriate, additional carbocation scavenger. The ratios of these components in the individual detritylation cocktails are shown for each synthetic example compiled in the table below.

[0195] Detritylation cocktails were typically mixed as follows: base (0.75-1.6%, w / v) and strong acid (0.5-1.0%, v / v), or alternatively preformed salt form (5.0%, w / v), were added to a solution of fluorinated alcohol (2-19%, v / v) and, optionally, carbocation scavenger (76-96%, v / v) in dichloromethane (DCM), toluene, or anisole. The following bases were used: 4-cyanopyridine (0.75-1.0%, w / v), 3-cyanopyridine (1.0%, w / v), 3-chloropyridine (1.6%, w / v). The following strong acids were used: trifluoroacetic acid (TFA) (0.5-1.0%, v / v), methanesulfonic acid (0.8%, w / v). The following preformed salts of bases and strong acids were used: 4-chloropyridinium hydrochloride (5.0%, w / v). The fluorinated alcohols 2,2,2-trifluoroethanol (TFE) (5–19%, v / v) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) (2–5%, v / v) were used. The following carbocation scavengers were used: ethanedithiol (EDT) (1.4%, v / v), 1,8-octanedithiol (DODT) (2.7%, v / v), 1,4-dithioerythrol (DTE) (2.3%, w / v), 1,4-benzenedimethanethiol (BDMT) (2.5%, w / v), HO (1%, v / v), and triethylsilane (TES) (1.9%, v / v).

[0196] As an example, a detritylation cocktail described as "1.0:0.7:19:80, 4-cyanopyridine:TFA.TFE:toluene, m:v:v:v" refers to a solution obtained by dissolving 4-cyanopyridine (1.0% (m / v)) in a mixture of TFE (19% (v / v)), TFA (0.7% (v / v)), and toluene (80.3% (v / v)). Unless otherwise indicated, ratios written as "m:v:v:v" refer to the ratio of mass g to volume mL.

[0197] As used herein, the terms "m:v" and "w:v" are used interchangeably, where m and w refer to mass (g) and v refers to volume (mL), unless otherwise indicated. Similarly, the terms "m / v" and "w / v" are used interchangeably, where again, unless otherwise indicated, m and w refer to mass (g) and v refers to volume (mL).

[0198] Prior to detritylation, the resin was typically washed with a solvent, e.g., DCM or toluene. Detritylation was performed using a detritylation cocktail, see above. The protected oligonucleotides on the resin were deprotected 1-4 times or 2-3 times, typically for 5-20 minutes each, and for UnyLinker™-loaded solid supports, typically for 30-70 minutes each. The resin was then typically washed with acetonitrile.

[0199] General Protocol (GP) C: Detritylation using 10% dichloroacetic acid (DCA) in toluene (v / v) Detritylation was typically carried out using commercially available 10% DCA in toluene (v / v) in one step for 1 min, or in two steps for 2×10 min (i.e., two repeats of 10 min each), and the resin was then typically washed with acetonitrile.

[0200] General Protocol (GP)D: Detritylation using 2% DCA and 0.1% ethanol (v / v) in dichloromethane (DCM) Detritylation was typically carried out once with a contact time of 310 seconds, or in two steps (i.e., two repeats) of 10+10, 5+5, 5+10, or 5+20 minutes. The resin was then typically washed with acetonitrile.

[0201] General Protocol (GP) E: Detritylation using 10% DCA in toluene (v / v) Tritylation with 10% DCA (v / v) in toluene was typically carried out in 2-4 steps (i.e., 2-4 iterations). Detritylation times were typically 5-20 min per iteration. For UnyLinker™-loaded solid supports, detritylation times were typically 5+5+20 min per iteration, indicating 3 steps (i.e., iterations) in which the detritylation solution was contacted with the resin for 5 min each in the first and second steps, and 20 min in the third step. The resin was typically washed with acetonitrile after completion of the detritylation procedure.

[0202] Standard protocol for the synthesis of oligonucleotides: Unless otherwise indicated, syntheses were carried out manually in 10 mL syringe reactors (R-1), manually in 1 L stirred bed reactors (R-2), or automatically in 25 mL sparged bed reactors (agitation by bubbling N2 gas into the filter reactor) (R-3) using 3'-phosphoramidite nucleosides according to the desired sequence in the 3' to 5' direction. All reactor types were equipped with filters, frits, or membranes of appropriate pore size to retain the resin in the reactor and to evacuate solvents and reagents.

[0203] The syntheses were typically carried out on 40 μmol, 2.5 mmol, or 0.04-5.0 mmol scales based on the resin weight and substitution (approximately 0.34-0.41 mmol / g or 0.2-0.4 mmol / g). Commercially available DMT-protected polystyrene starting resins (functionalized with either the first nucleoside or UnyLinker™) were typically used. The nucleoside and UnyLinker™ groups of the commercially available starting resins were typically linked to the polystyrene support by succinate esters or amides.

[0204] Prior to the first coupling cycle, the resin was transferred to a reaction vessel and washed with acetonitrile (ACN, 40-50 mL / g) for 10 min. For each added nucleoside phosphoramidite, one coupling cycle according to Table 1 was carried out unless otherwise indicated.

[0205] [Table 2]

[0206] Detritylation was typically performed using the detritylation cocktails described in GP B, 10% (v / v) DCA in toluene in GP C, 2% (v / v) DCA and 0.1% (v / v) ethanol in DCM as shown in GP D, or 10% DCA (v / v) in toluene in GP E.

[0207] Couplings were typically carried out using phosphoramidites in solution prepared with GPA and with the addition of 5-(ethylthio)-1H-tetrazole activator (ETT, 0.5M).

[0208] The oxidation was typically carried out using iodine solution (50 mM) in a mixture of water and pyridine (1:9, v / v) solution. In the examples, when a 25 mL sparged bed reactor (R-3) was used, the oxidation volume per resin mass was 17.7 mL / g.

[0209] Thiolation (i.e., sulfurization) was typically carried out using a 0.2 M xanthan hydride solution in pyridine or a 0.1 M xanthan hydride solution in a mixture of acetonitrile and pyridine (1:1, v / v). In the examples, the thiolation volume per resin mass was 17.7 mL / g when a 25 mL sparged bed reactor (R-3) was used.

[0210] It should be noted that a typical synthesis cycle (i.e., a coupling cycle) involves either an oxidation or a thiolation. In both cases, a phosphorus(III) linking group is converted to a phosphorus(V) linking group.

[0211] Capping was typically performed with a mixture (1:1, v / v) of Cap A (20% acetic anhydride in acetonitrile, v / v) and Cap B (N-methylimidazole, 2,6-lutidine, acetonitrile, 20:30:50, v / v / v). In the examples, the capping volume per resin mass was 8.7 mL / g when a 25 mL sparged bed reactor (R-3) was used.

[0212] Washing steps were typically performed using acetonitrile. Only before detritylation, DCM (e.g., when DCM is used as the solvent for the detritylation cocktail) or toluene (e.g., when toluene or anisole is used as the solvent for the detritylation cocktail) were used for the washing step. In the examples, when a 25 mL sparged bed reactor (R-3) was used, the DCM and toluene washing volume before detritylation was 14 mL / g, and the ACN washing volume per resin mass was 44 mL / g. Activator washes were only performed in some cases. Unless otherwise indicated, an activator wash was performed. This term refers to the washing step using the acetonitrile and activator solution specified for the coupling step (i.e., 0.5 M ETT in acetonitrile).

[0213] Unless otherwise indicated, each step of a typical coupling cycle involved contacting the solid support bearing the growing oligonucleotide chain with the respective reagent solution (e.g., detritylation cocktail for the detritylation step or neat solution for the washing step) for the indicated time under stirring (stirred bed reactor) or shaking (syringe reactor) or agitation by nitrogen bubbling (sparged bed reactor), followed by draining the respective solution through a filter so as to retain the growing oligonucleotide bound to the support inside the reactor. A step of a typical coupling cycle, e.g., the detritylation step, is repeated one or more times. In such a case, the aforementioned procedure is simply repeated, i.e., the reagent solution is drained from the reactor and the oligonucleotide on the resin is treated with another batch of the respective solution, followed by draining the solution. In Examples 1-10, the number of repetitions of each detritylation step and the contact time with the detritylation cocktail per repetition are specifically shown in tabular form. For example, two replicates with 10 minute contact times each would be referred to as 2×10 minutes or 10+10 minutes.

[0214] Unless otherwise indicated, syntheses were carried out at 22°C.

[0215] The synthesis cycle was repeated in sequence using the appropriate nucleoside phosphoramidites.

[0216] Final 5'-DMT detritylation was performed using the same procedure as used in the previous cycle, including the washing steps. The resin was then dried under vacuum for a minimum of 16 hours.

[0217] After the completion of all coupling cycles and the final detritylation step, the resin was weighed and a trial cleavage and deprotection of the sample was performed. For this purpose, typically 10-50 mg of dry resin was weighed into a suitable reaction vessel (e.g., a 2 mL PP tube). Aqueous ammonium hydroxide solution (i.e., aqueous ammonia, 25-28% (w / v), 1-2 mL) was added. The tube was closed, sealed, shaken and heated to 45-55 °C for 2-20 h depending on the length and sequence of the oligonucleotide. Optionally, treatment with diethylamine (DEA) in acetonitrile is performed before the treatment with aqueous ammonia. The supernatant was typically removed using a syringe and transferred to a second tube (2-5 mL). Ammonia was typically removed using a vacuum centrifuge or with a slight stream of nitrogen gas. The solution (containing the oligonucleotide cleaved from the support) was then diluted with HO to the desired concentration and subsequently analyzed via HPLC or HPLC-MS.

[0218] 4. Depurination Assay Assay Method A: The effect of increased (i.e., extended) contact times on depurination of A and G was assessed by comparing individual detritylation cocktails (with GP B) with a standard detritylation cocktail (10% (v / v) dichloroacetic acid (DCA) in toluene, GP C) using 2 x 10 min cleavage times. Adenosine was chosen as the preferred sequence since it is the most amenable to depurination.

[0219] The synthesis and results of a depurination assay using Assay Method A are listed below as Example 5.

[0220] Assay method B: The detritylation protocol GP B was compared with the prior art (WO9603417A1) which describes a detritylation protocol that specifically helps to reduce or eliminate depurination. From this prior art, a detritylation mixture consisting of 2% (v / v) DCA and 0.1% (v / v) ethanol in DCM was selected for comparison (GP D). The 5mer DNA 5'-AAA AT-3' was selected as the target oligonucleotide.

[0221] The depurination by-products were identified by HPLC-MS analysis of the cleaved oligonucleotides as products with masses corresponding to the depurinated abasic site and by-products generated as a result of the decomposition reaction of the depurinated abasic site during standard cleavage and deprotection with 25-28% (w / v) aqueous ammonia (see for example: S. Pourshahian et al., Mass Spectrometry Reviews 2021, 40, 75-109). Since a large number of different depurination products were observed, the UV traces of the HPLC-MS measurements were integrated without applying a minimum relative area threshold. This avoids the exclusion of smaller peaks for depurination-related by-products according to their individual masses.

[0222] The synthesis and results of the depurination assay using assay method B are listed below as Example 7. The chromatograms of these analyses are shown as Figures 6a and 6b. In these chromatograms, the peaks were assigned peak IDs. In Table B.1 below, these peak IDs are assigned to oligonucleotides or fragments based on the mass spectrometry data (providing the calculated and detected monoisotopic masses). This is followed by Table B.2, which shows the putative structures of these oligonucleotides or fragments. The extent of depurination was determined from the UV traces of the individual HPLC-MS measurements by dividing the sum of the peak areas of all identified depurination products by the peak area of ​​the product (i.e., the target oligonucleotide, also referred to as the full-length product), followed by multiplying by 100% to arrive at a percentage (%) value.

[0223] [Table 3]

[0224] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0225] Assay Method C: The detritylation protocols GP B and GP E were applied to the synthesis of oligonucleotides bearing electron-withdrawing substituents at the 2' position (eg, 2'-O-methylribose, 2'-F-deoxyribose).

[0226] The 23-mer oligonucleotide 5'-[mAs][fCs][mA][fA][fA][fA][mG][fC][mA][fA][mA][mA][mC][fA][mG][fG][mU][fC][mU][mA][mGs][mAs][mA]-3', which contains both 2'-F and 2'-OMe nucleosides and a mixed phosphodiester (PO) and phosphorothioate (PS) backbone, was selected as the targeted oligonucleotide, which represents a building block commonly found in oligonucleotide active pharmaceutical ingredients (APIs). The following notation was used: "m" indicates that the ribose moiety of the individual nucleoside subunits has a 2'-O-Me group; "f" indicates that the ribose moiety of the individual nucleoside subunits has a 2'-F (fluoro) substituent; and "s" indicates that the internucleoside linkage group at the 3' position of the individual nucleoside subunits is a phosphorothioate group instead of a phosphodiester group.

[0227] Overall purity was used to evaluate detritylation performance. To specifically determine the degree of depurination, the amount of abasic sites lacking an adenine nucleobase was quantified by HPLC-MS. Due to the stability of the abasic sites of 2'-F / 2'-OMe oligonucleotides during ammonia treatment, the abasic sites were used to directly quantify depurination. Since the abasic by-products co-elute with other by-products, the peaks in question were integrated in the UV trace (HPLC-MS) and the relative amounts of the different abasic by-products were determined by integration of the mass trace after applying charge analysis in Bruker data analysis software using the maximum entropy algorithm.

[0228] The synthesis and results of depurination assays using assay method C are listed below as Example 8. Chromatograms of these analyses are shown as Figures 7a and 7b. In these chromatograms, peaks have been assigned peak IDs. In Table C.1 below, these peak IDs are assigned to oligonucleotides or fragments based on mass spectrometry data (providing calculated and detected monoisotopic masses). This is followed by Table C.2 which shows the putative structures of these oligonucleotides or fragments.

[0229] To determine the degree of depurination, the relative content of C.1-2 in the mass trace was determined for the peak containing C.1-2 and multiplied by the relative area of ​​the respective peak in the UV trace. The degree of depurination was obtained by dividing the thus obtained corrected area of ​​C.1-2 in the UV trace by the area of ​​the product (i.e., target oligonucleotide or full-length product) in the UV trace, followed by multiplication by 100% to obtain a percentage (%) value.

[0230] [Table 5]

[0231] [Table 6]

[0232] Example 1: Comparative Example - Detritylation with 10% DCA (v / v) in toluene.

[0233] [Table 7]

[0234] Synthesis EOP 4148503 / 34, 37, 70, 73 and 4149467 / 01, 21 were carried out according to GPC.

[0235] Two different model sequences were synthesized using the standard detritylation cocktail of the prior art. Two or four syntheses were performed for each model sequence. GPC was used for detritylation with a deprotection time of 1×1 min or 2×10 min. The protocols of both syntheses differ only in terms of the deprotection time, all other synthesis parameters were kept constant. The comparison demonstrates that the standard detritylation cocktail used in the prior art (10% DCA in toluene, v / v) is disadvantageous when longer contact times of the detritylation cocktail with the oligonucleotide occur.

[0236] Example 2: Detritylation using a composition according to the invention

[0237] [Table 8]

[0238] Synthesis EOPs 4148503 / 35, 41, 48 and 4149467 / 02 were carried out using GP B as described above.

[0239] For EOP4148503 / 35 and 4149467 / 02, a detritylation cocktail was prepared by dissolving TFE (19 mL), EDT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (79 mL).

[0240] For EOP 4148503 / 48, a detritylation cocktail was prepared by dissolving TFE (19 mL), EDT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (79 mL).

[0241] For EOP4148503 / 41, a detritylation cocktail was prepared by dissolving TFE (0.23 L), EDT (17 mL), 4-cyanopyridine (12 g, 96 mM), TFA (8.4 mL, 92 mM) in DCM (0.95 L).

[0242] Two different model sequences were synthesized using the detritylation protocol according to the present invention. The synthesis parameters for each sequence were identical to Example 1, except for the fact that the detritylation protocol GP C was replaced by GP B with a detritylation time of 2 x 10 minutes. Comparison with the results of Example 1 (compare EOP 4148503 / 48 vs. EOP 4148503 / 34; EOP 4149467 / 02 vs. EOP 4149467 / 01; EOP 4148503 / 41 vs. EOP 4148503 / 37) demonstrates that the liquid composition C according to the present invention provides improved weight gain and purity compared to the standard detritylation method of the prior art, i.e., 10% (v / v) DCA in toluene with a deprotection time of 1 x 1 minute. This effect is particularly pronounced for the more stringent 20-mer sequence (EOP 4149467 / 02 vs. EOP 4149467 / 01): the sample synthesized according to the invention showed an overall more distinct impurity profile; the overall purity was increased by 11%. The corresponding analytical chromatograms are shown in FIG. 5. These observations suggest that the deprotection method and composition according to the invention allow improved removal of protecting groups while suppressing impurity formation. The disadvantages of the prior art protocols compared to the protocol of the invention are even more evident when longer contact times cannot be avoided (compare EOP 4148503 / 48 vs. EOP 4148503 / 70; EOP 4149467 / 02 vs. EOP 4149467 / 21; EOP 4148503 / 41 vs. EOP 4148503 / 73).

[0243] In EOP 4149467 / 02, the volume of the detritylation solution was 2.5 mL / 100 μmol starting material (i.e., 100 μmol DMT-protected OH groups on the starting resin) per iteration. For a synthesis scale of 40 μmol, the detritylation volume was 1.0 mL. This contains 92 μmol of acid per iteration, which represents 2.3 equivalents to the DMT group and 0.12 equivalents to the nucleobase during the final detritylation step. The detritylation cocktail contains 2.6 mmol of TFE per iteration, which is equivalent to 66 equivalents of TFE to the nucleobase in the first cycle and 3.3 equivalents of TFE to the nucleobase when the 5-mer oligonucleotide is finished.

[0244] Example 3: Detritylation using compositions according to the invention - Use of various carbocation scavengers

[0245] [Table 9-1] [Table 9-2]

[0246] The synthesis was carried out using GP B as described above.

[0247] For EOP4148503 / 53, a detritylation cocktail was prepared by dissolving TFE (19 mL), DODT (2.7 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (77.6 mL).

[0248] For EOP4148503 / 50, a detritylation cocktail was prepared by dissolving TFE (19 mL), DTE (2.3 g), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.70% (v / v), 0.7 mL, 92 mM) in DCM (78 mL).

[0249] For EOP4148503 / 52, a detritylation cocktail was prepared by dissolving TFE (19 mL), BDMT (2.5 g), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (78 mL).

[0250] For EOP4148503 / 55, a detritylation cocktail was prepared by dissolving TFE (19 mL), TES (1.9 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (78 mL).

[0251] For EOP4148503 / 54, a detritylation cocktail was prepared by dissolving TFE (19 mL), water (1.0 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (79 mL).

[0252] For EOP4149467 / 07, a detritylation cocktail was prepared by dissolving TFE (19 mL), water (1.0 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (79 mL).

[0253] For EOP4148503 / 51, a detritylation cocktail was prepared by dissolving TFE (19 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (80 mL).

[0254] For EOP4149467 / 06, a detritylation cocktail was prepared by dissolving TFE (19 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (80 mL).

[0255] This example shows that various types of common carbocation scavengers, such as silanes [e.g., triethylsilane (TES)], thiols or thiophenols [e.g., 3,6-dioxa-1,8-octanedithiol (DODT), 1,4-dithioerythrol (DTE), 1,4-benzenedimethanethiol (BDMT)], and water, can be used, for example.

[0256] Example 4: Detritylation using a composition according to the invention - Use of different fluorinated alcohols

[0257] [Table 10]

[0258] The synthesis was carried out as described above using GP B for detritylation.

[0259] For EOP4148503 / 63, a detritylation cocktail was prepared by dissolving HFIP (5 mL), EDT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (93 mL).

[0260] For EOP4148503 / 66, a detritylation cocktail was prepared by dissolving TFE (5 mL), EDT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (93 mL).

[0261] For EOP4148503 / 67, a detritylation cocktail was prepared by dissolving HFIP (2 mL), EDT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (96 mL).

[0262] This example shows that the concentration of fluorinated alcohol in the detritylation cocktail can be varied, and that fluorinated alcohols other than TFE can be used.

[0263] Example 5: Effect on depurination (depurination assay, assay method A)

[0264] [Table 11]

[0265] GP B vs. GP C used for detritylation: 2 x 10 min for EOP4149994 / 01, EOP4149994 / 02, EOP4149467 / 22 and 3 x 10 min for EOP4149467 / 24. Experiments were performed without capping as described in the general protocol "Depurination assay, assay method A".

[0266] For EOP 4149994 / 01 and 4149467 / 24, a detritylation cocktail was prepared by dissolving TFE (19 mL), DODT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in toluene (77.6 mL).

[0267] The benefit of the detritylation cocktail of the present invention is shown by comparison with standard conditions, i.e., 10% DCA in toluene (v / v) and 2 x 10 min detritylation time. For the sequence 5'-TTT TAT TTT T-3' (SEQ ID NO: 1), a 4% increase in purity was observed (see FIG. 3). In the case of the 10% DCA protocol, 1537 Da [MH] - species was detected by LC-MS. For the more challenging 20-mer DNA 5'-ATA CCG ATT AAG CGA AGT TT-3' (SEQ ID NO: 2), a 14% increase in purity was observed (see FIG. 4).

[0268] Example 6: Synthesis of oligonucleotides with electron-withdrawing substituents at the 2' position (e.g., RNA synthesis, GPB was used for detritylation)

[0269] [Table 12]

[0270] Synthesis EOP 4149469 / 01 and EOP 4149468 / 01 were carried out using GP B as described above.

[0271] A detritylation cocktail was prepared by dissolving TFE (19 mL), EDT (1.4 mL), 4-cyanopyridine (1.0 g, 96 mM), TFA (0.7 mL, 92 mM) in DCM (79 mL).

[0272] Both syntheses show the applicability of GPB to oligonucleotides with 2'-F and 2'-OMe substitutions. Furthermore, EOP 4149469 / 01 demonstrates the applicability to oligonucleotides with mixed phosphodiester (PO) and phosphorothioate (PS) backbones.

[0273] Example 7: Depurination Assay (Assay Method B)

[0274] [Table 13-1] [Table 13-2]

[0275] The syntheses in Example 7 (as compiled in Table E.7.1) were carried out on a 0.10 mmol scale using either GP B, GP D, or a protocol similar to GP B substituting ethanol for the fluorinated alcohol, as shown in section "3. Synthesis of Oligonucleotides," specifically Table 1.

[0276] Synthesis EOP4153156 / 03-1 to -5 was carried out according to GP D using different detritylation times as a reference to the prior art (WO96 / 03417) describing detritylation with reduced depurination. The detritylation mixture was prepared by dissolving DCA (9.0 mL, 0.24 M), ethanol (0.45 mL, 17 mM) in DCM (441 mL). The volume of the detritylation solution was 3.75 mL / 100 μmol starting material per iteration (i.e. 100 μmol DMT-protected OH groups on the starting resin). This volume contained 0.90 mmol of acid per iteration, which represents 9.0 equivalents for the DMT group and 1.8 equivalents for the nucleobase during the last detritylation step.

[0277] Synthesis EOP 4153156 / 04-1 to -5 was carried out according to GP B. The detritylation cocktail was prepared by dissolving TFE (85.5 mL), 4-cyanopyridine (4.50 g, 96 mM), TFA (3.2 mL, 92 mM) in DCM (361 mL). The volume of the detritylation solution was 3.75 mL / 100 μmol starting material per iteration (i.e., 100 μmol DMT-protected OH groups on the starting resin). This volume contained 0.35 mmol of acid per iteration, which represents 3.50 equivalents relative to the DMT group and 0.70 equivalents relative to the nucleobase during the last detritylation step. DCM was chosen as the aprotic solvent to ensure good comparability with the prior art and to exclude solvent effects. General synthesis conditions and detritylation times were comparable to the synthesis carried out using the detritylation cocktail described in EOP 4153156 / 03-1 to -5.

[0278] The shortest detritylation time of 310 seconds was selected in view of the prior art (WO96 / 03417), where 310 seconds is the preferred detritylation time in the flow-through protocol. For a batch reactor setup, this detritylation time results in incomplete detritylation and therefore low purity (EOP 4153156 / 03-1, 35% purity, and EOP 4153156 / 04-1, 51% purity, respectively). The detritylation cocktail of the present invention results in higher purity, probably as a result of faster detritylation. Extended detritylation procedures with two detritylation steps (i.e., two iterations) (10+10 min, 5+5 min, 5+10 min, 5+20 min) also all result in higher purity with the detritylation cocktail of the present invention.

[0279] To demonstrate that the improved purity achieved by the detritylation cocktail of the present invention is not simply a result of higher alcohol concentration, additional syntheses using 19% (v / v) ethanol were performed (synthesis EOP 4153156 / 20-1 to -5). The detritylation cocktail was prepared by dissolving ethanol (86 mL), 4-cyanopyridine (4.5 g, 96 mM), TFA (3.2 mL, 92 mM) in toluene (0.36 L). The volume of the detritylation solution was 3.75 mL / 100 μmol starting material (i.e., 100 μmol DMT-protected OH groups on the starting resin) per detritylation iteration. In all cases, little full-length product was observed, as detritylation was significantly hindered by the presence of 19% (v / v) ethanol. Since DCM and toluene have both been shown to be suitable for detritylation reactions using GP B (see, for example, Examples 2, 4, and 5), the very poor results obtained when replacing TFE with ethanol in EOP 4153156 / 20-1 to -5 are mainly attributable to the change of this alcohol.

[0280] The degree of detritylation was then determined by HPLC-MS (Figures 6.a, 6.b) for all syntheses listed in Table E.7.1 and the results are compiled in Table E.7.2. For this purpose, the peak areas of all identified depurination products (see Tables B.1 and B.2) were summed and divided by the peak area of ​​the product (i.e., the target oligonucleotide, also referred to as the full-length product), and subsequently multiplied by 100% to arrive at a percentage (%) value.

[0281] [Table 14]

[0282] For all detritylation times, the compositions / methods of the present invention gave a lower degree of depurination than the compositions / methods of WO 96 / 03417. Chromatograms of EOP4153156 / 03-5 and EOP4153156 / 04-5 are provided in FIG.

[0283] Example 8: Synthesis of oligonucleotides with electron-withdrawing substituents at the 2' position (depurination assay, assay method C)

[0284] [Table 15]

[0285] Synthesis EOP4150430 / 28-1 was carried out on a 0.1 mmol scale using GP B for detritylation. The detritylation cocktail was prepared by dissolving TFE (0.46 L), 4-cyanopyridine (24 g, 96 mM), TFA (17 mL, 92 mM) in toluene (1.9 L).

[0286] Synthesis EOP4150430-36-1 was carried out on a 0.1 mmol scale using GP E for detritylation. The detritylation cocktail was prepared by dissolving DCA (0.30 L) in toluene (2.7 L).

[0287] As can be concluded from Table E.8, the purity obtained in the synthesis using GP B for detritylation (EOP4150430 / 28-1) is higher and the degree of depurination is lower than with the usual detritylation solution of GP E, 10% DCA (v / v) in toluene (EOP4150430 / 36-1). With 10% DCA (v / v) in toluene, the detritylation is incomplete in some steps, resulting in a large amount of by-products that elute short before the target product (Figures 7a, 7b).

[0288] Example 9: Detritylation using a composition according to the invention - Use of different non-halogenated aprotic solvents

[0289] [Table 16-1] [Table 16-2]

[0290] Synthesis EOP 4153156 / 05-1, 3, 4 and 4153156 / 12-2, 5, as well as EOP 4153156 / 15-1 to -5 were carried out under the same conditions as EOP 4153156 / 04-1 to -5 in Example 7 (see Table E.7.1), except that DCM in the detritylation cocktail was replaced with either toluene or anisole. The volume of the detritylation solution was 3.75 mL / 100 μmol starting material (i.e. 100 μmol DMT-protected OH groups on the starting resin) per detritylation iteration. It contained 0.35 mmol of acid per iteration, and this volume represents 3.50 equivalents to the DMT group and 0.70 equivalents to the nucleobase during the last detritylation step. The detritylation cocktail contained 9.9 mmol of TFE per iteration, which is equivalent to 99 equivalents of TFE per nucleobase in the first cycle and 20 equivalents of TFE per nucleobase when the 5-mer oligonucleotide is completed.

[0291] A detritylation cocktail of synthetic EOPs 4153156 / 05-1, 3, 4 and 4153156 / 12-2, 5 was prepared by dissolving TFE (86 mL), 4-cyanopyridine (4.5 g, 96 mM), TFA (3.2 mL, 92 mM) in toluene (0.36 L). Similarly, a detritylation cocktail of EOPs 4153156 / 15-1 to -5 was prepared by dissolving TFE (86 mL), 4-cyanopyridine (4.5 g, 96 mM), TFA (3.2 mL, 92 mM) in anisole (0.36 L).

[0292] The examples listed in Table E.9 show that both detritylation cocktails containing toluene and detritylation cocktails containing anisole lead to successful synthesis of the 5mer DNA 5'-AAA AT3' following the detritylation procedure GP B. The use of non-halogenated solvents presents great environmental benefits for industrial scale synthesis.

[0293] Example 10: Detritylation using compositions according to the invention - Use of different bases and strong acids

[0294] [Table 17-1] [Table 17-2] [Table 17-3]

[0295] The synthesis EOP 4153156 / 06-1 to -5 was carried out on a 0.1 mmol scale according to GP B. A detritylation cocktail was prepared by dissolving TFE (86 mL), 3-chloropyridine (7.0 g, 0.14 M), TFA (4.5 mL, 0.13 M) in toluene (0.36 L).

[0296] The synthesis EOP 4153156 / 11-1 to -5 was carried out on a 0.1 mmol scale according to GP B. A detritylation cocktail was prepared by dissolving TFE (86 mL), 3-cyanopyridine (4.5 g, 96 mM), TFA (3.2 mL, 92 mM) in toluene (0.36 L).

[0297] The synthesis EOP4153156 / 13-1 to -5 was carried out on a 0.1 mmol scale according to GP B. A detritylation cocktail was prepared by dissolving TFE (86 mL), 4-cyanopyridine (4.5 g, 96 mM), methanesulfonic acid (MSA) (2.7 mL, 92 mM) in toluene (0.36 L).

[0298] The synthesis EOP4153156 / 14-1 to -3 was carried out on a 0.1 mmol scale according to GP B. A detritylation cocktail was prepared by dissolving TFE (49 mL), 4-chloropyridine-HCl (13 g, 0.33 M) in toluene (0.20 L).

[0299] The 5-mer DNA 5'-AAA AT-3' was successfully synthesized with all detritylation cocktails described in this example, showing that the method of the present invention allows good variability of bases and strong acids. The pKa values ​​of the protonated forms of the pyridine bases used (i.e., pyridinium ions / cations of the salts) can be taken from the literature (Fischer, A., Galloway, WJ, Vaughan, J., Journal of the Chemical Society (Abstract) 1964, pp. 3591-3596): 3-cyanopyridine (protonated form: pKa=1.35), 4-cyanopyridine (protonated form: pKa=1.86), 3-chloropyridine (protonated form: pKa=2.81), 4-chloropyridine (protonated form: pKa=3.83). Thus, the pKa values ​​of the pyridinium ions cover a range of pKa values ​​from 1.35 to 3.83. Similarly, the strong acids used cover a broad range of acid classes: TFA is a carboxylic acid, MSA is a sulfonic acid, and HCl (used in the form of a preformed HCl salt) is a mineral acid.

Claims

1. A method for the synthesis of oligonucleotides comprising the steps of: a) providing a nucleoside or oligonucleotide bound to a support moiety, the nucleoside or oligonucleotide comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group; b) cleaving the di(p-methoxyphenyl)phenylmethyl protecting group from the nucleoside or oligonucleotide by incubating said compound with liquid composition C, thereby generating a free backbone hydroxyl moiety; c) reacting the free backbone hydroxyl group resulting from step b) with a phosphorus moiety of a nucleoside or oligonucleotide building block, the building block further comprising a hydroxyl group protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby producing a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus atom of said building block; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b)-c) or b)-d); and f) cleaving the oligonucleotide from the support moiety Including, Liquid composition C comprises an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, and a salt of a base and a strong acid, the cation of the salt having a pKa in the range of 1 to 4, and the strong acid having a pKa of less than 1; method.

2. the phosphorus moiety of the nucleoside or oligonucleotide building block used in step c) is a phosphorus(III) moiety; 2. The method according to claim 1, wherein the method comprises a further step d) of oxidizing or sulfurizing the phosphorus(III) atom to a phosphorus(V) atom, thereby creating an internucleoside linkage of the desired type.

3. step a) comprises reacting a compound according to formula I 【Chemistry 1】 [In the formula, R １ is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y, independently for each repeat unit n, is selected from the group consisting of S and O; Z, independently for each repeat unit n, is selected from the group consisting of O and S; R ２ are the same or different protecting groups for each repeat unit n; Each of the nucleosides x0 to xn is the same or different; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting portion. providing a step c) comprises reacting the free hydroxyl group resulting from step b) with a nucleoside or oligonucleotide phosphoramidite building block comprising a backbone hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent linkage between the oxygen atom of said free hydroxyl group and the phosphorus(III) atom of said building block, The method of claim 1.

4. The nucleoside or oligonucleotide building block is a compound according to formula II 【Chemistry 2】 [In the formula, R １ is a di(p-methoxyphenyl)phenylmethyl protecting group; m is an integer equal to or greater than 0; Y, independently for each repeat unit m, is selected from the group consisting of S and O; Z is independently at each occurrence selected from the group consisting of O and S; R ２ are the same or different protecting groups for each position; R ３ and R ４ are the same or different protecting groups; Each of the nucleosides xm0 to xm is the same or different. That is, The method of claim 1.

5. step a) comprises reacting a compound according to formula III 【Chemistry 3】 [In the formula, R １ is a di(p-methoxyphenyl)phenylmethyl protecting group; n is an integer equal to or greater than 0; Y is O; Z is H; Each of the nucleosides x0 to xn is the same or different; CA is a capping moiety or a single bond; L is a linker moiety or a single bond; SM is the supporting portion. providing a step c) comprises reacting the free hydroxyl group resulting from step b) with an H-phosphonate building block comprising a main chain hydroxyl moiety protected by a di(p-methoxyphenyl)phenylmethyl protecting group, thereby generating a covalent link between the oxygen atom of said free hydroxyl group and the phosphorus(III) atom of said building block; step e) optionally comprises the step of constructing the desired oligonucleotide sequence by repeating the order of steps b) and c) before carrying out step d); The method of claim 1.

6. 2. The method of claim 1, further comprising a step g) after step c or after step d) of blocking unreacted free hydroxyl groups.

7. 10. The method of claim 1, wherein at least step b) is carried out in a batch reactor.

8. 2. The process of claim 1, wherein the process is carried out in a column reactor and the flow rate of liquid composition C through the column reactor is below 300 cm / hr in at least one repetition of step b).

9. The method of claim 1, wherein the synthesis is carried out on a scale of 100 mmol or more of oligonucleotide product.

10. The method comprises the following step h): h) isolating the oligonucleotide cleaved from the support The method of claim 1 further comprising:

11. 2. The method according to claim 1, wherein the aprotic solvent contained in composition C is selected from the group consisting of halogenated hydrocarbon solvents, (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers.

12. The method of claim 1 , wherein the aprotic solvent contained in composition C is non-halogenated.

13. 1. A liquid composition C for use in cleaving a di(p-methoxyphenyl)phenylmethyl protecting group from a hydroxyl group, the composition comprising an aprotic solvent, at least one alcohol selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, pentafluoropropanol, 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, and nonafluoro-tertiary butyl alcohol, a base, and a salt of a strong acid, wherein the cation of the salt has a pKa in the range of 1 to 4, and the strong acid has a pKa of less than 1, the aprotic solvent is non-halogenated and is selected from the group consisting of (hetero)aromatic solvents, alkyl(hetero)aromatic solvents, (hetero)aromatic ethers, and alkyl(hetero)aryl ethers.

14. The method of claim 1 , wherein the base contained in composition C is a cyclic amine.

15. The base contained in the composition C is one or more selected from the group consisting of a halogen atom, a cyano group, an aldehyde group, a keto group, a carboxy ester group, and a carboxamide group.

2. The method of claim 1, wherein said pyridine is substituted with the above electron-withdrawing substituent.

16. 2. The method of claim 1, wherein the base is selected from the group consisting of 4-cyanopyridine, 3-cyanopyridine, 4-chloropyridine, 3-chloropyridine, and mixtures thereof.

17. 2. The method of claim 1, wherein the strong acid contained in composition C is selected from the group consisting of carboxylic acids, mineral acids, sulfonic acids, and mixtures thereof.

18. 2. The method of claim 1, wherein the strong acid contained in composition C is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfonic acid, and mixtures thereof.

19. 2. The method of claim 1, wherein the aprotic solvent contained in composition C is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, mesitylene, anisole, and mixtures thereof.

20. 2. The method of claim 1, wherein the at least one alcohol contained in liquid composition C is selected from the group consisting of trifluoroethanol, hexafluoroisopropanol, and mixtures thereof.

21. the total volume of the aprotic solvents contained in liquid composition C accounts for 60 to 99% of the total volume of liquid composition C; the total volume of the alcohol contained in the liquid composition C occupies from 1 to 40% of the total volume of the liquid composition C; The method of claim 1.

22. 2. The method of claim 1, wherein the molar concentration of the alcohol in Composition C is at least twice as high as the molar concentration of the base contained in Composition C.

23. In the liquid composition C, the total molar amount of said strong acids is in the range of 0.80 to 15.0 equivalents relative to the total molar amount of di(p-methoxyphenyl)phenylmethyl groups; the total molar amount of the alcohol is in the range of 2.0 to 100.0 equivalents relative to the total molar amount of the nucleobase; The method of claim 1.

24. The base contained in composition C is a cyclic amine selected from the group consisting of pyridine, pyrimidine, pyrazine, thiazole, pyridazine, pyrazole, and triazole; The method of claim 1.

25. the phosphorus moiety of the nucleoside or oligonucleotide building block used in step c) is a phosphorus(III) moiety; the method comprises a further step d) of oxidizing or sulfurizing the phosphorus(III) atom to a phosphorus(V) atom, thereby creating an internucleoside linkage of the desired type; step e) comprises either repeating the sequence of steps b) to d) or repeating the sequence of steps b) and c) before carrying out step d); The method of claim 1.

26. Step e) comprises repeating steps b) to d) in sequence; The method according to claim 3.

Images