LNA-G method

Abstract

Recent advances in LNA oligonucleotides include the use of amine linkers to attach LNA antisense oligonucleotides to conjugation groups. See, for example, WO2014 / 118267. This invention stems from the identification of a problem when deprotecting LNA oligonucleotides containing aliphatic amine groups and DMF-protected LNA G nucleotides, resulting in a +28 Da impurity. This problem was solved by using an acyl protecting group on the exocyclic nitrogen of the LNA-G residue, instead of the standard DMF protecting group.

Description

[0001] This application is a divisional application of PCT application PCT / EP2016 / 069765, filed on August 22, 2016, entitled "LNA-G Method". The date of entry into the national phase of the PCT application in China was February 24, 2018, and the application number was 201680049158.5. Invention Field

[0002] This invention relates to the field of LNA antisense oligonucleotide conjugates and their synthesis methods. Background of the Invention

[0003] Recent advances in LNA oligonucleotides include the use of amine linkers to attach LNA antisense oligonucleotides to conjugation groups. See, for example, WO2014 / 118267. This invention stems from the identification of a problem when deprotecting LNA oligonucleotides containing aliphatic amine groups and DMF-protected LNA G nucleotides, resulting in a +28 Da impurity. This problem was solved by using an acyl protecting group on the exocyclic nitrogen of the LNA-G residue, instead of the standard DMF protecting group. Summary of the Invention

[0004] This invention provides a method for preparing LNA oligonucleotides, the method comprising the following steps: a) Introducing at least one LNA-G monomer containing an exocyclic nitrogen protected by an acyl group into an oligonucleotide; b) Introduce at least one optionally protected aliphatic amine group into the oligonucleotide; c) Deprotecting the exocyclic nitrogen of the at least one LNA-G monomer by removing the acyl protecting group; Steps a) and b) can occur in any order.

[0005] Suitably, when other G monomers such as DNA G-monomers are present to be introduced into the LNA oligonucleotide, they may also be acyl-protected on their exocyclic nitrogen, for example by introducing G-monomers such as DNA G-monomers or 2'-substituted G-monomers (e.g., 2'-O-methoxyethyl G-monomers or 2'-methyl G-monomers) into the oligonucleotide, wherein the G monomers contain an exocyclic nitrogen protected by an acyl group, for example by employing an acyl protecting group as described herein, such as the R group as defined in Formula I.

[0006] This invention provides a method for preparing LNA oligonucleotides that are essentially free of +28 adducts, the method comprising the following steps: a) Introducing at least one LNA-G monomer containing an exocyclic nitrogen protected by an acyl group into an oligonucleotide; b) Introduce at least one optionally protected aliphatic amine group into the oligonucleotide; c) Deprotecting the exocyclic nitrogen of the at least one LNA-G monomer by removing the acyl protecting group; Steps a) and b) can occur in any order.

[0007] This invention provides a method for preparing LNA oligonucleotides, the method comprising the following steps: a) Introducing at least one G monomer containing an exocyclic nitrogen protected by an acyl group into an oligonucleotide; b) Introduce at least one optionally protected aliphatic amine group into the oligonucleotide; c) Deprotecting the exocyclic nitrogen of the at least one G monomer by removing the acyl protecting group; Steps a) and b) can occur in any order.

[0008] The present invention provides an LNA oligonucleotide comprising at least one LNA-G monomer containing an exocyclic nitrogen protected by an acyl group and at least one optionally protected aliphatic amine group, wherein the LNA oligonucleotide is linked to a solid support.

[0009] The present invention provides an LNA oligonucleotide comprising at least one G monomer containing an exocyclic nitrogen protected by an acyl group and at least one optionally protected aliphatic amine group, wherein the LNA oligonucleotide is linked to a solid support.

[0010] The present invention provides an LNA oligonucleotide comprising at least one LNA-G monomer and at least one optionally protected aliphatic amine group, wherein the LNA oligonucleotide is substantially free of +28 adducts.

[0011] The present invention provides an LNA oligonucleotide comprising at least one G monomer and at least one optionally protected aliphatic amine group, wherein the LNA oligonucleotide is substantially free of +28 adducts.

[0012] The present invention provides a pharmaceutical composition comprising an LNA oligomer conjugate and a pharmaceutically acceptable diluent, carrier or adjuvant, wherein the LNA oligomer conjugate comprises an LNA-G monomer and an aliphatic amine linker located between the 5' nucleotide of the LNA oligomer and the conjugate moiety, wherein the composition is substantially free of +28 adducts.

[0013] The present invention provides a pharmaceutical composition comprising an LNA oligomer conjugate and a pharmaceutically acceptable diluent, carrier or adjuvant, wherein the LNA oligomer conjugate comprises a G monomer and an aliphatic amine linker located between the 5' nucleotide of the LNA oligomer and the conjugate moiety, wherein the composition is substantially free of +28 adducts.

[0014] This invention provides the use of LNA-G monomers containing acyl-protected exocyclic nitrogen in the synthesis of LNA oligonucleotides containing aliphatic amines.

[0015] This invention provides the use of LNA-G monomers containing acyl-protected exocyclic nitrogen in the synthesis of LNA oligonucleotide conjugates containing aliphatic amines.

[0016] In some implementations, the LNA-G monomer is a monomer of formula I:

[0017] R can be selected from optionally substituted alkyl-, alkenyl-, alkynyl-, cycloalkyl-, or aryl- groups, and is preferably selected from optionally substituted C groups. 1-6 -alkyl-, C 2-6 -Alkenyl-, C 2-6 -alkynyl-, C 3-7 -cycloalkyl- or phenyl- groups.

[0018] If substituted, the R group can be mono- or poly-substituted, for example, substituted by one or more substituents selected from: halogens, C 1-6 -alkyl, C 2-6 -Alkenyl, C 2-6 -Alynyl group, C 1-6 -alkoxy, optionally substituted aryloxy, or optionally substituted aryl. Aryl groups include phenyl groups, and the optional substituents for aryl groups are as described above.

[0019] This disclosure also discloses the following specific implementation plans 1-26.

[0020] 1. A method for preparing LNA oligonucleotides, comprising the following steps: a) Introducing at least one LNA-G monomer containing an exocyclic nitrogen protected by an acyl group into an oligonucleotide; b) Introduce at least one optionally protected aliphatic amine group into the oligonucleotide; c) Deprotecting the exocyclic nitrogen of the at least one LNA-G monomer by removing the acyl protecting group; Steps a) and b) can occur in any order.

[0021] 2. The method according to embodiment 1, wherein the optionally protected aliphatic amine group is a primary amine or a secondary amine.

[0022] 3. The method according to either embodiment 1 or 2, wherein the optionally protected aliphatic amine group is a non-nucleoside amine group.

[0023] 4. The method according to any one of embodiments 1-3, wherein the optional aliphatic amine group is selected from aminoalkyl, alkylaminoalkyl, piperidine, piperazine, pyrrolidine and imidazole.

[0024] 5. The method according to any one of embodiments 1-3, wherein the optional aliphatic amine group is selected from 5'-TFA-amino-modified group-C5-CE phosphoramidamide, 5'-TFA-amino-modified group-C6-CE phosphoramidamide, 11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidamide, 5'-TFA-amino-modified group-C12-CE phosphoramidamide, amino-modified group-C2-dT-CE phosphoramidamide, amino-modified group-C6-dA-CE phosphoramidamide, amino-modified group-C6-dA-CE phosphoramidamide, amino-modified group-C6-dT-CE phosphoramidamide, N2-amino-modified group-C6 dG, Fmoc amino-modified group-C6 dT, 3'-amino-modified group-C7 CPG 1000, 3'-amino-modified group-C6-dC CPG, 3'-amino-modified C6-dC CPG, 3'-PT-amino-modified C6 CPG, 3'-amino-modified C6-dT CPG, PC, 5'-amino-modified CE phosphoramide, 5'-amino-modified C6-PDA, 5'-amino-modified C12-PDA, 5'-amino-modified TEG PDA, amino-modified serine alcohol, and 3'-amino-modified serine alcohol CPG.

[0025] 6. The method according to any one of embodiments 1-3, wherein the optionally protected aliphatic amine group is an aminohexyl linker.

[0026] 7. The method according to any one of embodiments 1-6, wherein the aliphatic amine group is introduced into the oligonucleotide via an amino-modified monomer.

[0027] 8. The method according to embodiment 7, wherein the monomer modified with aliphatic amino group is phosphorous amide, H-phosphonate or triphosphate monomer.

[0028] 9. The method according to embodiment 7, wherein the amino-modified monomer is phosphorous amide.

[0029] 10. The method according to any one of embodiments 1-9, wherein the acyl protecting group on the exocyclic nitrogen of the LNA-G monomer is composed of or includes groups selected from the group consisting of: optionally substituted alkyl-, alkenyl-, alkynyl-, cycloalkyl-, or aryl- groups, preferably selected from optionally substituted C- groups. 1-6 -alkyl-, C 2-6 -Alkenyl-, C2-6 -alkynyl-, C 3-7 - Cycloalkyl- or phenyl- group; wherein when substituted, the R group can be mono- or poly-substituted, for example, substituted by one or more substituents selected from: halogen, C 1-6 -alkyl, C 2-6 -Alkenyl, C 2-6 -Alynyl group, C 1-6 -alkoxy, optionally substituted aryloxy, or optionally substituted aryl.

[0030] 11. The method according to any one of embodiments 1-10, wherein the acyl protecting group on the outer nitrogen of the LNA-G monomer is selected from isobutyryl (iBu), acetyl (Ac), phenoxyacetyl (PAC), p-isopropylphenoxyacetyl (iPrPAC), phenylacetyl, isopropyloxyacetyl, methoxyacetyl, benzoyl, p-methoxyphenylacetyl, diphenylacetyl, cyclohexylcarbonyl, 1,1-dimethylpropionyl and p-tert-butylphenoxyacetyl.

[0031] 12. The method according to any one of embodiments 1-10, wherein the acyl protecting group on the outer nitrogen of the LNA-G monomer is selected from isobutyryl (iBu), acetyl (Ac), phenoxyacetyl (PAC) and p-isopropylphenoxyacetyl (iPrPAC).

[0032] 13. The method according to any one of embodiments 1-12, wherein the other G residues introduced into the oligonucleotide, if present, also include an acyl protecting group, such as the acyl protecting group of any one of embodiments 10-12.

[0033] 14. The method according to any one of embodiments 1-13, wherein the LNA-G monomer and, when present, the optional G monomer are phosphorous amide, H-phosphonate or phosphate triester monomers.

[0034] 15. The method according to any one of embodiments 1-14, wherein the LNA-G monomer and, if present, optionally the G monomer, are phosphorous amides.

[0035] 16. The method according to any one of embodiments 1-15, wherein the LNA-G monomer includes a 2'-O-CH2-4' bigroup in the furanose ring.

[0036] 17. The method according to any one of embodiments 1-16, wherein step c) further includes the deprotection of the primary amine group.

[0037] 18. The method according to any one of embodiments 1-17, wherein step c) comprises the deprotection of oligonucleotides, carried out in the presence of ammonia, for example using a solution containing ammonium hydroxide.

[0038] 19. The method according to any one of embodiments 1-18, wherein step (c) is followed by a further step (d), said further step (d) comprising introducing a conjugate group onto an aliphatic primary amine group.

[0039] 20. The method according to embodiment 19, wherein the conjugation group is a nonnucleotide portion selected from lipids, sterols, carbohydrates, peptides and proteins.

[0040] 21. The method according to any one of embodiments 1-20, wherein at least steps a)-c) are performed on a solid support, followed by cleavage of the oligonucleotide from the solid support, the cleavage being performed during or after step c).

[0041] 22. The method according to any one of embodiments 1-21, wherein the acyl protecting group is isobutyryl and the aliphatic primary amine group is an aminohexyl linker.

[0042] 23. An LNA oligonucleotide comprising at least one LNA-G monomer containing an exocyclic nitrogen protected by an acyl group and at least one optionally protected aliphatic amine group, wherein the LNA oligonucleotide is linked to a solid support.

[0043] 24. A pharmaceutical composition comprising an LNA oligomer conjugate and a pharmaceutically acceptable diluent, carrier, or adjuvant, said LNA oligomer conjugate comprising an LNA-G monomer and an aliphatic amine linker located between the 5' nucleotide of the LNA oligomer and the conjugate moiety, wherein said composition is substantially free of +28 adducts.

[0044] 25. Use of LNA-G monomers containing acyl-protected exocyclic nitrogen in the synthesis of LNA oligonucleotides containing aliphatic amines.

[0045] 26. Use of LNA-G monomers containing acyl-protected exocyclic nitrogen in the synthesis of LNA oligonucleotide conjugates containing aliphatic amines. Attached Figure Description

[0046] Figure 1 Examples of commercially available amino linkages containing aliphatic amine groups.

[0047] Figure 2 Examples of GalNAc conjugates.

[0048] Figure 3 Mass spectra of FL and FL+28 (showing 5-fold charged ions).

[0049] Figure 4MS / MS (MS2MS3 fragmentation mode) of b4 ions (except for the mass range of 500–1500 Da). Indicates a peak with a mass difference of +28 Da and the corresponding peak in the unmodified molecule.

[0050] Figure 5 Structures of the b4-fraction and the a2-base (a2-B) and b4-cytosine (b4-C) fragments obtained from the MS3 experiment.

[0051] Figure 6 Formamide was formed at one location. Detailed description

[0052] This invention provides a method for preparing oligonucleotides comprising at least one LNA-G nucleoside and an aliphatic amine group. The inventors have discovered that when DMF-protected LNA-G is deprotected in the presence of an aliphatic amine, a +28 Da adduct (referred to herein as the +28 adduct) is formed. The formation of the +28 adduct can be avoided by using an acyl protecting group. The presence of the +28 adduct can be identified by mass spectrometry using contaminants in oligonucleotides with a molecular weight of +28. As explained in the examples, the +28 adduct can be determined by mass spectrometry and can have a molecular weight of approximately +28, for example, a molecular weight of +27.5 to +28.5 (Da), or for example, +27.9 to +28.1 (Da). This impurity is difficult to separate from the desired oligonucleotide product, requiring additional downstream purification steps, thus increasing production costs and significantly reducing oligonucleotide yield. Therefore, it is highly desirable to avoid the formation of +28 adducts. The inventors have discovered that this can be achieved by using a G-protecting group that is not DMF, particularly an acyl or carbamate protecting group.

[0053] LNA oligonucleotides are oligonucleotides containing at least one LNA nucleoside. Therefore, this invention relates to a method for preparing LNA antisense oligonucleotides, said LNA antisense oligonucleotides containing at least one LNA-G monomer and at least one aliphatic amine group. LNA oligonucleotides can be antisense oligonucleotides. As used herein, the term oligonucleotide is defined as a molecule containing two or more covalently linked nucleosides, as is generally understood by those skilled in the art. For use as antisense oligonucleotides, oligonucleotides are typically synthesized to a length of 7-30 nucleotides.

[0054] As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide capable of regulating the expression of a target gene by hybridization with a target nucleic acid, particularly with adjacent sequences on the target nucleic acid. Antisense oligonucleotides can also be defined by their complementarity with the target nucleic acid. Antisense oligonucleotides are single-stranded. Antisense oligonucleotides are not inherently double-stranded and are therefore not siRNAs. Antisense oligonucleotides contain adjacent nucleotides complementary to the target nucleic acid. Antisense oligonucleotides typically contain one or more modified internucleotide links, which, by way of non-limiting example, can be in the form of LNA gapmers or mixed-wing gapmers. In other embodiments, the oligonucleotide can be LNA mixmers (LNA and non-LNA nucleotides, such as LNA and DNA (see, for example, WO2007 / 112754, incorporated herein by reference) or LNA and 2'-O-MOE nucleotides or LNA, DNA, and 2'-O-MOE nucleotides) or LNA totalmers (LNA nucleotides only—see, for example, WO2009 / 043353, incorporated herein by reference).

[0055] The term "modified internucleotide link" is defined as a link other than a phosphodiester (PO) link that covalently couples two nucleosides together, as commonly understood by those skilled in the art. Modified internucleotide links can be used particularly to stabilize oligonucleotides for in vivo application and to protect against nuclease cleavage. Phosphothioester internucleotide links are particularly useful due to nuclease resistance, beneficial pharmacokinetics, and ease of production. In some embodiments, at least 70%, for example, at least 80%, or for example, at least 90% of the internucleotide links in the oligonucleotide or its adjacent nucleotide sequence are phosphothioesters. In some embodiments, all internucleotide links in the oligonucleotide or its adjacent nucleotide sequence are phosphothioesters. Other internucleotide linkers are disclosed in WO2009 / 124238 (incorporated herein by reference).

[0056] The term "substantially free" is defined as an oligonucleotide composition prepared by the method of the present invention containing less than 5%, for example less than 1%, for example less than 0.5%, for example less than 0.1%, of the +28 adduct level. Therefore, an oligonucleotide "substantially free" of +28 adducts may contain small amounts of +28 adducts, and in some embodiments, the level of +28 adducts may be below the level detectable by mass spectrometry. The term "substantially free" includes embodiments in which the oligonucleotide product does not contain +28 adducts.

[0057] The term nucleobase includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of this invention, the term nucleobase also encompasses modified nucleobases, which may differ from naturally occurring nucleobases but are useful during nucleic acid hybridization. In some embodiments, the nucleobase moieties are modified by modifying or replacing nucleobases. Hereinafter, “nucleobase” refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Supplement 37 1.4.1.

[0058] Nucleotides are building blocks of oligonucleotides and polynucleotides, and for the purposes of this invention, they include naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, include a ribose moiety, a nucleobase moiety, and one or more phosphate ester groups (which are absent in nucleosides). Modified nucleosides and nucleotides are modified compared to equivalent DNA or RNA nucleosides / nucleotides by introducing modifications into the ribose moiety, nucleobase moiety, or, when the nucleotide is modified, introducing inter-nucleoside linkages. Nucleosides and nucleotides may also be interchangeably referred to as "units" or "monomers".

[0059] As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified by introducing one or more sugar or (nucleo)base moieties compared to an equivalent DNA or RNA nucleoside. The term "modified nucleoside" may also be used interchangeably herein with the terms "nucleoside analogue," "modified unit," or "modified monomer." Examples of modified nucleosides are described in the separate section "Oligomer Modification" and its subsections.

[0060] Exocyclic nitrogen protected by acyl group

[0061] The following explanation describes the exocyclic nitrogen group (surrounding) of guanine. This group is protected by an acyl group during steps a) and b) of the method of the present invention, and is removed during the deprotection step c).

[0062]

[0063] Aliphatic amine groups

[0064] Aliphatic amines are amines in which there is no aromatic ring directly on the nitrogen atom, and are therefore typically non-nucleoside amine groups. Nucleoside amine groups are amine groups in which the nitrogen atom of the amine is directly attached to an aromatic ring of a purine or pyrimidine base. Aliphatic amine groups can be primary or secondary amines. In some embodiments, the aliphatic amine group is selected from aminoalkyl, alkylaminoalkyl, piperidine, piperazine, pyrrolidine, and imidazole. In some embodiments, the aliphatic amine group is selected from 5'-TFA-amino-modified-C5-CE phosphoramidamide, 5'-TFA-amino-modified-C6-CE phosphoramidamide, 11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidamide, 5'-TFA-amino-modified-C12-CE phosphoramidamide, amino-modified-C2-dT-CE phosphoramidamide, amino-modified-C6-dA-CE phosphoramidamide, amino-modified-C6-dA-CE phosphoramidamide, amino-modified-C6-dT-CE phosphoramidamide, N2-amino-modified-C6-dG, Fmoc-amino-modified-C6-dT, 3'-amino-modified-C7 CPG 1000, 3'-amino-modified-C6-dCCPG, 3'-amino-modified-C6-dC CPG, 3'-PT-amino-modified C6 CPG, 3'-amino-modified C6-dTCPG, PC, 5'-amino-modified CE phosphoramide, 5'-amino-modified C6-PDA, 5'-amino-modified C12-PDA, 5'-amino-modified TEG PDA, amino-modified serine alcohol, 3'-amino-modified serine alcohol CPG.

[0065] In some embodiments, the aliphatic amine group is in the form of an amino linker (i.e., the amino linker includes an aliphatic amine group). Examples of commercially available amino linkers are shown in [the following text is missing from the original]. Figure 1 In some embodiments, the amino linker is an aminoalkyl linker, such as C... 2-12 Aminoalkyl linkers, such as aminohexyl linkers.

[0066] In some embodiments, the aliphatic amino group is protected, for example by a protecting group selected from: trifluoroacetyl (TFA), trichloroacetyl (TCA), monomethoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), fluorenemethoxycarbonyl (Fmoc), phthalimide, or 2-(methylaminocarbonyl)-benzoate. In some embodiments, the amine protecting group, if present, is removed before or during deprotection step c).

[0067] It is acknowledged that some aliphatic amine protecting groups may remain after step c), thus providing a method for avoiding +28 adducts. Therefore, this invention provides a method for preparing LNA oligonucleotides, comprising the following steps: a) Introduce at least one protected exocyclic nitrogen LNA-G monomer into an oligonucleotide; b) Introduce at least one protected aliphatic amine group into the oligonucleotide; c) Deprotecting oligonucleotides containing at least one exocyclic nitrogen LNA-G monomer by removing the exocyclic nitrogen protecting group; d) After step c), deprotect the aliphatic amine groups.

[0068] Deprotection step c) may include exposing the oligonucleotide to ammonium hydroxide, and suitably, the aliphatic amine protecting group is not cleaved under the deprotection conditions of step c) (i.e., treatment with ammonium hydroxide). In the above method, the exocyclic nitrogen protecting group may be an acyl group or other protecting groups such as dimethylformamide (DMF). The aliphatic amine protecting group may be, for example, selected from TFA, monomethoxytriphenylmethyl (MMT), DMT, Fmoc, phthalimide, or 2-(methylaminocarbonyl)-benzoate.

[0069] In some embodiments, an aliphatic amine group, such as an amino linker, is attached to a solid support for oligonucleotide synthesis. Therefore, the cleavage of the oligonucleotide from the solid support (which may occur during step c) will result in the cleavage of the aliphatic amine group from the solid support and thus the release of the oligonucleotide from the solid support. In some embodiments, the aliphatic amine group is introduced into the oligonucleotide via a monomer with an amino-modified group. In some embodiments, the amino-modified monomer is a phosphorus amide, H-phosphonate, or phosphate triester monomer. In some embodiments, the amino-modified monomer is a phosphorus amide. Examples of such amino-modified monomers are shown in... Figure 1 middle.

[0070] Aliphatic amine groups can be introduced into oligonucleotides via any suitable oligonucleotide synthesis method, such as H-phosphonate synthesis, phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, or phosphorus amide oligonucleotide synthesis. In some embodiments, the aliphatic amine group is introduced into the oligonucleotide as a phosphorus amide, H-phosphonate, or phosphotriester. In some embodiments, the aliphatic amine group is introduced into the oligonucleotide during phosphorus amide oligonucleotide synthesis.

[0071] Acyl protecting group

[0072] Using an acyl protecting group on the exocyclic nitrogen of the G residue allows for the avoidance of +28 adducts in methods for synthesizing oligonucleotides containing aliphatic amines.

[0073] Some non-limiting examples of suitable acyl protecting groups on the nitrogen atom of the LNA-G monomer may be selected from isobutyryl (iBu), acetyl (Ac), phenoxyacetyl (PAC), p-isopropylphenoxyacetyl (iPrPAC), phenylacetyl, isopropyloxyacetyl, methoxyacetyl, benzoyl, p-methoxyphenylacetyl, diphenylacetyl, cyclohexylcarbonyl, 1,1-dimethylpropionyl and p-tert-butylphenoxyacetyl.

[0074] In some embodiments, the acyl protecting group on the exocyclic nitrogen of the LNA-G monomer is selected from isobutyryl (iBu), acetyl (Ac), phenoxyacetyl (PAC), and p-isopropylphenoxyacetyl (iPrPAC).

[0075] In an alternative embodiment, the acyl protecting group can be replaced by a carbamate protecting group.

[0076] LNA G monomer

[0077] The term LNA-G refers to a nucleoside containing a 2'-4' bigroup in the furanose ring and guanine nucleobase. LNA-G monomers can be introduced into oligonucleotides via any suitable oligonucleotide synthesis method, such as H-phosphonate synthesis, phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, or phosphoramidite oligonucleotide synthesis. In some embodiments, LNA-G monomers are introduced into oligonucleotides as phosphoramidite, H-phosphonate, or phosphotriester. In some embodiments, LNA-G monomers are introduced into oligonucleotides during phosphoramidite oligonucleotide synthesis.

[0078] In some implementations, the LNA oligonucleotide contains at least one G monomer, such as at least two G monomers, such as at least three G monomers, such as at least four G monomers.

[0079] Oligonucleotide synthesis (step a or step a) and b))

[0080] The preparation of oligonucleotides can be carried out using any suitable oligonucleotide synthesis method, such as H-phosphonate synthesis, phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, or phosphoramide oligonucleotide synthesis. The LNA-G monomer and optional aliphatic amine group (optionally protected) can be in a form that allows the introduction of oligonucleotides (e.g., as phosphoramide) during this standard oligonucleotide method.

[0081] Deprotection step c)

[0082] Step c) of the method of the present invention includes removing the acyl protecting group from the exocyclic nitrogen of the LNA-G monomer introduced into the oligonucleotide. Deprotection may further include removing other base protecting groups and optionally removing aliphatic amine protecting groups (when present). During the solid-phase synthesis of the oligonucleotide, the deprotection step may further result in the cleavage of the oligonucleotide from the solid support. The deprotection (and optional cleavage) of the oligonucleotide can be carried out in the presence of ammonia, for example, using a solution containing ammonium hydroxide. For example, concentrated ammonium hydroxide (e.g., a 28-33% solution of NH3 in water) or a 1:1 mixture of ammonium hydroxide and aqueous methylamine (AMA) can be used. Other deprotection methods are known in the art.

[0083] Joining steps

[0084] Oligonucleotides synthesized according to the method of the present invention are particularly useful for preparing oligonucleotide conjugates because aliphatic amine groups provide suitable conjugation sites. Oligonucleotide conjugates comprise oligonucleotides covalently linked to a non-nucleoside moiety, which may be, for example, lipids, sterols, carbohydrates, peptides, and proteins. Examples of conjugate moieties are disclosed in WO2014 / 076195 and WO2014 / 179620, which are incorporated herein by reference.

[0085] In some embodiments, the method of the present invention includes an additional step performed after step c), which includes introducing a conjugate group onto an aliphatic primary amine group.

[0086] Therefore, the present invention provides a method for preparing LNA oligonucleotide conjugates, the method comprising the following steps: a) Introducing at least one nitrogen LNA-G monomer containing an exocyclic nitrogen protected by an acyl group into an oligonucleotide; b) Introduce at least one optionally protected aliphatic amine group into the oligonucleotide; c) Deprotecting the at least one acyl-protected exocyclic nitrogen of the at least one LNA-G monomeric oligonucleotide by removing the acyl protecting group; d) Introducing a conjugation group onto an aliphatic amine group, Steps a) and b) can occur in any order or simultaneously.

[0087] In some embodiments, the conjugate moiety is a carbohydrate, such as an N-acetylgalactosamine (GalNAc) conjugate, see WO2014 / 118267, which is incorporated herein by reference. GalNaC conjugates can be used to increase uptake into cells such as hepatocytes and are commonly used as GalNAc clusters, such as trivalent GalNAc clusters. Figure 2 The explanation illustrates examples of how the method of the present invention can be used to introduce GalNAc conjugates into oligonucleotides.

[0088] Connecting base

[0089] Linkers or linkers are connections between two atoms that link one or more chemical groups or segments of interest via one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or through a linker (e.g., a linker or ligand). Linkers are used to covalently link a third region, such as the conjugate moiety, to a first region, such as the oligonucleotide (region A).

[0090] In the context of this invention, the linker may include an aliphatic amine group, such as a primary or secondary aliphatic amine group. In some embodiments, the linker is an aliphatic aminoalkyl group, such as a C2-C group. 36 Aliphatic aminoalkyl groups, including, for example, C6 to C6 groups. 12 Aliphatic aminoalkyl group. In some embodiments, the linker is a C6 aliphatic aminoalkyl group. In some embodiments, the oligonucleotide comprises a DNA phosphodiester nucleotide region, such as 1-5 DNA PO nucleotides, located between the antisense oligonucleotide and the aliphatic amino linker—see WO2014 / 076195, which is incorporated herein by reference.

[0091] Locked nucleoside (LNA)

[0092] LNA nucleotides are modified nucleotides that contain a linker group (called a bigroup or bridge) between the C2' and C4' of the ribose ring of the nucleotide. These nucleotides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA).

[0093] In some embodiments, the modified nucleosides or LNA nucleosides of the oligomers of the present invention have the general formula structure of formula I or II:

[0094] Where W is selected from -O-, -S-, -N(R) a )-、-C(R a R b -, for example, -O- in some implementations; B refers to a nucleobase or a modified nucleobase portion; Z refers to the inter-nucleoside link or 5'-terminal group that connects to an adjacent nucleoside. Z Refers to the inter-nucleosyl linkage or 3'-terminal group that connects to adjacent nucleosyl groups; X refers to the selection from -C(R) a R b )-、-C(R a )=C(R b ), -C(Ra )=N-、-O-、-Si(R a -2-, -S-, -SO2-, -N(R) a )- and >C=Z groups; In some implementations, X is selected from: -O-, -S-, NH-, NR a R b -CH2-, CR a R b -C(=CH2)- and -C(=CR) a R b )-; In some implementations, X is -O-; Y refers to the selection from -C(R) a R b )-、-C(R a )=C(R b )-、-C(R a )=N-、-O-、-Si(R a -2-, -S-, -SO2-, -N(R) a )- and >C=Z groups; In some implementations, Y is selected from: -CH2-, -C(R) a R b -CH2CH2- -C(R) a R b )-C(R a R b -CH2CH2CH2- -C(R) a R b )C(R a R b )C(R a R b )-、-C(R a )=C(R b )- and -C(R a )=N-; In some implementations, Y is selected from: -CH2-, -CHR a -、-CHCH3-、CR a R b - Alternatively, -XY- together refers to a divalent linker group (also called a radical group), or together refers to one, two, or three radicals selected from -C(R). a R b )-、-C(R a )=C(R b )-、-C(R a )=N-、-O-、-Si(R a-2-, -S-, -SO2-, -N(R) a Divalent linker groups composed of )- and >C=Z groups / atoms; In some implementations, -XY- refers to a bigroup selected from the following: -X-CH2-, -X-CR a R b -、-X-CHR a -, -XC(HCH3)-, -OY-, -O-CH2-, -S-CH2-, -NH-CH2-, -O-CHCH3-, -CH2-O-CH2, -O-CH(C H3CH3)-, -O-CH2-CH2-, OCH2-CH2-CH2-, -O-CH2OCH2-, -O-NCH2-, -C(=CH2)-CH2-, -NR a -CH2-, NO-CH2, -S-CR a R b -and-S-CHR a -; In some implementations, -XY- refers to -O-CH2- or -O-CH(CH3)-; Z is selected from -O-, -S-, and -N(R) a )-; and R a and when R exists b Each is independently selected from hydrogen, and the C atoms are optionally substituted. 1-6 -alkyl, optionally substituted C 2-6 -Alkenyl group, optionally substituted C 2-6 -Alkyne, hydroxyl, optional substituted C 1-6 -alkoxy group, C 2-6 -alkoxyalkyl, C 2-6 -Alkenyloxy group, carboxyl group, C 1-6 -alkoxycarbonyl, C 1-6 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C 1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl)-amino-carbonyl, amino-C 1-6 -alkyl-aminocarbonyl, mono- and di(C) 1-6 -alkyl)amino-C 1-6 -alkyl-aminocarbonyl, C 1-6 -alkyl-carbonylamino, urea, C 1-6 -alkyl acyloxy group, sulfonyl group, C 1-6 -alkylsulfonyloxy, nitro, azide, thioalkyl, C1-6 -alkylthio, halogen, wherein the aryl and heteroaryl groups may be optionally substituted, and wherein the two twin substituents R a and R b Together can refer to the arbitrarily substituted methylene (=CH2), where for all chiral centers, an R or S-oriented asymmetric group can be found; Where R 1 R 2 R 3 R 5 and R 5 Independently selected from: hydrogen, optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -Alkenyl group, optionally substituted C 2-6 -Alynyl group, hydroxyl group, C 1-6 -alkoxy group, C 2-6 -alkoxyalkyl, C 2-6 -Alkenyloxy group, carboxyl group, C 1-6 -alkoxycarbonyl, C 1-6 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C 1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl)-amino-carbonyl, amino-C 1-6 -alkyl-aminocarbonyl, mono- and di(C) 1-6 -alkyl)amino-C 1-6 -alkyl-aminocarbonyl, C 1-6 -alkyl-carbonylamino, urea, C 1-6 -alkyl acyloxy group, sulfonyl group, C 1-6 -alkylsulfonyloxy, nitro, azide, thioalkyl, C 1-6 -alkylthio, halogen, wherein the aryl and heteroaryl groups may be optionally substituted, and wherein the two twin substituents together may refer to an oxo group, a thio group, an imino group, or an optionally substituted methylene group.

[0095] In some implementation schemes, R 1 R 2 R 3 R 5 and R 5 Selected independently from C 1-6 Alkyl groups, such as methyl and hydrogen.

[0096] In some implementation schemes, R 1 R 2 R3 R 5 and R 5 Both are hydrogen.

[0097] In some implementation schemes, R 1 R 2 R 3 Both are hydrogen, R 5 and R 5 One of them is also hydrogen and R 5 and R 5 Another one in it is not hydrogen, for example, C 1-6 Alkyl groups, such as methyl groups.

[0098] In some implementation schemes, R a It is hydrogen or methyl. In some embodiments, R, when present, b It is either hydrogen or methyl.

[0099] In some implementation schemes, R a and R b One or both of them are hydrogen.

[0100] In some implementation schemes, R a and R b One of them is hydrogen, and the other is not hydrogen.

[0101] In some implementation schemes, R a and R b One of them is a methyl group, and the other is hydrogen.

[0102] In some implementation schemes, R a and R b Both are methyl groups.

[0103] In some embodiments, the di-XY- is -O-CH2-, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. These types of LNA nucleosides are disclosed in WO99 / 014226, WO00 / 66604, WO98 / 039352 and WO2004 / 046160, all of which are incorporated herein by reference, including those commonly referred to as β-D-oxyLNA and α-L-oxyLNA nucleosides.

[0104] In some embodiments, the di-XY- is -S-CH2-, W is O, and R 1 R 2 R3 R 5 and R 5 All are hydrogen. This type of thio-LNA nucleoside is disclosed in WO99 / 014226 and WO2004 / 046160, which are incorporated herein by reference.

[0105] In some embodiments, the di-XY- is -NH-CH2-, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. This type of amino-LNA nucleoside is disclosed in WO99 / 014226 and WO2004 / 046160, which are incorporated herein by reference.

[0106] In some embodiments, the di-XY- is -O-CH2-CH2- or -O-CH2-CH2-CH2-, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. This type of LNA nucleoside has been disclosed in WO00 / 047599 and Morita et al. Bioorganic & Med. Chem. Lett. J. Org. Chem. In 12 73-76, they are incorporated herein by reference, including those commonly referred to as 2'-O-4'C-ethylene-bridged nucleic acids (ENA).

[0107] In some embodiments, the di-XY- is -O-CH2-, W is O, and R 1 R 2 R 3 All and R 5 and R 5 One of them is hydrogen, and R 5 and R 5 Another one in it is not hydrogen, such as C. 1-6 Alkyl groups, such as methyl groups. Such 5'-substituted LNA nucleosides are disclosed in WO2007 / 134181, which is incorporated herein by reference.

[0108] In some implementations, the di-XY- group is -O-CR a R b -, where R a and R b One or both of them are not hydrogen, for example, methyl, W is O, and R 1R 2 R 3 All and R 5 and R 5 One of them is hydrogen, and R 5 and R 5 Another one in it is not hydrogen, such as C. 1-6 Alkyl groups, such as methyl groups. This type of dual-modified LNA nucleoside is disclosed in WO2010 / 077578, which is incorporated herein by reference.

[0109] In some implementations, the di-XY- group refers to the divalent linker group -O-CH(CH2OCH3)-(2'O-methoxyethyl bicyclic nucleic acid -Seth et al., 2010, J. Org. Chem , 2010, 75 (5), pp. 1569-1581). In some embodiments, the di-XY- refers to the divalent linker group -O-CH(CH2CH3)-(2'O-ethyl bicyclic nucleic acid-Seth et al., 2010, 75 (5), pp. 1569-1581). J. Org. Chem In some embodiments, the di-XY- group is -O-CHR. a -, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. Such 6'-substituted LNA nucleosides are disclosed in WO10036698 and WO07090071, both of which are incorporated herein by reference.

[0110] In some embodiments, the di-XY- is -O-CH(CH2OCH3)-, W is O, and R 1 R 2 R 3 R 5 and R 5 All of them are hydrogen. This type of LNA nucleoside is also known in the art as cyclic MOEs (cMOE), which is disclosed in WO07090071.

[0111] In some embodiments, the divalent group -XY- refers to the divalent linker group -O-CH(CH3)-, with an R- or S- configuration. In some embodiments, the divalent group -XY- together refers to the divalent linker group -O-CH2-O-CH2- (Seth et al., 2010, J. Org. Chem In some embodiments, the di-XY- is -O-CH(CH3)-, W is O, and R 1 R 2R 3 R 5 and R 5 All are hydrogen. These 6'-methyl-LNA nucleosides are also known in the art as cET nucleosides, which can be (S)cET or (R)cET stereoisomers, as disclosed in WO07090071 (β-D) and WO2010 / 036698 (α-L), both of which are incorporated herein by reference.

[0112] In some implementations, the di-XY- group is -O-CR a R b -, where R a Or R b Neither of them is hydrogen, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. In some implementations, R a and R b All are methyl groups. This type of 6'-disubstituted LNA nucleoside is disclosed in WO 2009006478, which is incorporated herein by reference.

[0113] In some implementations, the di-XY- group is -S-CHR a -, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. This type of 6'-substituted thio-LNA nucleoside is disclosed in WO11156202, which is incorporated herein by reference. In some 6'-substituted thio-LNA embodiments, R a It is a methyl group.

[0114] In some embodiments, the di-XY- is -C(=CH2)-C(R) a R b -, for example -C(=CH2)-CH2- or -C(=CH2)-CH(CH3)-, where W is O and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. This type of vinyl carbon LNA nucleoside is disclosed in WO08154401 and WO09067647, both of which are incorporated herein by reference.

[0115] In some implementations, the di-XY- group is -N(-OR) a )-, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. In some implementations, R a It is C 1-6 Alkyl groups, such as methyl groups. These LNA nucleosides are also known as N-substituted LNAs, disclosed in WO2008 / 150729, which is incorporated herein by reference. In some embodiments, the di-XY- group together refers to the divalent linker group -O-NR. a -CH3- (Seth et al., 2010, Figure 1 In some embodiments, the di-XY- group is -N(R) a )-, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. In some implementations, R a It is C 1-6 Alkyl groups, such as methyl groups.

[0116] In some implementation schemes, R 5 and R 5 One or both of them are hydrogen and R when substituted 5 and R 5 The other one is C. 1-6 Alkyl groups, such as methyl groups. In these embodiments, R... 1 R 2 R 3 Both groups can be hydrogen, and the digroup -XY- can be selected from -O-CH2- or -OC(HCR). a )-like-OC(HCH3)-.

[0117] In some implementations, the digroup is -CR a R b -O-CR a R b For example, CH2-O-CH2-, W is O, and R 1 R 2 R 3 R 5 and R 5 All are hydrogen. In some implementations, R aIt is C 1-6 Alkyl groups, such as methyl groups. These LNA nucleotides are also known as conformation-restricted nucleotides (CRNs), disclosed in WO2013036868, which is incorporated herein by reference.

[0118] In some implementations, the digroup is -O-CR a R b -O-CR a R b For example, in O-CH2-O-CH2-, W is O, and R... 1 R 2 R 3 R 5 and R 5 All are hydrogen. In some implementations, R a It is C 1-6 Alkyl groups, such as methyl groups. This type of LNA nucleoside is also known as a COC nucleotide, published in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which is incorporated herein by reference.

[0119] Unless otherwise specified, it is understood that LNA nucleosides can be either β-D or α-L stereoisomeric.

[0120] Some examples of LNA nucleosides are given in Procedure 1.

[0121] Process 1

[0122] As explained in the examples, in some embodiments of the invention, the LNA nucleotide in the oligonucleotide is or contains β-D-oxy-LNA nucleotide.

[0123] Gapmer

[0124] As used herein, the term gapmer refers to an antisense oligonucleotide containing an oligonucleotide region (gap) that recruits RNase H, whose 5' and 3' flanks are one or more affinity-enhancing modified nucleosides (flanks). Various gapmer designs are described herein. Headmers and tailmers are oligonucleotides capable of recruiting RNase H that lack one of their flanks; that is, the oligonucleotide contains an affinity-enhancing modified nucleoside at only one end. For headmers, the 3' flank is missing (i.e., the 5' flank contains an affinity-enhancing modified nucleoside), and for tailmers, the 5' flank is missing (i.e., the 3' flank contains an affinity-enhancing modified nucleoside).

[0125] LNA Gapmer

[0126] The term LNA gapmer refers to a gapmer oligonucleotide in which at least one of the modified nucleosides that enhances affinity is an LNA nucleoside.

[0127] Mixed Wing Gapmer

[0128] The term "mixed-wing gapmer" refers to an LNA gapmer as defined below, wherein the flanking region contains at least one LNA nucleoside and at least one non-LNA modified nucleoside, such as at least one 2'-substituted modified nucleoside like 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleotide (ANA), 2'-fluoro-ANA, and 2'-F-ANA nucleoside. In some embodiments, the mixed-wing gapmer has one flanking region containing an LNA nucleoside (e.g., 5' or 3') and another flanking region (3' or 5', respectively) containing a 2'-substituted modified nucleoside.

[0129] length

[0130] When referring to the length of a nucleotide molecule as described herein, length corresponds to the number of monomer units, i.e., nucleotides, regardless of whether those monomer units are nucleotides or nucleotide analogs. For nucleotides, the terms monomer and unit are used interchangeably herein.

[0131] The method of the present invention is particularly suitable for purifying short oligonucleotides, such as short oligonucleotides consisting of 7-30 nucleotides, for example 7-10, for example 7, 8, 9, 10 or 10-20 nucleotides, for example 12-18 nucleotides, for example 12, 13, 14, 15, 16, 17 or 18 nucleotides.

[0132] Example

[0133] Example 1

[0134] Koshkin et al., Tetrahedron (1998), 54(14), 3607-3630, described the synthesis of isobutyryl-protected LNA-G phosphoramidides.

[0135] Crude phosphate thioester oligonucleotides were synthesized in DMT-OFF mode at a scale of 20 µmol on a NittoPhase UnyLinker 200 polystyrene support via standard phosphorous amide chemistry, with the exception of oligonucleotides in entries 17-18, which were synthesized on a 3'-amino-modified C7 CPG support. 4,5-dicyanimidazole was used as the activator, and xanthane hydride was used for thiooxidation. Standard DNA phosphorous amides with benzyl-protected A and C were used. LNA phosphorous amides with benzyl-protected A and 5-methyl-C were used. LNA-G was either DMF- or iBu-protected, as shown in the table below.

[0136] 5'-TFA-amino-modifying group C6-CE phosphoramide, available from Link Technologies, Lanakshire, Scotland, is used to introduce a 6-aminohexyl linker (AM-C6) at the 5'-end.

[0137] The amino-modified C6-dT-CE phosphoramidamide (5'-dimethoxytriphenylmethyl-5-[N-(trifluoroacetylaminohexyl)-3-propenylimino]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidamide), available from Glen Research, Sterling, VA, is used to introduce a 5-[N-(aminohexyl)-3-propenylimino]-2'-deoxyuridine linker (t AMC6 ).

[0138] 11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, available from Link Technologies, Lanakshire, Scotland, for introducing an 11-amino-3,6,9-trioxaundecan-1-yl group at the 5'-terminus (AM-TEG).

[0139] The 3'-amino-modifying group C7 CPG (2-dimethoxytriphenylmethyloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinyl)-long-chain alkylamino-CPG, available from Glen Research, Sterling, VA, is used to introduce a 6-amino-2-(hydroxymethyl)-hexyl group (AM-C7) at the 3'-terminus. In this case, after synthesis, the support is first treated with a diethylamine solution and then with a 20% solution of piperidine in DMF to remove the Fmoc group.

[0140] After synthesis, the support was washed with diethylamine solution and then suspended in 1 mL of concentrated ammonium hydroxide at 60°C overnight. The support was filtered off, and the solution was evaporated to dryness under vacuum.

[0141] The crude material was analyzed by UPLC-MS, and the proportion of +28Da impurities compared to the correct product was evaluated by the peak area at 260 nm.

[0142]

[0143] Uppercase letters = LNA, lowercase letters = DNA. AM-C6 = 6-aminohexyl, t AMC6 = 5-[N-(aminohexyl)-3-propionylimino]-2'-deoxyuridine, AM-TEG = 11-amino-3,6,9-trioxaundecane-1-yl, AM-C7 = 6-amino-2-(hydroxymethyl)-hexyl. All examples are phosphate thioester oligonucleotides, wherein the LNA monomer is β-D-oxyLNA. The LNA-C monomer is a 5-methylcytosine LNA monomer.

[0144]

[0145] Example 2

[0146] As described in Example 1, a 5'-aminohexyl-linked phosphate-thioester oligonucleotide with the sequence 5'-AM-C6-caGCGtaaagagAGG-3' was prepared using DMF-protected LNA-G and iBu-protected DNA-G phosphoramide. The crude product contained the full-length product (FL) and approximately 20% formyl (HCO,+28) impurities (FL+28).

[0147] We determined two [M-5H] samples using ultra-high resolution mass spectrometry (FT-ICR-MS; Thermo LTQ-FT Ultra). 5- The exact mass difference of the peak is 27.9968 Da (1044.11945 Da for FL and 1049.71881 Da for FL+28). Figure 3 The mass difference of 5.59936 Da for 5-fold charged ions results in a mass difference of 5.59936 Da for uncharged molecules. 5 = 27.9968 Da ( Figure 4 ).

[0148] The measured mass is within the accuracy range of Δ = 1.885 mmu for the carbonyl group (CO), and the next possible modification with a nominal mass of +28 Da is "N2", where the accurate mass difference is -9.348 mmu, which is outside the accuracy range of the instrument used.

[0149] To determine the precise location of the CO-modification, the molecules were sequenced by mass spectrometry using the same instrument via MS / MS. For the nomenclature of the oligonucleotide fragments, see McLuckey et al., J. Am. Chem. Soc., 115, 25, 12085-12095.

[0150] In MS / MS experiments, two 5-fold charged ions (w / and w / o modified) were fragmented. Here, the modification on the ion was placed from the 5' end to the eighth nucleotide, while the nucleotide from the 3' end was broken at the same position without modification. No other 3' fragments with longer sequences were detected. In the resulting spectra, b4-ions were observed at FL+28. Figure 4 ).

[0151] Further fragmentation with MS3 of b4- ions ( Figure 5 The spectrum produced, among other signals, showed the presence of an a2-B ion and a b4-cytosine ion (b4-C) at the first nucleotide. These two ions led to a proposal to modify the C6-amino linker. Figure 6 ).

[0152] Since only amino linker groups exist in both structures as possible reaction partners for modification, we conclude that formamide is formed at this position. ​ ).

Claims

1. A method for preparing LNA oligonucleotides that are essentially free of +28 adducts, comprising the following steps: a) Introducing at least one LNA-G monomer containing an exocyclic nitrogen protected by an isobutyryl group into an oligonucleotide; b) Introduce at least one optionally protected aliphatic amine group into the oligonucleotide; c) Deprotecting the isobutyryl-protected exocyclic nitrogen of the at least one LNA-G monomer by removing the isobutyryl protecting group; Step a) occurs before step b).

2. The method of claim 1, wherein the optionally protected aliphatic amine group is a primary amine or a secondary amine.

3. The method according to claim 1 or 2, wherein the optionally protected aliphatic amine group is a non-nucleoside amine group.

4. The method according to any one of claims 1-3, wherein the optionally protected aliphatic amine group is selected from aminoalkyl, alkylaminoalkyl, piperidine, piperazine, pyrrolidine, and imidazole.

5. The method according to any one of claims 1-3, wherein the optionally protected aliphatic amine group is selected from 5'-TFA-amino-modified-C5-CE phosphoramidamide, 5'-TFA-amino-modified-C6-CE phosphoramidamide, 11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidamide, 5'-TFA-amino-modified-C12-CE phosphoramidamide, amino-modified-C2-dT-CE phosphoramidamide, amino-modified-C6-dA-CE phosphoramidamide, amino-modified-C6-dA-CE phosphoramidamide, amino-modified-C6-dT-CE phosphoramidamide, N2-amino-modified-C6 dG, Fmoc amino-modified-C6 dT, 3'-amino-modified-C7 CPG 1000, 3'-amino-modified-C6-dC CPG, 3'-amino-modified C6-dC CPG, 3'-PT-amino-modified C6 CPG, 3'-amino-modified C6-dT CPG, PC, 5'-amino-modified CE phosphoramide, 5'-amino-modified C6-PDA, 5'-amino-modified C12-PDA, 5'-amino-modified TEG PDA, amino-modified serine alcohol, and 3'-amino-modified serine alcohol CPG.

6. The method according to any one of claims 1-3, wherein the optionally protected aliphatic amine group is an aminohexyl linker.

7. The method according to any one of claims 1-6, wherein the aliphatic amine group is introduced into the oligonucleotide via a monomer in which an amino group is introduced.

8. The method of claim 7, wherein the monomer modified with the aliphatic amino group is a phosphorus amide, an H-phosphonate, or a triphosphate monomer.

9. The method of claim 7, wherein the amino-modified monomer is phosphoramide.

10. The method according to any one of claims 1-9, wherein the other G residues introduced into the oligonucleotide, if present, also include an isobutyryl protecting group.

11. The method according to any one of claims 1-10, wherein the LNA-G monomer and, when present, optionally the G monomer, are phosphorous amide, H-phosphonate, or phosphate triester monomer.

12. The method according to any one of claims 1-11, wherein the LNA-G monomer and, when present, optionally the G monomer, are phosphorous amides.

13. The method according to any one of claims 1-12, wherein the LNA-G monomer comprises a 2'-O-CH2-4' bigroup in the furanose ring.

14. The method according to any one of claims 1-13, wherein step c) further comprises deprotection of the primary amine group.

15. The method according to any one of claims 1-14, wherein step c) comprises the deprotection of oligonucleotides, carried out in the presence of ammonia, for example using a solution containing ammonium hydroxide.

16. The method according to any one of claims 1-15, wherein step (d) is performed after step c), said additional step (d) comprising introducing a conjugate group onto an aliphatic primary amine group.

17. The method of claim 16, wherein the conjugation group is a nonnucleotide portion selected from lipids, sterols, carbohydrates, peptides, and proteins.

18. The method according to any one of claims 1-17, wherein at least steps a)-c) are performed on a solid support, followed by cleavage of the oligonucleotide from the solid support, the cleavage being performed during or after step c).

19. The method according to any one of claims 1-18, wherein the aliphatic primary amine group is an aminohexyl linker.

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