LNA-G Process

Abstract

Recent advancements in LNA oligonucleotides include the use of amine linkers to link an LNA antisense oligonucleotide to a conjugate group. For example please see WO2014/118267. The present invention originates from the identification of a problem when de-protecting LNA oligonucleotides which comprise an aliphatic amine group and DMF protected LNA G nucleoside, which results in the production of a +28 Da impurity. This problem is solved by using acyl protection groups on the exocyclic nitrogen of the LNA-G residue, rather than the standard DMF protection group.

1 . A method of preparing a LNA oligonucleotide comprising the steps of:
 a) Incorporating at least one LNA-G monomer comprising an acyl protected exocyclic nitrogen into an oligonucleotide   b) Incorporating at least one optionally protected aliphatic amine group into the oligonucleotide   c) deprotecting the acyl protected exocyclic nitrogen of the at least one LNA-G monomer by removal of the acyl protection group.   
       wherein steps a) and b) can occur in either order. 
     
     
         2 . The method according to  claim 1  wherein the optionally protected aliphatic amine group is a primary or secondary amine. 
     
     
         3 . The method according to any one of  claim 1  or  2 , wherein the optionally protected aliphatic amine group is a non nucleosidic amine group. 
     
     
         4 . The method according to any one of  claims 1 - 3 , wherein the optionally aliphatic amine group is selected from the group consisting of an amino alkyl, alkylamino alkyl, piperidine, piperazine, pyrrolidine, & imidazole. 
     
     
         5 . The method according to any one of  claims 1 - 3 , wherein the optionally aliphatic amine group is selected from the group consisting of 5′-TFA-Amino-Modifier-C5-CE Phosphoramidite, 5′-TFA-Amino-Modifier C6-CE Phosphoramidite, 11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite, 5′-TFA-Amino-Modifier-C12-CE Phosphoramidite, Amino-Modifier C2-dT-CE Phosphoramidite, Amino-Modifier C6-dA-CE Phosphoramidite, Amino-Modifier C6-dA-CE Phosphoramidite, Amino-Modifier C6-dT-CE Phosphoramidite, N2-Amino-Modifier C6 dG, Fmoc Amino-Modifier C6 dT, 3′-Amino-Modifier C7 CPG 1000, 3′-Amino-Modifier C6-dC CPG, 3′-Amino-Modifier C6-dC CPG, 3′-PT-Amino-Modifier C6 CPG, 3′-Amino-Modifier C6-dT CPG, PC 5′-Amino-Modifier-CE Phosphoramidite, 5′-Amino-Modifier C6-PDA, 5′-Amino-Modifier C12-PDA, 5′-Amino-Modifier TEG PDA, Amino-Modifier Serinol, & 3′-Amino-Modifier Serinol CPG. 
     
     
         6 . The method according to any one of  claims 1 - 3 , wherein the optionally protected aliphatic amine group is an amino hexyl linker. 
     
     
         7 . The method according to any one of  claims 1 - 6 , wherein the aliphatic amine group is incorporated into the oligonucleotide via the incorporation of an amino-modified monomer. 
     
     
         8 . The method according to  claim 7 , wherein the aliphatic amino-modified monomer is a phosphoramidite, a H phosphonate or a phosphotriester monomer. 
     
     
         9 . The method according to  claim 7 , wherein the amino-modified monomer is a phosphoramidite. 
     
     
         10 . The method according to any one of  claims 1 - 9 , wherein the acyl protection group on the exocyclic nitrogen of the LNA-G monomer(s) consists or comprises a group selected from the group consisting of an optionally substituted alkyl-, alkenyl-, alkynyl-, cycloalkyl- or aryl-group, preferably from an optionally substituted C 1-6 -alkyl-, C 2-6 -alkenyl-, C 2-6 -alkinyl-, C 3-7 -cycloalkyl- or phenyl-group; wherein when substituted, the R group may be mono or poly substituted, e.g. with one or more substituents selected from the group consisting of halogen, C 1-6 -alkyl, C 2-6 -alkenyl, C m -alkynyl, C 1-6 -alkoxy, optionally substituted aryloxy or optionally substituted aryl. 
     
     
         11 . The method according to any one of  claims 1 - 10 , wherein the acyl protection group on the exocyclic nitrogen of the LNA-G monomer(s) is selected from the group consisting of Isobuturyl (iBu), Acetyl (Ac), Phenoxyacetyl (PAC), p-Isopropylphenoxyacetyl (iPrPAC), phenylacetyl, Isopropyloxyacetyl, methoxyacetyl, benzoyl, p-methoxyphenylacetyl, diphenylacetyl, cyclohexylcarbonyl, 1,1-dimethylpropanoyl, and p-tert-Butyl-phenoxyacetyl. 
     
     
         12 . The method according to any one of  claims 1 - 10 , wherein the acyl protection group on the exocyclic nitrogen of the LNA-G monomer(s) is selected from the group consisting of Isobuturyl (iBu), Acetyl (Ac), Phenoxyacetyl (PAC), & p-Isopropylphenoxyacetyl (iPrPAC). 
     
     
         13 . The method according to any one of  claims 1 - 12 , wherein, if present, other G residues incorporated into the oligonucleotide also comprise an acyl protection group, such as the acyl protection groups of any one of  claims 10 - 12 . 
     
     
         14 . The method according to any one of  claims 1 - 13 , wherein the LNA-G monomer(s), and optionally when present other G monomers, is a phosphoramidite, a H-phosphonate or a phosphotriester monomer. 
     
     
         15 . The method according to any one of  claims 1 - 14 , wherein the LNA-G monomer(s), and optionally when present other G monomers, is a phosphoramidite. 
     
     
         16 . The method according to any one of  claims 1 - 15  wherein the LNA-G monomer comprises a 2′-O—CH2-4′ biradical in the furanose ring. 
     
     
         17 . The method according to any one of  claims 1 - 16 , wherein step c) further comprises deprotection of the primary amine group. 
     
     
         18 . The method according to any one of  claims 1 - 17 , wherein step c) comprises deprotection of the oligonucleotide is performed in the presence of ammonia, such as using a solution comprising ammonium hydroxide. 
     
     
         19 . The method according to any one of  claims 1 - 18 , wherein step c) is followed by an additional step (d) which comprises incorporating a conjugate group onto the aliphatic primary amine group. 
     
     
         20 . The method according to  claim 19 , wherein the conjugate group is a non-nucleotide moiety, selected from the group consisting of a lipid, a sterol, a carbohydrate, a peptide and a protein. 
     
     
         21 . The method according to any one of  claims 1 - 20 , wherein at least steps a)-c) are performed on a solid support and are followed by the cleavage of the oligonucleotide from the solid support which may be performed during step c) or subsequent to step c). 
     
     
         22 . The method according to any one of  claims 1 - 21 , wherein the acyl protection group(s) is isobuturyl and the aliphatic primary amine group(s) is an aminohexyl linker. 
     
     
         23 . An LNA oligonucleotide which comprises at least one LNA-G monomer comprising an acyl protected exocyclic nitrogen and at least one optionally protected aliphatic amine group, wherein said LNA oligonucleotide is attached to a solid support. 
     
     
         24 . A pharmaceutical composition comprising an LNA oligomer conjugate which comprises an LNA-G monomer and an aliphatic amine linker positioned between the 5′ nucleotide of LNA oligomer and a conjugate moiety, and a pharmaceutically acceptable diluent, carrier or adjuvant, wherein said composition is essentially free of +28 adduct. 
     
     
         25 . Use of an LNA-G monomer comprising an acyl protected exocyclic nitrogen for use in the synthesis of an aliphatic amine containing LNA oligonucleotide. 
     
     
         26 . Use of an LNA-G monomer comprising an acyl protected exocyclic nitrogen for use in the synthesis of an aliphatic amine containing LNA oligonucleotide conjugate.