Simple chemical approaches to introduce 2,6-diaminopurine and 2-aminoadenine conjugates into oligonucleotides

A compound of Formula (III):

• wherein:
• RH is halogen;
• R32 is hydrogen, hydroxy, halogen protected hydroxy, phosphate group, reactive phosphorous group, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, —O—C4-30alkyl-ON(CH2R8)(CH2R9), —O—C4-30alkyl-ON(CH2R8)(CH2R9), a solid support, or a linker covalently bonded (e.g., —C(O)CH2CH2C(O)—) to a solid support;
• R33 is hydrogen, hydroxy, halogen protected hydroxy, phosphate group, a reactive phosphorous group, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, —O—C4-30alkyl-ON(CH2R8)(CH2R9), —O—C4-30alkyl-ON(CH2R8)(CH2R9), a solid support, or a linker covalently bonded (e.g., —C(O)CH2CH2C(O)—) to a solid support, and optionally, only one of R32 and R33 is a phosphate group, a reactive phosphorous group, a solid support or a linker to a solid support;
• R4 is hydrogen, optionally substituted C1-6alkyl, optionally substituted C2-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C1-6alkoxy;
• or R4 and R32 taken together are 4′-C(R10R11)v—Y-2′ or 4′-Y—C(R10R11)v-2′;
• Y is —O—, —CH2—, —CH(Me)-, —C(CH3)2—, —S—, —N(R12)—, —C(O)—, —C(S)—, —S(O)—, —S(O)2—, —OC(O)—, —C(O)O—, —N(R12)C(O)—, or —C(O)N(R2)—;
• R10 and R11 independently are H, optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl or optionally substituted C2-C6alkynyl;
• R12 is hydrogen, optionally substituted C1-30alkyl, optionally substituted C1-C30alkoxy, C1-4haloalkyl, optionally substituted C2-4alkenyl, optionally substituted C2-4alkynyl, optionally substituted C1-30alky-CO2H, or a nitrogen-protecting group;
• v is 1, 2 or 3;
• or R4 and R33 taken together with the atoms to which they are attached form an optionally substituted C3-8cycloalkyl, optionally substituted C3-8cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl;
• R35 is hydroxy, protected hydroxy, phosphate group, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, —O—C4-30alkyl-ON(CH2R8)(CH2R9), —O—C4-30alkyl-ON(CH2R8)(CH2R9), vinylphosphonate (VP) group, C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO)2(O)P—O-5′), diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′), triphosphate ((HO)2(O)P-0-(HO)(O)P—O—P(HO)(O)—O-5′);
• monothiophosphate (phosphorothioate, (HO)2(S)P—O-5′), monodithiophosphate (phosphorodithioate;
• (HO)(HS)(S)P—O-5′), phosphorothiolate ((HO)2(O)P—S-5′);
• alpha-thiotriphosphate;
• beta-thiotriphosphate;
• gamma-thiotriphosphate;
• phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), alkylphosphonates (R(OH)(O)P—O-5′, R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc. . . . ), alkyletherphosphonates (R(OH)(O)P—O-5′, R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc. . . . ), (HO)2(X)P—O[—(CH2)a—O—P(X)(OH)—O]b-5′ or (HO)2(X)P—O[—(CH2)a—P(X)(OH)—O]b-5′ or (HO)2(X)P—[—(CH2)a—O—P(X)(OH)—O]b-5′, where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[—(CH2)a—O—P(X)(OH)—O]b-5′, H2N[—(CH2)a—O—P(X)(OH)—O]b-5′, H[—(CH2)a—O—P(X)(OH)—O]b-5′, Me2N[—(CH2)a—O—P(X)(OH)—O]b-5′, HO[—(CH2)a—P(X)(OH)—O]b-5′, H2N[—(CH2)a—P(X)(OH)—O]b-5′, H[—(CH2)a—P(X)(OH)—O]b-5′, Me2N[—(CH2)a—P(X)(OH)—O]b-5′, wherein a and b are each independently 1-10);
• and
• each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30alkynyl.

A method for preparing an oligonucleotide comprising a nucleoside of Formula (X):

• the method comprising reacting an oligonucleotide comprising a nucleoside of Formula (XI):
• with an alkali hydroxide or alkali earth hydroxide (e.g., NaOH);
• wherein:
• RH is halogen (e.g., chloro or fluoro);
• R2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, —O—C4-30alkyl-ON(CH2R8)(CH2R9), or —O—C4-30alkyl-ON(CH2R8)(CH2R9), a bond to an internucleotide linkage to a subsequent nucleotide, a 3′-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, a linker covalently bonded to one or more ligands (e.g., N-acetylgalactosamine (GalNac)), a solid support, or a linker covalently bonded (e.g., —C(O)CH2CH2C(O)—) to a solid support;
• R3 is a bond to an internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxy, protected hydroxy, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, —O—C4-30alkyl-ON(CH2R8)(CH2R9), —O—C4-30alkyl-ON(CH2R8)(CH2R9), a 3′-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, a linker covalently bonded to one or more ligands (e.g., N-acetylgalactosamine (GalNac)), a solid support, or a linker covalently bonded (e.g., —C(O)CH2CH2C(O)—) to a solid support;
• R4 is hydrogen, optionally substituted C1-6alkyl, optionally substituted C2-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C1-6alkoxy;
• or R4 and R2 taken together are 4′-C(R10R11)v—Y-2′ or 4′-Y—C(R10R11)v-2′;
• Y is —O—, —CH2—, —CH(Me)-, —C(CH3)2—, —S—, —N(R12)—, —C(O)—, —C(S)—, —S(O)—, —S(O)2—, —OC(O)—, —C(O)O—, —N(Ra13)C(O)—, or —C(O)N(Ra13)—;
• R10 and R11 and Ra13 independently are H, optionally substituted C1-C6alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl;
• R12 is hydrogen, optionally substituted C1-30alkyl, optionally substituted C1-C30alkoxy, C1-4haloalkyl, optionally substituted C2-4alkenyl, optionally substituted C2-4alkynyl, optionally substituted C1-30alkyl-CO2H, or a nitrogen-protecting group;
• v is 1, 2 or 3;
• or R4 and R3 taken together with the atoms to which they are attached form an optionally substituted C3-8cycloalkyl, optionally substituted C3-8cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl;
• R5 represents a bond to an internucleotide linkage to a preceding nucleotide, hydrogen, hydroxy, protected hydroxy, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, optionally substituted 3-8 membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, —O—C4-30alkyl-ON(CH2R8)(CH2R9), —O—C4-30alkyl-ON(CH2R8)(CH2R9), vinylphosphonate (VP) group, C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO)2(O)P—O-5′), diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′), triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);
• monothiophosphate (phosphorothioate, (HO)2(S)P—O-5′), monodithiophosphate (phosphorodithioate;
• (HO)(HS)(S)P—O-5′), phosphorothiolate ((HO)2(O)P—S-5′);
• alpha-thiotriphosphate;
• beta-thiotriphosphate;
• gamma-thiotriphosphate;
• phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), alkylphosphonates [(RP)(OH)(O)P—O-5′, RP is optionally substituted C1-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(RP)(OH)(O)P—O-5′, RP1 is alkoxyalkyl, e.g., methoxymethyl (CH2OMe) or ethoxymethyl], (HO)2(X)P—O[—(CH2)a—O—P(X)(OH)—O]b-5′ or (HO)2(X)P—O[—(CH2)a—P(X)(OH)—O]b-5′ or (HO)2(X)P—[—(CH2)a—O—P(X)(OH)—O]b-5′, or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[—(CH2)a—O—P(X)(OH)—O]b-5′, H2N[—(CH2)a—O—P(X)(OH)—O]b-5′, H[—(CH2)a—O—P(X)(OH)—O]b-5′, Me2N[—(CH2)a—O—P(X)(OH)—O]b-5′, HO[—(CH2)a—P(X)(OH)—O]b-5′, H2N[—(CH2)a—P(X)(OH)—O]b-5′, H[—(CH2)a—P(X)(OH)—O]b-5′, Me2N[—(CH2)a—P(X)(OH)—O]b-5′, wherein
• X is 0 or S;
• a and b are each independently 1-10;
• each R6 and R7 is independently hydrogen or -L-RL, provided that at least one of R6 and R7 is not H;
• or R6 and R7taken together with the nitrogen atom to which they are attached form a 3-10 membered heterocyclyl or a 3-10 membered heteroaryl group, the heterocyclyl or heteroaryl comprising one -L-RL group,
• wherein
• L is absent or a linker;
• each RL is a ligand, (e.g., selected independently from the group consisting of carbohydrates, peptides, lipids, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, vitamins, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl);
• each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30alkynyl,
• provided that,
• (i) no more than one of R2 and R3 is a bond to an internucleotide linkage to a subsequent nucleotide;
• and
• (ii) when both of R2 and R3 are not a bond, then R5 is a bond to an internucleotide linkage to a preceding nucleotide.