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WO2025021654A1 - Phosphoramidite linkers - Google Patents

Phosphoramidite linkers Download PDF

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Publication number
WO2025021654A1
WO2025021654A1 PCT/EP2024/070454 EP2024070454W WO2025021654A1 WO 2025021654 A1 WO2025021654 A1 WO 2025021654A1 EP 2024070454 W EP2024070454 W EP 2024070454W WO 2025021654 A1 WO2025021654 A1 WO 2025021654A1
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compound
alkyl
alkenyl
alkynyl
trialkylsilyl
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French (fr)
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Ysobel BAKER
Sritama BOSE
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United Kingdom Research and Innovation
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United Kingdom Research and Innovation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the invention relates to phosphoramidite linkers, oligonucleotides bound to these linkers, and to oligonucleotide conjugates connected via the linkers of the invention. Methods of preparing 5 oligonucleotide conjugates are also provided.
  • Oligonucleotide conjugates are an emerging technology, and it has been demonstrated that conjugated moieties can be used in therapeutics to successfully treat disease.
  • conjugated oligonucleotides represent an important class of laboratory tools, including labelled oligonucleotides 10 for use in understanding intracellular trafficking.
  • Conjugated oligonucleotides can also be broadly used to modulate prokaryotic and eukaryotic gene expression.
  • Peptide ⁇ oligonucleotide conjugates are, in particular, a field of increasing interest.
  • the conjugation of an oligonucleotide to a carrier protein provides a method of increasing cellular uptake, tissue delivery, and bioavailability of therapeutic oligonucleotides such as antisense 15 oligonucleotides or small interfering RNAs (described in Klabenkova et al. Molecules 2021, 26(17), 5420). Accordingly, this conjugation improves the overall efficiency of these oligonucleotides in vivo.
  • the conjugation of these therapeutic oligonucleotides to an antigen binding fragment, nanobody, or antibody allows for greatly increased specificity of action for the oligonucleotide.
  • linker joining the peptide and oligonucleotide 20 it is necessary for the linker joining the peptide and oligonucleotide 20 to be both biologically stable and possible to synthesise/attach in conditions suitable for both peptides and oligonucleotides.
  • these disulfides are not stable in vivo and are known to undergo thiol exchange.
  • linkers have been developed 25 for use in oligonucleotides and peptides.
  • One standard type of linker is the triazole linker. These linkers, however, require additional modification of the protein with either an azide or alkyne group, and therefore frequently require post ⁇ synthetic oligonucleotide modification. These linkers also typically require the presence of a metal catalyst, such as copper, in order to provide the desired linked molecule. These metals can be 30 detrimental to drug molecules and can lead to the degradation of oligonucleotides.
  • Triazole linkers 1 11459075 v1 are also frequently not stable during extended storage periods, and triazole derivatives can display biological activity, which may lead to unintended effects when the oligonucleotide conjugates are provided in vivo.
  • Maleimides are the most frequently used reagent in oligonucleotide conjugation reactions. However, maleimide linkers have a number of significant disadvantages.
  • Maleimide modified oligonucleotides can be challenging to synthesise as the maleimide group is generally not stable to the conditions used in oligonucleotide synthesis.
  • two approaches are used to mitigate this, often the oligonucleotide must be synthesised with an amine modification and it is only after the oligonucleotide has been cleaved from the solid support and purified that this amine is reacted with a bifunctional small molecule linker with both an N ⁇ hydroxysuccinimide (NHS) ester and maleimide group.
  • the alternative approach is to make use of commercially available maleimide modifiers, which are both expensive and require additional processing steps.
  • maleimide functionalised oligonucleotides also generally display a relatively short half ⁇ life in pH neutral conditions. They also display a narrow pH range for selective reaction with thiols over amines, and a lack of selectivity between thiols and disulfide bonds. [0008] Accordingly, there is a need to provide new linkers for oligonucleotides.
  • a compound of formula (I) wherein: A is mono ⁇ or bicyclic heteroaryl group in which at least one carbon ring ⁇ atom is replaced with N; X, Y, and Z are each independently selected from C 1 ⁇ 30 alkyl, C 1 ⁇ 30 alkenyl, (S) n , (O) n , NR 5 , (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, C 5 ⁇ 10 heteroaryl, or absent; wherein at least one of X, Y and Z is present; wherein each C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl or C 5 ⁇ 10 heteroaryl is optional
  • a modified oligonucleotide wherein the 5’ end of the oligonucleotide is attached to a compound as described above via a covalent bond which replaces the W moiety to give a modified oligonucleotide of formula (II): wherein Q is S or O.
  • a modified oligonucleotide conjugate wherein the modified oligonucleotide described above is conjugated to a thiol ⁇ containing moiety (M ⁇ SH) via the vinyl group to provide a conjugate of formula (II): wherein Q is S or O.
  • a method of conjugating the modified oligonucleotide described in the second aspect of the invention to a thiol ⁇ containing molecule (M ⁇ SH) to form the modified oligonucleotide conjugate described in the third aspect of the invention wherein the method comprises a thiol ⁇ ene reaction between the thiol and the vinyl group of the modified oligonucleotide the second aspect of the invention.
  • M ⁇ SH thiol ⁇ containing molecule
  • Figure 1a shows hours 0 ⁇ 3 for the time course monitoring via HPLC for the formation of a glutathione ⁇ linker ⁇ oligonucleotide conjugate of the invention.
  • Figure 1b shows hours 3.5 ⁇ 6 for the time course monitoring via HPLC for the formation of a glutathione ⁇ linker ⁇ oligonucleotide conjugate of the invention.
  • Figure 2 is a graph showing the area under the curve for the formation of a glutathione ⁇ linker ⁇ oligonucleotide conjugate of the invention.
  • FIG. 3 shows the SDS ⁇ PAGE for the formation of a human serum albumen (HSA) ⁇ linker ⁇ oligonucleotide conjugate of the invention.
  • HSA human serum albumen
  • FIG. 3 shows the SDS ⁇ PAGE for the formation of a human serum albumen (HSA) ⁇ linker ⁇ oligonucleotide conjugate of the invention.
  • HSA human serum albumen
  • FIG. 3 shows the SDS ⁇ PAGE for the formation of a human serum albumen (HSA) ⁇ linker ⁇ oligonucleotide conjugate of the invention.
  • Halo or “halogen” refers to bromo, chloro, fluoro or iodo radical.
  • “Hydroxy” or “hydroxyl” refers to the ⁇ OH radical.
  • “Nitro” refers to the ⁇ NO2 radical.
  • Alkyl or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain radical having from one to thirty carbon atoms unless otherwise specified, and which is attached to the rest of the molecule by a single bond.
  • Alkyls comprising any number of carbon atoms from 1 to 30 are included.
  • An alkyl comprising up to 10 carbon atoms is a C 1 ⁇ C 10 alkyl
  • an alkyl comprising up to 6 carbon atoms is a C 1 ⁇ C 6 alkyl
  • an alkyl comprising up to 5 carbon atoms is a C 1 ⁇ C 5 alkyl.
  • a C 1 ⁇ C 5 alkyl includes C 5 alkyls, C 4 alkyls, C 3 alkyls, C 2 alkyls and C 1 alkyl (i.e., methyl).
  • a C 1 ⁇ C 6 alkyl includes all moieties described above for C 1 ⁇ C 5 alkyls but also includes C 6 alkyls.
  • a C 1 ⁇ C 10 alkyl includes all moieties described above for C 1 ⁇ C 5 alkyls and C 1 ⁇ C 6 alkyls, but also includes C 7 , C 8 , C 9 and C 10 alkyls.
  • Non ⁇ limiting examples of C 1 ⁇ C 10 alkyl include methyl, ethyl, n ⁇ propyl, i ⁇ propyl, sec ⁇ propyl, n ⁇ butyl, i ⁇ butyl, sec ⁇ butyl, t ⁇ butyl, n ⁇ pentyl, t ⁇ amyl, n ⁇ hexyl, n ⁇ heptyl, n ⁇ octyl, n ⁇ nonyl, and n ⁇ decyl.
  • alkenyl or “alkenyl group” refers to a straight or branched hydrocarbon chain radical having from two to thirty carbon atoms unless otherwise specified, and having one or more carbon ⁇ carbon double bonds.
  • alkenyl or “alkenyl group” refer to straight or branched hydrocarbon chain radicals comprising E and/or Z (trans and/or cis) alkenes. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups comprising any number of carbon atoms from 2 to 30 are included.
  • An alkenyl group comprising up to 10 carbon atoms is a C 2 ⁇ C 10 alkenyl
  • an alkenyl group comprising up to 6 carbon atoms is a C 2 ⁇ C 6 alkenyl
  • an alkenyl group comprising up to 5 carbon atoms is a C 2 ⁇ C 5 alkenyl.
  • a C 2 ⁇ C 5 alkenyl includes C 5 alkenyls, C 4 alkenyls, C 3 alkenyls, and C 2 alkenyls.
  • a C 2 ⁇ C 6 alkenyl includes all moieties described above for C 2 ⁇ C 5 alkenyls but also includes C 6 alkenyls.
  • a C 2 ⁇ C 10 alkenyl includes all moieties described above for C 2 ⁇ C 5 alkenyls and C 2 ⁇ C 6 alkenyls, but also includes C 7 , C 8 , C 9 and C 10 alkenyls.
  • Non ⁇ limiting examples of C 2 ⁇ C 10 alkenyl include ethenyl (vinyl), 1 ⁇ propenyl, 2 ⁇ propenyl (allyl), iso ⁇ propenyl, 2 ⁇ methyl ⁇ 1 ⁇ propenyl, 1 ⁇ butenyl, 2 ⁇ butenyl, 3 ⁇ butenyl, 1 ⁇ pentenyl, 2 ⁇ pentenyl, 3 ⁇ pentenyl, 4 ⁇ pentenyl, 1 ⁇ hexenyl, 2 ⁇ hexenyl, 3 ⁇ hexenyl, 4 ⁇ hexenyl, 5 ⁇ hexenyl, 1 ⁇ heptenyl, 2 ⁇ heptenyl, 3 ⁇ heptenyl, 4 ⁇ heptenyl, 5 ⁇ heptenyl, 6 ⁇ heptenyl, 1 ⁇ octenyl, 2 ⁇ octenyl, 3 ⁇ octenyl, 4 ⁇ octenyl, 5 ⁇ octenyl, 6 ⁇ octenyl, 7 ⁇ octen
  • Alkynyl or “alkynyl group” refers to a straight or branched hydrocarbon chain radical having from two to thirty carbon atoms unless otherwise specified, and having one or more carbon ⁇ carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl groups comprising any number of carbon atoms from 2 to 30 are included.
  • An alkynyl group comprising up to 10 carbon atoms is a C 2 ⁇ C 10 alkynyl
  • an alkynyl group comprising up to 6 carbon atoms is a C 2 ⁇ C 6 alkynyl
  • an alkynyl group comprising up to 5 carbon atoms is a C 2 ⁇ C 5 alkynyl.
  • a C 2 ⁇ C 5 alkynyl includes C 5 alkynyls, C 4 alkynyls, C 3 alkynyls, and C 2 alkynyls.
  • a C 2 ⁇ C 6 alkynyl includes all moieties described above for C 2 ⁇ C 5 alkynyls but also includes C 6 alkynyls.
  • a C 2 ⁇ C 10 alkynyl includes all moieties described above for C 2 ⁇ C 5 alkynyls and C 2 ⁇ C 6 alkynyls, but also includes C 7 , C 8 , C 9 and C 10 alkynyls.
  • Non ⁇ limiting examples of C 2 ⁇ C 10 alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like.
  • “Aryl” refers to a hydrocarbon ring system radical comprising 6 to 18 carbon atoms (C 6 ⁇ C 18 ) and at least one aromatic ring.
  • the aryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems.
  • Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as ⁇ indacene, Sindacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
  • aryl is meant to include aryl radicals that are optionally substituted.
  • Cycloalkyl refers to a stable non ⁇ aromatic monocyclic or polycyclic fully saturated hydrocarbon radical consisting solely of carbon and hydrogen atoms, which can include fused or bridged ring systems, having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7 ⁇ dimethyl ⁇ bicyclo[2.2.1]heptanyl, and the like.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2 ⁇ trifluoroethyl, 1,2 ⁇ difluoroethyl, 3 ⁇ bromo ⁇ 2 ⁇ fluoropropyl, 1,2 ⁇ dibromoethyl, and the like.
  • Heterocyclyl refers to a stable 3 ⁇ to 20 ⁇ membered (also denoted as “C 3 ⁇ C 20 ”) non ⁇ aromatic, partially aromatic, or aromatic ring radical which may consist of two to fourteen carbon atom and from one to six heteroatoms.
  • a heterocyclyl, heterocyclic ring, or heterocycle can also refer to a non ⁇ aromatic, partially aromatic, or aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium.
  • Heterocyclyl or heterocyclic rings include heteroaryls as defined below.
  • the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon, sulfur or selenium atoms in the heterocyclyl radical can be optionally oxidised; the nitrogen atom can be optionally quaternised; and the heterocyclyl radical can be partially or fully saturated.
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2 ⁇ oxopiperazinyl, 2 ⁇ oxopiperidinyl, 2 ⁇ oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4 ⁇ piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 ⁇ oox
  • Heteroaryl refers to a 5 ⁇ to 20 ⁇ membered (also denoted as “C 5 ⁇ C 20 ”) ring system radical comprising hydrogen atoms, one to fourteen carbon atom and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium, and at least one aromatic ring.
  • a heteroaryl can also refer to a ring system radical comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium, and at least one aromatic ring.
  • the heteroaryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon, sulfur or selenium atoms in the heteroaryl radical can be optionally oxidised; the nitrogen atom can be optionally quaternised.
  • Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4 ⁇ benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2 ⁇ a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, fur
  • a 5 ⁇ to 20 ⁇ membered heteroaryl may be denoted herein as “C 5 ⁇ C 20 heteroaryl”, meaning 5 ⁇ to 20 ⁇ membered ring system radical comprising hydrogen atoms, one to fourteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium, and at least one aromatic ring.
  • a 5 ⁇ to 10 ⁇ membered heteroaryl radical may be denoted herein as “C 5 ⁇ C 10 heteroaryl”.
  • Heterocycloalkyl refers to a stable non ⁇ aromatic monocyclic or polycyclic fully saturated or unsaturated hydrocarbon radical which consists of carbon and hydrogen atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium.
  • An “N ⁇ heterocycloalkyl” is a heterocycloalkyl comprising at least one ring nitrogen.
  • the heterocycloalkyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon, sulfur or selenium atoms in the heterocycloalkyl radical can be optionally oxidised; the nitrogen atom can be optionally quaternised; and the heterocycloalkyl radical can be partially or fully saturated.
  • heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2 ⁇ oxopiperazinyl, 2 ⁇ oxopiperidinyl, 2 ⁇ oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4 ⁇ piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 ⁇ oxo ⁇ thiomorpholin
  • a 3 ⁇ to 10 ⁇ membered heterocycloalkyl may be denoted herein as “C 3 ⁇ C 10 heterocycloalkyl”, meaning a stable 3 ⁇ to 10 ⁇ membered non ⁇ aromatic monocyclic or polycyclic fully saturated or unsaturated hydrocarbon radical which consists of carbon and hydrogen atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium.
  • substituted used herein means any of the above groups (e.g.
  • alkyl alkenyl, alkynyl, aryl, cycloalkyl, haloalkyl, heterocyclyl, heterocycloalkyl and/or heteroaryl
  • “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher ⁇ order bond (e.g., a double ⁇ or triple ⁇ bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles.
  • a point of attachment bond denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond.
  • the term “protecting group” refers to a reversibly formed derivative of an existing functional group in a molecule, wherein the functional group is derivatised to decrease its reactivity (i.e. the functional group is “protected”). This is done to prevent the protected functional group from reacting under the synthetic conditions to which the molecule comprising the protected functional group is subjected in one or more subsequent steps.
  • a commonly protected functional group is an alcohol, with common protecting groups being acetyl, trihaloacetyl, benzoyl, benzyl, methoxyethoxymethyl ether, dimethoxytrityl, methoxymethyl ether, p ⁇ methoxybenxyl ether, p ⁇ methoxyphenyl ether, methylthiomethyl ether, pivaloyl, t ⁇ butyl ethers, tetrahydropyranyl, tetrahydrofuran, trityl, silyl ethers such as trimethylsilyl (TMS), t ⁇ butyldimethylsilyl (TBDMS or TBS), tri ⁇ i ⁇ propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers, methyl ethers, and ethoxyethyl
  • Amines are another commonly protected functional group, with common protecting groups being carbobenxyloxy, p ⁇ methoxybenzyl carbonyl, t ⁇ butyloxycarbonyl, 9 ⁇ fluorenylmethyloxycarbonyl (Fmoc), acetyl, trihaloacetyl, benzoyl, benzyl, carbamate, p ⁇ methoxybenzyl, 3,4 ⁇ dimethoxybenzyl, p ⁇ methoxyphenyl, tosyl, troc (tricholoroethyl chloroformate), nosyl, and phthalimidyl.
  • common protecting groups being carbobenxyloxy, p ⁇ methoxybenzyl carbonyl, t ⁇ butyloxycarbonyl, 9 ⁇ fluorenylmethyloxycarbonyl (Fmoc), acetyl, trihaloacetyl, benzoyl, benzyl, carbamate, p ⁇ methoxybenzyl, 3,4 ⁇
  • base labile refers to a group which is removable from a molecule when subjected to basic reaction conditions.
  • a “base labile protecting group” is a protecting group as defined above which can be removed from the relevant functional group via the application of a base.
  • base labile protecting groups include acetyl, benzoyl, Fmoc, trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, cyanoethyl, and phthalimidyl groups.
  • oligonucleotide refers to an oligomer comprising multiple nucleotide monomeric units and may be a nucleic acid or nucleic acid analogue.
  • a “nucleic acid analogue” is a compound that has an arrangement of nucleobases that mimics the arrangement of nucleobases in nucleic acids containing a 2’ deoxyribose 5’ monophosphate or ribose 5’ monophosphate backbone, wherein the nucleic acid analogue is capable of base pairing with a complementary nucleic acid.
  • backbone moieties include amino acids as in peptide nucleic acids, glycol molecules as in glycol nucleic acids, threofuranosyl sugar molecules as in threose nucleic acids, morpholine rings and phosphorodiamidate groups as in morpholinos, and cyclohexenyl molecules as in cyclohexenyl nucleic acids.
  • a “furanose” or “furanosyl” is a carbohydrate containing a 5 ⁇ membered ring system consisting of four carbon atoms and one oxygen atom, i.e. a tetrahydrofuran derivative.
  • furanoses include ribose, deoxyribose and dideoxyribose.
  • Further furanoses include arabinofuranose (arabinose), xylofuranose (xylose), lyxofuranose (lyxose), xylulofuranose (xylulose), ribulofuranose (ribulose), erythrofuranose (erythrose), and threofuranose (threose).
  • furanoses where the tetrahydrofuran is substituted with groups other than OH and CH 2 OH.
  • the furanose may comprise OH protecting groups such as those defined above, e.g.
  • furanose OH may be protected to provide an OMe, OAc, or OBn group.
  • Other substitutions may be any of those described herein.
  • the furanose may be substituted with one or more halo, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, cyano, or nitro groups.
  • Another possible substitution would be a furanose OH being replaced with an ⁇ O ⁇ CH 2 ⁇ CH 2 ⁇ O ⁇ CH 3 group.
  • the oligonucleotides of the invention may comprise a nucleoside or nucleosides that have been modified, for instance the modifications may be to the sugar moiety.
  • the 2’ ⁇ position of the sugar moiety may be modified.
  • a modification may be to any moiety that is not “ ⁇ H” for DNA or not “ ⁇ OH” for RNA. Examples of such 2’ modifications are ⁇ O ⁇ CH 3 or ⁇ O ⁇ CH 2 ⁇ CH 2 ⁇ O ⁇ CH 3 , and further examples are provided herein.
  • the 4’ position of the sugar moiety may be modified, for instance to result in a bridge between the 2’ position and the 4’ position.
  • the oligonucleotide may comprise one, two, three, four, or more types of nucleosides.
  • the oligonucleotide may comprise a combination of modified nucleosides and unmodified nucleosides.
  • the oligonucleotide may comprise only modified nucleosides.
  • the nucleotides of the oligonucleotide may all be modified in the same manner or may be modified in two or more different manners. [0044] Oligonucleotides display directionality in the internucleoside linkages, and as such, the ends of the oligonucleotide are designated the 5’ (five prime) and 3’ (three prime) end.
  • oligonucleotide has the fifth carbon of the sugar ring at its terminus, and the 3’ end terminates in the hydroxyl group of the third carbon in the sugar ring.
  • An exemplary nucleoside is provided below, with the 5’ and 3’ carbons labelled: .
  • oligonucleotides are prepared from the 3’ end to the 5’ end, i.e. with each additional nucleotide attached to the 5’ end of the oligonucleotide. Often, oligonucleotides are prepared making use of solid phase synthesis.
  • the 3’ end of the first nucleotide is bonded to a solid support and each additional nucleotide is reacted in turn to provide the desired oligonucleotide.
  • the 3’ end of the oligonucleotide is cleaved from the solid support so that the oligonucleotide may be isolated.
  • the oligonucleotide may comprise a modification to one or more internucleoside linkages.
  • the oligonucleotide may comprise one or more phosphorothioate linkages.
  • all of the internucleotide linkages within the oligonucleotide are phosphorothioate linkages.
  • the oligonucleotide may be or may comprise an oligonucleotide phosphorothioate.
  • the oligonucleotide may comprise one or more phosphorodiamidate linkage.
  • all of the intermonomer linkages within the oligonucleotide are phosphorodiamidate linkages.
  • the oligonucleotide may comprise one or more of a deoxyribonucleotide, a ribonucleotide, an arabinonucleotide, a 2′ ⁇ Fluoroarabinonucleotide (FANA), a 2′ ⁇ O ⁇ methyl (2’OMe) nucleotide, a phosphorothioate 2′ ⁇ O ⁇ methyl (PS ⁇ 2’OMe) nucleotide, a 2' ⁇ O ⁇ methoxyethyl (MOE) nucleotide, a phosphorothioate 2’ ⁇ O ⁇ methoxyethyl (PS ⁇ MOE) nucleotide, a phosphorodiamidate morpholino monomer, a locked nucleotide, a P ⁇ alkyl phosphonate nucleotide, a threose nucleotide, a hexitol nucleotide, a 2’ hydroxy ⁇ he
  • the oligonucleotide may be or may comprise a DNA oligomer, an RNA oligomer, an arabinonucleic acid (ANA) oligomer, a 2′ ⁇ Fluoroarabinonucleic acid (FANA) oligomer, a 2′ ⁇ O ⁇ methyl ribonucleic acid (2’OMe) oligomer, a phosphorothioate 2′ ⁇ O ⁇ methyl ribonucleic acid (PS ⁇ 2’OMe) oligomer, a 2' ⁇ O ⁇ methoxyethyl (MOE) nucleic acid oligomer, a phosphorothioate 2’ ⁇ O ⁇ methoxyethyl (PS ⁇ MOE) nucleic acid oligomer, a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic acid (LNA) oligomer, a P ⁇ alkyl phosphonate nucleic acid (phNA)
  • any of the oligonucleotides of the present disclosure may be present as a pharmaceutically acceptable salt, ester, salt of said ester, or hydrate of said oligonucleotide, and references to an oligonucleotide encompass such compounds.
  • the oligonucleotide of the present disclosure may be present as a prodrug.
  • the oligonucleotide is an antisense oligonucleotide.
  • An antisense oligonucleotide in the context of the present disclosure, is an oligonucleotide comprising subunits that comprise moieties capable of binding to nucleobases.
  • antisense oligonucleotides can be designed to be capable of hybridising to specific nucleic acid sequences.
  • the subunits may be monomers, each monomer comprising a moiety capable of binding to a nucleobase.
  • the moieties capable of binding to a nucleobase may bind by base ⁇ specific hydrogen bonding, such as Watson ⁇ Crick base pairing.
  • the term “antisense” refers to oligonucleotides that are at least partially complementary to a region of a sense strand of a nucleic acid. The degree of complementarity may not be exact, as long as the antisense oligonucleotide and the pre ⁇ mRNA can hybridise under physiological conditions.
  • the antisense oligonucleotide and the pre ⁇ mRNA may be complementary apart from 5, 4, 3, 2, or 1 mismatches.
  • the antisense oligonucleotide is perfectly complementary to a region of the pre ⁇ mRNA.
  • the antisense oligonucleotide comprises a region that is perfectly complementary to a region of the pre ⁇ mRNA, wherein the complementary regions are of a sufficient length to allow binding by hybridisation.
  • aptamer refers to oligonucleotides that are short, single stranded nucleic acids or nucleic acid analogues, such as single stranded DNA (ssDNA) or single stranded RNA (ssRNA), which selectively bind to a specific target.
  • This target may be a protein, peptide, carbohydrate, small molecule, toxin, or potentially a cell.
  • Aptamers are highly versatile as their binding is determined by their tertiary structure. Target recognition and binding results from the aptamer “fitting” the desired target through base stacking, intercalation, hydrophobic interactions and sterics.
  • siRNA short interfering RNA
  • siRNAs may be synthetic or naturally occurring. Typically, siRNAs are 20 ⁇ 24 base pairs long. Typically, siRNAs include a number of overhanging nucleotides (for example, 1, 2, or 3) at each end. These siRNAs are therefore typically 22 ⁇ 30 nucleotides long. siRNAs are used in gene silencing as these siRNAs will bind to their complementary sequence and prevent gene transcription.
  • splice ⁇ switching antisense oligonucleotide refers to synthetic antisense nucleic acid oligomers which are configured to inhibit or prevent protein production relating to a specific gene. Splice ⁇ switching antisense oligonucleotides bind to pre ⁇ mRNA via base pairing, preventing the pre ⁇ mRNA from interacting with the splicing machinery of the cell. This disrupts the normal splicing process of gene transcription by blocking either RNA ⁇ RNA base pairing or protein ⁇ RNA binding resulting from the pre ⁇ mRNA/splicing machinery interactions.
  • miRNA refers to oligonucleotides that are short, single stranded RNA molecules which are non ⁇ coding. Typically miRNAs are 21 ⁇ 23 nucleotides long. miRNAs may be synthetic or naturally occurring. These miRNAs are used to base pair to mRNA molecules comprising the complementary nucleotide sequence. These mRNA molecules are then gene silenced.
  • xeno nucleic acid (XNA) oligomers are synthetic nucleic acid analogues where the naturally occurring sugar backbone (2’ deoxyribose 5’ monophosphate or ribose 5’ monophosphate) of an RNA or DNA sequence has been replaced with a synthetic alternative resulting in an arrangement of nucleobases that mimics the arrangement of nucleobases in nucleic acids containing 2’ deoxyribose 5’ monophosphate or ribose 5’ monophosphate.
  • These nucleic acid analogues are generally capable of base pairing with a complementary nucleic acids.
  • This synthetic alternative may be an alternate sugar or may be a non ⁇ sugar moiety.
  • Exemplary sugar replacing/backbone moieties include amino acids as in peptide nucleic acids, glycol molecules as in glycol nucleic acids, threofuranosyl sugar molecules as in threose nucleic acids, morpholine rings and phosphorodiamidate groups as in morpholinos, cyclohexenyl molecules as in cyclohexenyl nucleic acids, 1,5 ⁇ anhydrohexitol nucleic acids, locked nucleic acids, and fluoro arabino nucleic acids.
  • Locked nucleic acids are nucleic acids where a carbon bridge has been introduced to ribose to connect the 2’ oxygen and the 4’ carbon, “locking” the sugar conformation.
  • PNAs are nucleic acids where the sugar ⁇ phosphate backbone has been replaced with a synthetic peptide, i.e. a polymer prepared from amino acid monomers.
  • Gapmers are short antisense oligonucleotide structures comprising a DNA sequence with RNA ⁇ like sequences on both sides of the DNA sequence. These gapmers are designed to take part in gene silencing and are designed to hybridise to a target piece of RNA. Once hybridised to the target RNA, RNase H cleavage is induced. [0059] As used in this disclosure the terms “expanded RNA” (xRNA) and “expanded DNA” (xDNA) refer to nucleotide systems which comprise size ⁇ expanded nucleotides.
  • Size ⁇ expanded nucleotides are nucleotides where the naturally occurring bases are fused with a benzene ring to provide the four expanded bases xA, xG, xT, and xC, which have the structures shown below. These expanded bases generally form base pairs with the associated natural base, i.e. xA ⁇ T, xG ⁇ C, xT ⁇ A, and xC ⁇ G.
  • Physiological conditions are the conditions (such as temperature, pH, concentration of various ions, etc) found in natural, in vivo, situations, or conditions that correspond such a situation.
  • the antisense oligonucleotide may bind by hybridisation in intracellular conditions, such as the intracellular conditions of human cells.
  • the physiological conditions may be those during pre ⁇ mRNA splicing in cells found in the liver, for instance the intracellular conditions of human hepatocytes. Confirmation of hybridisation in physiological conditions may be performed in vitro, for instance at a temperature, pH, and salt concentration that approximates intracellular conditions.
  • A is mono ⁇ or bicyclic heteroaryl group in which at least one carbon ring ⁇ atom is replaced with N;
  • X, Y, and Z are each independently selected from C 1 ⁇ 30 alkyl, C 1 ⁇ 30 alkenyl, (S) n , (O) n , NR 5 , (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, C 5 ⁇ 10 heteroaryl, or absent; wherein at least one of X, Y and Z is present; wherein each C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl or C 5 ⁇ 10 heteroaryl is optionally substituted with at least one group selected from halo, C 1
  • X ⁇ Y ⁇ Z is not a furanosyl group.
  • X ⁇ Y ⁇ Z does not comprise a furanosyl group.
  • A is able to undertake Watson ⁇ Crick base pairing.
  • A is a natural or artificial nucleobase.
  • a nucleobase, or nitrogenous base is a nitrogen ⁇ containing biological compound which is bonded to a five ⁇ carbon sugar (ribose or 2’ ⁇ deoxyribose) to form a nucleoside.
  • Nucleobases generally have ring structures which are derived from purine (purine bases) or pyrimidine (pyrimidine bases). There are five “primary” or “canonical” nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). These five primary nucleobases are natural nucleobases. Non ⁇ primary nucleobases may also be natural nucleobases, i.e. modified nucleobases which occur in nature, such as aminoadenine (Z).
  • nucleobases include the modified nucleobases 5 ⁇ methylcytosine (m 5 C), pseudouridine ( ⁇ ), dihydrouridine (D), inosine (I), and 7 ⁇ methylguanosine (m 7 G).
  • Artificial nucleobases are capable of forming hydrogen bonds and therefore are capable of taking part in Watson ⁇ Crick base pairing via these hydrogen bonds.
  • Other artificial nucleobases include aminoallyl nucleobases such as aminoallyl uracil or aminoallyl cytosine which are used in post ⁇ labelling of nucleic acids by fluorescence detection, i.e. providing a non ⁇ isotopic tag.
  • both and X may be attached to A at any appropriate point of connection.
  • A is monocyclic, and X are attached in a meta or para configuration.
  • A is monocyclic, and X are attached in a meta configuration.
  • A is monocyclic, and X are attached in a para configuration.
  • A is selected from: be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration.
  • A is monocyclic, and X are attached in a meta configuration.
  • A is selected from: appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration.
  • A is monocyclic
  • X are attached in a meta configuration.
  • A is monocyclic
  • X are attached in a para configuration.
  • . [0080] In some embodiments, .
  • A is selected from: embodiments, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration.
  • A is monocyclic
  • X are attached in a meta configuration.
  • A is monocyclic
  • X are attached in a para configuration.
  • A is selected from: embodiments, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration.
  • A is monocyclic
  • X are attached in a meta configuration.
  • A is monocyclic
  • X are attached in a para configuration.
  • [0089] In some embodiments, [0090] In some embodiments, [0091] In some embodiments, [0092] In some embodiments, [0093] In some embodiments, [0094] In some embodiments, [0095] In some embodiments, .
  • R 1 is selected from H, cyanoethyl, trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, or phthalimidyl.
  • R 1 is H.
  • R 1 is cyanoethyl.
  • R 1 is trialkylsilyl.
  • R 1 is tert ⁇ butyldiphenylsilyl.
  • R 1 is acetyl.
  • R 1 is trihaloacetyl. In some embodiments, R 1 is phthalimidyl.
  • R 2 is selected from H, cyanoethyl, trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, or phthalimidyl. In some embodiments, R 2 is H. In some embodiments, R 2 is cyanoethyl. In some embodiments, R 2 is trialkylsilyl. In some embodiments, R 2 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 2 is acetyl.
  • R 2 is trihaloacetyl.
  • R 1 is phthalimidyl.
  • R 3 and R 4 are each an optionally substituted C 1 ⁇ 10 alkyl.
  • R 3 and R 4 are each an unsubstituted branched chain C 1 ⁇ 10 alkyl.
  • R 3 and R 4 are each isopropyl.
  • R 5 is absent, or is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 5 is absent. In some embodiments, R 5 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 5 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 5 is trialkylsilyl.
  • R 5 is tert ⁇ butyldiphenylsilyl.
  • R 5 is acetyl.
  • R 5 is trihaloacetyl.
  • R 5 is phthalimidyl.
  • R 5 is H.
  • R 5 is C 1 ⁇ 10 alkyl.
  • R 5 is C 2 ⁇ 10 alkenyl.
  • R 5 is C 2 ⁇ 10 alkynyl.
  • R 6 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 6 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 6 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 6 is trialkylsilyl. In some embodiments, R 6 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 6 is acetyl. In some embodiments, R 6 is trihaloacetyl. In some embodiments, R 6 is phthalimidyl. In some embodiments, R 6 is H. In some embodiments, R 6 is C 1 ⁇ 10 alkyl. In some embodiments, R 6 is C 2 ⁇ 10 alkenyl. In some embodiments, R 6 is C 2 ⁇ 10 alkynyl.
  • R 7 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 7 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 7 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 7 is trialkylsilyl. In some embodiments, R 7 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 7 is acetyl. In some embodiments, R 7 is trihaloacetyl. In some embodiments, R 7 is phthalimidyl. In some embodiments, R 7 is H. In some embodiments, R 7 is C 1 ⁇ 10 alkyl. In some embodiments, R 7 is C 2 ⁇ 10 alkenyl. In some embodiments, R 7 is C 2 ⁇ 10 alkynyl.
  • R 8 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 8 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 8 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 8 is trialkylsilyl. In some embodiments, R 8 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 8 is acetyl. In some embodiments, R 8 is trihaloacetyl. In some embodiments, R 8 is phthalimidyl. In some embodiments, R 8 is H. In some embodiments, R 8 is C 1 ⁇ 10 alkyl. In some embodiments, R 8 is C 2 ⁇ 10 alkenyl. In some embodiments, R 8 is C 2 ⁇ 10 alkynyl.
  • R 12 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 12 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 12 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 12 is trialkylsilyl. In some embodiments, R 12 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 12 is acetyl. In some embodiments, R 12 is trihaloacetyl. In some embodiments, R 12 is phthalimidyl. In some embodiments, R 12 is H. In some embodiments, R 12 is C 1 ⁇ 10 alkyl. In some embodiments, R 12 is C 2 ⁇ 10 alkenyl. In some embodiments, R 12 is C 2 ⁇ 10 alkynyl.
  • R 13 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 13 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 13 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 13 is trialkylsilyl. In some embodiments, R 12 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 13 is acetyl. In some embodiments, R 13 is trihaloacetyl. In some embodiments, R 13 is phthalimidyl. In some embodiments, R 13 is H. In some embodiments, R 13 is C 1 ⁇ 10 alkyl. In some embodiments, R 13 is C 2 ⁇ 10 alkenyl. In some embodiments, R 13 is C 2 ⁇ 10 alkynyl.
  • R 14 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 14 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 14 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 14 is trialkylsilyl. In some embodiments, R 14 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 14 is acetyl. In some embodiments, R 14 is trihaloacetyl. In some embodiments, R 14 is phthalimidyl. In some embodiments, R 14 is H. In some embodiments, R 14 is C 1 ⁇ 10 alkyl. In some embodiments, R 14 is C 2 ⁇ 10 alkenyl. In some embodiments, R 14 is C 2 ⁇ 10 alkynyl.
  • R 18 when present, is selected from H, an optionally substituted C 1 ⁇ 10 alkyl group, trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 18 is selected from H and an optionally substituted C 1 ⁇ 10 alkyl group. In some embodiments, R 18 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 18 is H. In some embodiments, R 18 is an optionally substituted C 1 ⁇ 10 alkyl group.
  • R 18 is trialkylsilyl. In some embodiments, R 18 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 18 is acetyl. In some embodiments, R 18 is trihaloacetyl. In some embodiments, R 18 is phthalimidyl. [0110] In some embodiments, R 20 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 20 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 20 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 20 is trialkylsilyl. In some embodiments, R 20 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 20 is acetyl. In some embodiments, R 20 is trihaloacetyl. In some embodiments, R 20 is phthalimidyl. In some embodiments, R 20 is H.
  • R 20 is C 1 ⁇ 10 alkyl. In some embodiments, R 20 is C 2 ⁇ 10 alkenyl. In some embodiments, R 20 is C 2 ⁇ 10 alkynyl. [0111] In some embodiments, R 21 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 21 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 21 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 21 is trialkylsilyl. In some embodiments, R 21 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 21 is acetyl. In some embodiments, R 21 is trihaloacetyl. In some embodiments, R 21 is phthalimidyl. In some embodiments, R 21 is H.
  • R 21 is C 1 ⁇ 10 alkyl. In some embodiments, R 21 is C 2 ⁇ 10 alkenyl. In some embodiments, R 21 is C 2 ⁇ 10 alkynyl. [0112] In some embodiments, R 22 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 22 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 22 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 22 is trialkylsilyl. In some embodiments, R 22 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 22 is acetyl. In some embodiments, R 22 is trihaloacetyl. In some embodiments, R 22 is phthalimidyl. In some embodiments, R 22 is H.
  • R 22 is C 1 ⁇ 10 alkyl. In some embodiments, R 22 is C 2 ⁇ 10 alkenyl. In some embodiments, R 22 is C 2 ⁇ 10 alkynyl. [0113] In the preceding embodiments, the trialkylsilyl group, if present, may have the formula Si(C 1 ⁇ C 4 alkyl) 3 . In further embodiments, trialkylsilyl group, if present, is selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). In further embodiments, the trialkylsilyl group is trimethylsilyl (TMS).
  • the trialkylsilyl group is triethylsilyl (TES). In further embodiments, the trialkylsilyl group is tert ⁇ butyldimethylsilyl (TBS). In further embodiments, the trialkylsilyl group is triisopropylsilyl (TIPS).
  • the trihaloacetyl group if present, is trichloroacetyl or trifluoroacetyl. In some embodiments, the trihaloacetyl group is trichloroacetyl. In some embodiments, the trihaloacetyl group is trifluoroacetyl.
  • the base labile protecting group may be selected from trialkylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trihalo acetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trichloroacetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trihalo acetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trichloroacetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trifluoroacetyl, and phthalimidyl.
  • R 9 is H.
  • X is selected from NR 5 , C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl; wherein each is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6 , C(O)NR 7 R 8 , SR 6 , S(O)R 6 , S(O) 2 R 6 , P(O)(OR 6 ) 2 , CN, NO 2 , and N 3 .
  • X is NR 5 .
  • X is C 3 ⁇ 10 cycloalkyl. In some embodiments, X is C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is C 5 ⁇ 10 cycloalkenyl. In some embodiments, X is C 6 ⁇ 10 aryl. In some embodiments, X is C 5 ⁇ 10 heteroaryl. [0123] In some embodiments, X is selected from NR 5 , C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl. In some embodiments, X is NR 5 . In some embodiments, X is C 3 ⁇ 10 cycloalkyl.
  • X is C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is C 5 ⁇ 10 cycloalkenyl. In some embodiments, X is C 6 ⁇ 10 aryl. In some embodiments, X is C 5 ⁇ 10 heteroaryl. [0124] In some embodiments, X is selected from NR 5 and C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is NR 5 . In some embodiments, X is C 3 ⁇ 10 heterocycloalkyl. [0125] In some embodiments, X is selected from NR 5 , pyrrolidine, and piperidine. In some embodiments, X is NR 5 . In some embodiments, X is pyrrolidine.
  • X is piperidine.
  • Y is selected from C 1 ⁇ 30 alkyl, (CH 2 CH 2 O) m , C(O)n, C 3 ⁇ 10 cycloalkyl, and C 3 ⁇ 10 heterocycloalkyl; wherein each C 3 ⁇ 10 cycloalkyl or C 3 ⁇ 10 heterocycloalkyl is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6 , C(O)NR 7 R 8 , SR 6 , S(O)R 6 , S(O) 2 R 6 , P(O)(OR 6 ) 2 , CN, NO 2 , and N 3 .
  • Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is (CH 2 CH 2 O) m . In some embodiments, Y is C(O)n. In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. In some embodiments, Y is C 3 ⁇ 10 heterocycloalkyl. [0127] In some embodiments, Y is selected from C 1 ⁇ 30 alkyl, (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, and C 3 ⁇ 10 heterocycloalkyl. In some embodiments, Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is (CH 2 CH 2 O) m .
  • Y is C(O). In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. In some embodiments, Y is C 3 ⁇ 10 heterocycloalkyl. [0128] In some embodiments, Y is selected from C 1 ⁇ 30 alkyl, C(O), and C 3 ⁇ 10 cycloalkyl. In some embodiments Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is C(O). In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. [0129] In some embodiments, X ⁇ Y ⁇ Z comprises at least one tertiary amine.
  • X ⁇ Y ⁇ Z comprises at least one of NR 5 , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 N ⁇ heterocycloalkyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl.
  • X ⁇ Y ⁇ Z comprises at least one of NR 5 , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 N ⁇ heterocycloalkyl, and C 6 ⁇ 10 aryl.
  • X ⁇ Y ⁇ Z comprises NR 5 .
  • X ⁇ Y ⁇ Z comprises C(O).
  • X ⁇ Y ⁇ Z comprises C 3 ⁇ 10 cycloalkyl. [0135] In some embodiments, X ⁇ Y ⁇ Z comprises C 3 ⁇ 10 N ⁇ heterocycloalkyl. [0136] In some embodiments, X ⁇ Y ⁇ Z comprises C 6 ⁇ 10 aryl. [0137] In some embodiments, X ⁇ Y ⁇ Z comprises C 5 ⁇ 10 heteroaryl. [0138] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0139] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0144] In some embodiments, . [0145] In some [0146] In some [0147] In some embodiments, . [0148] In some embodiments, .
  • X ⁇ Y ⁇ Z is selected from: , [0154] In some embodiments, X ⁇ Y ⁇ Z is selected from: , [0155] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0156] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0161] In some embodiments, . [0162] In some embodiments, . [0163] In some embodiments, . [0164] In some embodiments, . [0165] In some embodiments, . [0166] In some embodiments, . [0167] In some embodiments, . [0168] In some embodiments, .
  • the compound is selected from: , [0173] In some embodiments, the compound is selected from: . [0174] In some embodiments, the compound is selected from: [0175] In some embodiments, the compound is selected from: . [0176] In some embodiments, the compound is: . [0177] In some embodiments, the compound . [0178] In some embodiments, the compound is: . [0179] In some embodiments, the compound is: . [0180] In some embodiments, the compound is: . [0181] In some embodiments, the compound is: . [0182] In some embodiments, the compound is: . [0183] In some embodiments, the compound is: .
  • the compound is: . [0185] In some embodiments, the compound is: . [0186] In some embodiments, the compound is selected from: [0187] In some embodiments, the compound is selected from: . [0189] In some embodiments, the compound is selected from: [0191] In some embodiments, the compound is: . [0193] In some embodiments, the compound is: . [0194] In some embodiments, the compound is: . [0195] In some embodiments, the compound is: . [0196] In some embodiments, the compound is: . [0197] In some embodiments, the compound is: . [0198] In some embodiments, the compound is: .
  • the compound is: . [0201] In some embodiments, the compound is: . [0202] In some embodiments, the compound is: . [0203] In some embodiments, the compound . [0205] In some embodiments, the compound is selected from: [0206] In some embodiments, the compound is selected from: [0207] In some embodiments, the compound is selected [ [0209] In some embodiments, the compound i [0210] In some embodiments, the compound i [0211] In some embodiments, the compound i [0212] In some embodiments, the compound i [0213] In some embodiments, the compound . [0214] In some embodiments, the compound is: .
  • the compound is: . [0217] In some embodiments, the compound is: . [0218] In some embodiments, the compound is: . [0221] In some embodiments, the compound is: . [0222] In some embodiments, the compound is: . [0223] In some embodiments, the compound is . [0224] In some embodiments, the compound is: . Modified oligonucleotide [0225] The compounds described above are “building blocks” for use in the conjugation of oligonucleotides to other structures of interest, such as small molecules or peptides.
  • the phosphite/phosphoramidite portion of these compounds is suitable for reaction with the 5’ end of an oligonucleotide (i.e. with the 5’ sugar hydroxyl group), such that the linker is incorporated into the oligonucleotide backbone.
  • the phosphite/phosphoramidite portion of the compounds of the invention may be reacted with an oligonucleotide under the same or similar synthetic conditions as used to build the oligonucleotide itself. This approach minimises the risk of degrading the oligonucleotides when introducing the linker moiety.
  • the linker molecule will be reacted with the oligonucleotide first to provide a modified oligonucleotide.
  • This modified oligonucleotide may then be reacted with a molecule containing at least one thiol group.
  • a modified oligonucleotide wherein the 5’ end of the oligonucleotide is attached to any of the compounds described above via a covalent bond which replaces the W moiety to give a modified oligonucleotide of formula (II): ; wherein Q is S or O.
  • the oligonucleotide is an antisense oligonucleotide. In some embodiments, the oligonucleotide is an RNA interference (RNAi) oligonucleotide. In some embodiments, the oligonucleotide is an aptamer. In some embodiments, the oligonucleotide is a short interfering RNA (siRNA) oligonucleotide. In some embodiments, the oligonucleotide is a splice ⁇ switching antisense oligonucleotide. In some embodiments, the oligonucleotide is an microRNA (miRNA) oligonucleotide.
  • miRNA microRNA
  • the oligonucleotide is a DNA oligonucleotide. In some embodiments, the oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is an XNA. In some embodiments, the oligonucleotide is an xRNA. In some embodiments, the oligonucleotide is an xDNA. In some embodiments, the oligonucleotide is an saRNA. [0228] The features of the linker portion of the above modified oligonucleotide correspond to the features of the above ⁇ described compounds.
  • A is mono ⁇ or bicyclic heteroaryl group in which at least one carbon ring ⁇ atom is replaced with N;
  • X, Y, and Z are each independently selected from C 1 ⁇ 30 alkyl, C 1 ⁇ 30 alkenyl, (S) n , (O) n , NR 5 , (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, C 5 ⁇ 10 heteroaryl, or absent; wherein at least one of X, Y and Z is present; wherein each C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl or C 5 ⁇ 10 heteroaryl is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6
  • X ⁇ Y ⁇ Z is not a furanosyl group.
  • X ⁇ Y ⁇ Z does not comprise a furanosyl group.
  • A is able to undertake Watson ⁇ Crick base pairing.
  • A is a natural or artificial nucleobase.
  • a nucleobase, or nitrogenous base is a nitrogen ⁇ containing biological compound which are bonded to a five ⁇ carbon sugar (ribose or 2’ ⁇ deoxyribose) to form a nucleoside.
  • Nucleobases generally have ring structures which are derived from purine (purine bases) or pyrimidine (pyrimidine bases). There are five “primary” or “canonical” nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). These five primary nucleobases are natural nucleobases. Non ⁇ primary nucleobases may also be natural nucleobases, i.e. modified nucleobases which occur in nature, such as aminoadenine (Z).
  • nucleobases include the modified nucleobases 5 ⁇ methylcytosine (m 5 C), pseudouridine ( ⁇ ), dihydrouridine (D), inosine (I), and 7 ⁇ methylguanosine (m 7 G).
  • Artificial nucleobases are capable of forming hydrogen bonds and therefore are capable of taking part in base pairing via these hydrogen bonds.
  • Other artificial nucleobases include aminoallyl nucleobases such as aminoallyl uracil or aminoallyl cytosine which are used in post ⁇ labelling of nucleic acids by fluorescence detection, i.e. providing a non ⁇ isotopic tag.
  • both and X may be attached to A at any appropriate point of connection.
  • A is monocyclic, and X are attached in a meta or para configuration.
  • A is monocyclic, and X are attached in a meta configuration.
  • A is monocyclic, and X are attached in a para configuration.
  • A is selected from: be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration.
  • A is monocyclic, and X are attached in a meta configuration.
  • A is selected from: and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is monocyclic, and X are attached in a meta or para configuration.
  • A is selected from: embodiments, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is monocyclic, and X are attached in a meta or para configuration.
  • A is monocyclic
  • X are attached in a meta configuration. In some embodiments where A is monocyclic, and X are attached in a para configuration.
  • A is selected from: embodiments, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration. In some embodiments where A is monocyclic, and X are attached in a meta configuration. In some embodiments where A is monocyclic, and X are attached in a para configuration.
  • R 5 is absent, or is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 5 is absent.
  • R 5 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 5 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 5 is trialkylsilyl.
  • R 5 is tert ⁇ butyldiphenylsilyl.
  • R 5 is acetyl.
  • R 5 is trihaloacetyl.
  • R 5 is phthalimidyl.
  • R 5 is H. In some embodiments, R 5 is C 1 ⁇ 10 alkyl. In some embodiments, R 5 is C 2 ⁇ 10 alkenyl. In some embodiments, R 5 is C 2 ⁇ 10 alkynyl. [0267] In some embodiments, R 6 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 6 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 6 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 6 is trialkylsilyl. In some embodiments, R 6 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 6 is acetyl. In some embodiments, R 6 is trihaloacetyl. In some embodiments, R 6 is phthalimidyl. In some embodiments, R 6 is H.
  • R 6 is C 1 ⁇ 10 alkyl. In some embodiments, R 6 is C 2 ⁇ 10 alkenyl. In some embodiments, R 6 is C 2 ⁇ 10 alkynyl. [0268] In some embodiments, R 7 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 7 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 7 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 7 is trialkylsilyl.
  • R 7 is tert ⁇ butyldiphenylsilyl.
  • R 7 is acetyl.
  • R 7 is trihaloacetyl.
  • R 7 is phthalimidyl.
  • R 7 is H.
  • R 7 is C 1 ⁇ 10 alkyl.
  • R 7 is C 2 ⁇ 10 alkenyl.
  • R 7 is C 2 ⁇ 10 alkynyl.
  • R 8 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 8 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 8 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 8 is trialkylsilyl. In some embodiments, R 8 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 8 is acetyl. In some embodiments, R 8 is trihaloacetyl. In some embodiments, R 8 is phthalimidyl. In some embodiments, R 8 is H. In some embodiments, R 8 is C 1 ⁇ 10 alkyl. In some embodiments, R 8 is C 2 ⁇ 10 alkenyl. In some embodiments, R 8 is C 2 ⁇ 10 alkynyl.
  • R 12 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 12 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 12 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 12 is trialkylsilyl. In some embodiments, R 12 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 12 is acetyl. In some embodiments, R 12 is trihaloacetyl. In some embodiments, R 12 is phthalimidyl. In some embodiments, R 12 is H. In some embodiments, R 12 is C 1 ⁇ 10 alkyl. In some embodiments, R 12 is C 2 ⁇ 10 alkenyl. In some embodiments, R 12 is C 2 ⁇ 10 alkynyl.
  • R 13 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 13 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 13 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 13 is trialkylsilyl. In some embodiments, R 13 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 13 is acetyl. In some embodiments, R 13 is trihaloacetyl. In some embodiments, R 13 is phthalimidyl. In some embodiments, R 13 is H. In some embodiments, R 13 is C 1 ⁇ 10 alkyl. In some embodiments, R 13 is C 2 ⁇ 10 alkenyl. In some embodiments, R 13 is C 2 ⁇ 10 alkynyl.
  • R 14 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 14 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 14 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 14 is trialkylsilyl. In some embodiments, R 14 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 14 is acetyl. In some embodiments, R 14 is trihaloacetyl. In some embodiments, R 14 is phthalimidyl. In some embodiments, R 14 is H. In some embodiments, R 14 is C 1 ⁇ 10 alkyl. In some embodiments, R 14 is C 2 ⁇ 10 alkenyl. In some embodiments, R 14 is C 2 ⁇ 10 alkynyl.
  • R 18 when present, is selected from H, an optionally substituted C 1 ⁇ 10 alkyl group, trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 18 is selected from H and an optionally substituted C 1 ⁇ 10 alkyl group. In some embodiments, R 18 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 18 is H. In some embodiments, R 18 is an optionally substituted C 1 ⁇ 10 alkyl group.
  • R 18 is trialkylsilyl. In some embodiments, R 18 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 18 is acetyl. In some embodiments, R 18 is trihaloacetyl. In some embodiments, R 18 is phthalimidyl. [0274] In some embodiments, R 20 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 20 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 20 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 20 is trialkylsilyl. In some embodiments, R 20 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 20 is acetyl. In some embodiments, R 20 is trihaloacetyl. In some embodiments, R 20 is phthalimidyl. In some embodiments, R 20 is H.
  • R 20 is C 1 ⁇ 10 alkyl. In some embodiments, R 20 is C 2 ⁇ 10 alkenyl. In some embodiments, R 20 is C 2 ⁇ 10 alkynyl. [0275] In some embodiments, R 21 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 21 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 21 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 21 is trialkylsilyl. In some embodiments, R 21 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 21 is acetyl. In some embodiments, R 21 is trihaloacetyl. In some embodiments, R 21 is phthalimidyl. In some embodiments, R 21 is H.
  • R 21 is C 1 ⁇ 10 alkyl. In some embodiments, R 21 is C 2 ⁇ 10 alkenyl. In some embodiments, R 21 is C 2 ⁇ 10 alkynyl. [0276] In some embodiments, R 22 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 22 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 22 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 22 is trialkylsilyl. In some embodiments, R 22 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 22 is acetyl. In some embodiments, R 22 is trihaloacetyl. In some embodiments, R 22 is phthalimidyl. In some embodiments, R 22 is H.
  • R 22 is C 1 ⁇ 10 alkyl. In some embodiments, R 22 is C 2 ⁇ 10 alkenyl. In some embodiments, R 22 is C 2 ⁇ 10 alkynyl. [0277] In the preceding embodiments, the trialkylsilyl group, if present, may have the formula Si(C 1 ⁇ C 4 alkyl) 3 . In further embodiments, trialkylsilyl group, if present, is selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). In further embodiments, the trialkylsilyl group is trimethylsilyl (TMS).
  • the trialkylsilyl group is triethylsilyl (TES). In further embodiments, the trialkylsilyl group is tert ⁇ butyldimethylsilyl (TBS). In further embodiments, the trialkylsilyl group is triisopropylsilyl (TIPS).
  • the trihaloacetyl group if present, is trichloroacetyl or trifluoroacetyl. In some embodiments, the trihaloacetyl group is trichloroacetyl. In some embodiments, the trihaloacetyl group is trifluoroacetyl.
  • the base labile protecting group may be selected from trialkylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trihalo acetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trichloroacetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trihalo acetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trichloroacetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trifluoroacetyl, and phthalimidyl.
  • R 9 is H.
  • X is selected from NR 5 , C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl; wherein each is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6 , C(O)NR 7 R 8 , SR 6 , S(O)R 6 , S(O) 2 R 6 , P(O)(OR 6 ) 2 , CN, NO 2 , and N 3 .
  • X is NR 5 .
  • X is C 3 ⁇ 10 cycloalkyl. In some embodiments, X is C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is C 5 ⁇ 10 cycloalkenyl. In some embodiments, X is C 6 ⁇ 10 aryl. In some embodiments, X is C 5 ⁇ 10 heteroaryl. [0287] In some embodiments, X is selected from NR 5 , C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl. In some embodiments, X is NR 5 . In some embodiments, X is C 3 ⁇ 10 cycloalkyl.
  • X is C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is C 5 ⁇ 10 cycloalkenyl. In some embodiments, X is C 6 ⁇ 10 aryl. In some embodiments, X is C 5 ⁇ 10 heteroaryl. [0288] In some embodiments, X is selected from NR 5 and C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is NR 5 . In some embodiments, X is C 3 ⁇ 10 heterocycloalkyl. [0289] In some embodiments, X is selected from NR 5 , pyrrolidine, and piperidine. In some embodiments, X is NR 5 . In some embodiments, X is pyrrolidine.
  • X is piperidine.
  • Y is selected from C 1 ⁇ 30 alkyl, (CH 2 CH 2 O) m , C(O)n, C 3 ⁇ 10 cycloalkyl, and C 3 ⁇ 10 heterocycloalkyl; wherein each C 3 ⁇ 10 cycloalkyl or C 3 ⁇ 10 heterocycloalkyl is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6 , C(O)NR 7 R 8 , SR 6 , S(O)R 6 , S(O) 2 R 6 , P(O)(OR 6 ) 2 , CN, NO 2 , and N 3 .
  • Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is (CH 2 CH 2 O) m . In some embodiments, Y is C(O)n. In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. In some embodiments, Y is C 3 ⁇ 10 heterocycloalkyl. [0291] In some embodiments, Y is selected from C 1 ⁇ 30 alkyl, (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, and C 3 ⁇ 10 heterocycloalkyl. In some embodiments, Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is (CH 2 CH 2 O) m .
  • Y is C(O). In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. In some embodiments, Y is C 3 ⁇ 10 heterocycloalkyl. [0292] In some embodiments, Y is selected from C 1 ⁇ 30 alkyl, C(O), and C 3 ⁇ 10 cycloalkyl. In some embodiments Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is C(O). In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. [0293] In some embodiments, X ⁇ Y ⁇ Z comprises at least one tertiary amine.
  • X ⁇ Y ⁇ Z comprises at least one of NR 5 , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 N ⁇ heterocycloalkyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl.
  • X ⁇ Y ⁇ Z comprises at least one of NR 5 , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 N ⁇ heterocycloalkyl, and C 6 ⁇ 10 aryl.
  • X ⁇ Y ⁇ Z comprises NR 5 .
  • X ⁇ Y ⁇ Z comprises C(O).
  • X ⁇ Y ⁇ Z comprises C 3 ⁇ 10 cycloalkyl. [0299] In some embodiments, X ⁇ Y ⁇ Z comprises C 3 ⁇ 10 N ⁇ heterocycloalkyl. [0300] In some embodiments, X ⁇ Y ⁇ Z comprises C 6 ⁇ 10 aryl. [0301] In some embodiments, X ⁇ Y ⁇ Z comprises C 5 ⁇ 10 heteroaryl. [0302] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0303] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0306] In some embodiments, . [0307] In some embodiments, . [0308] In some embodiments, . [0309] In some embodiments, .
  • X ⁇ Y ⁇ Z is selected from:
  • X ⁇ Y ⁇ Z is selected from: [0326] In some embodiments, . [0327] In some embodiments, . [0328] In some embodiments, . [0329] In some embodiments, . [0330] In some embodiments, . [0331] In some embodiments, . [0332] In some embodiments, . [0333] In some embodiments, . [0334] In some embodiments, . [0335] In some embodiments, . [0336] In some embodiments, the modified oligonucleotide is selected from: , . [0337] In some embodiments, the modified oligonucleotide is selected from: , . [0338] In some embodiments, the modified oligonucleotide is selected from: , , and . [0339] In some embodiments, the modified oligonucleotide is selected from: , , , , and . [0339] In some embodiments, the modified oligonucle
  • the modified oligonucleotide is: . [0343] In some embodiments, the modified oligonucleotide is: . [0346] In some embodiments, the modified oligonucleotide is: . [0348] In some embodiments, the modified oligonucleotide is: . [0350] In some embodiments, the modified oligonucleotide is selected from: , . [0351] In some embodiments, the modified oligonucleotide is selected from: [0352] In some embodiments, the modified oligonucleotide is selected from: [0354] In some embodiments, the modified oligonucleotide .
  • the modified oligonucleotide is: . [0357] In some embodiments, the modified oligonucleotide is: . [0359] In some embodiments, the modified oligonucleotide is: . [0361] In some embodiments, the modified oligonucleotide is: . [0363] In some embodiments, the modified oligonucleotide is: . [0364] In some embodiments, the modified oligonucleotide is: . modified oligonucleotide .
  • the modified oligonucleotide is selected from: [0370] In some embodiments, the modified oligonucleotide is selected from: . [0371] In some embodiments, the modified oligonucleotide is selected from: . [0372] In some embodiments, the modified oligonucleotide is selected from: the modified oligonucleotide is . [0374] In some embodiments, the modified oligonucleotide . [0376] In some embodiments, the modified oligonucleotide is . [0377] In some embodiments, the modified oligonucleotide is .
  • the modified oligonucleotide is . [0381] In some embodiments, the modified oligonucleotide is . [0383] In some embodiments, the modified oligonucleotide is . [0384] In some embodiments, the modified oligonucleotide is . [0387] In some embodiments, the modified oligonucleotide is . [0389] In some embodiments, the modified oligonucleotide is . [0390] In some embodiments, the modified oligonucleotide is .
  • Oligonucleotide conjugates [0393]
  • the modified oligonucleotides described above can be reacted with other structures of interest, such as small molecules or peptides, which contain at least one thiol group.
  • the resulting structures are oligonucleotide conjugates.
  • a modified oligonucleotide conjugate wherein a modified oligonucleotide as described herein is conjugated to a thiol ⁇ containing moiety (M ⁇ SH) via the vinyl group to provide a conjugate of formula (II): wherein Q is S or O.
  • the oligonucleotide is an antisense oligonucleotide. In some embodiments, the oligonucleotide is an RNA interference (RNAi) oligonucleotide. In some embodiments, the oligonucleotide is an aptamer. In some embodiments, the oligonucleotide is a short interfering RNA (siRNA) oligonucleotide. In some embodiments, the oligonucleotide is a splice ⁇ switching antisense oligonucleotide. In some embodiments, the oligonucleotide is an microRNA (miRNA) oligonucleotide.
  • miRNA microRNA
  • the oligonucleotide is a DNA oligonucleotide. In some embodiments, the oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is an XNA. In some embodiments, the oligonucleotide is an xRNA. In some embodiments, the oligonucleotide is an xDNA. In some embodiments, the oligonucleotide is an saRNA. [0396] In some embodiments, M is a small molecule. For example, in some embodiments, M is a pharmacologically active small molecule. In some embodiments, M is a probe.
  • a probe is a molecule which is used to investigate biological mechanisms or validate targets for drug discovery.
  • Probes may comprise a moiety which allows for their detection by an external source.
  • a probe may be radiolabelled, i.e. may comprise radioisotopes.
  • the probe may comprise a moiety which displays fluorescence or phosphorescence.
  • M is a fluorophore.
  • M is fluorescein or a fluorescein derivative. Fluorescein is a common diagnostic contrast agent with many derivatives which are well known in the art.
  • Exemplary derivatives include fluorescein 5 ⁇ isothiocyanate, fluorescein 6 ⁇ isothiocyanate, mixtures of fluorescein 5 ⁇ isothiocyanate and fluorescein 6 ⁇ isothiocyanate, fluorescein succinimidyl esters, carboxyfluorescein, carboxyfluorescein succinimidyl ester, fluorescein pentafluorophenyl esters, fluorescein tetrafluorophenyl esters, and fluorescein amidites.
  • M is rhodamine or a rhodamine derivative.
  • Rhodamine derivatives are a well ⁇ known family of dyes which feature the rhodamine core structure.
  • rhodamine derivatives include carboxytetramethylrhodamine, tetramethylrhodamine, tetramethylrhodamine isothiocyanate, sulforhodamine 101, sulforhodamine 101 acid chloride (Texas Red), and rhodamine red.
  • M is 4,4 ⁇ difluoro ⁇ 4 ⁇ bora ⁇ 3a,4a ⁇ diaza ⁇ s ⁇ indacene (BODIPY).
  • BODIPY 4,4 ⁇ difluoro ⁇ 4 ⁇ bora ⁇ 3a,4a ⁇ diaza ⁇ s ⁇ indacene
  • M is a radiolabelled molecule.
  • Radiolabelled molecules are typically small molecules which are non ⁇ biologically active and which are isotopically enriched with a radioactive isotope such as 32P, 35S, 14C, and 3H.
  • M is a polypeptide.
  • M is a protein.
  • M is a polypeptide or protein connected to the modified oligonucleotide via a cysteine residue.
  • the polypeptide or protein of the invention may be a carrier protein.
  • a carrier protein is a protein or polypeptide which is capable of transporting an ion, small molecule, or macromolecule across a biological membrane, e.g. into a cell.
  • the carrier protein is selected to transport the modified oligonucleotide into the cell.
  • the oligonucleotide is an antisense oligonucleotide, RNAi oligonucleotide, aptamer, siRNA oligonucleotide, splice ⁇ switching antisense oligonucleotide, miRNA oligonucleotide, DNA oligonucleotide, gapmer, XNA oligonucleotide, xRNA oligonucleotide, xDNA oligonucleotide, or saRNA oligonucleotide which is intended to be transported into cells or a specific cell type.
  • the polypeptide or protein is an antigen binding fragment, a nanobody, or an antibody.
  • Antibodies comprise antigen binding fragments.
  • Antigen binding fragments are polypeptide sequences which bind to antigens.
  • Antigen binding fragments are antigen ⁇ specific, i.e. can only react to and bind to one specific antigen, or can cross ⁇ react, i.e. can react to and bind more than one antigen.
  • Nanobodies, or single ⁇ domain antibodies are a type of antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies generally have a molecular weight around 12 ⁇ 15 kDa and so are significantly lighter than common antibodies which generally have a molecular weight around 150 ⁇ 160 kDa.
  • Nanobodies are capable of selectively binding to a specific antigen.
  • an oligonucleotide By linking an oligonucleotide to an antigen binding fragment, nanobody, or an antibody, it is possible to provide a modified oligonucleotide conjugate which targets, for example, cells displaying a specific antigen. Delivery of a therapeutic oligonucleotide can, therefore, be targeted to only the cells in which the oligonucleotide is desired to have an effect.
  • the polypeptide or protein is a cell ⁇ penetrating peptide (CPP).
  • CPPs are usually short peptides, for example, of less than 30 ⁇ 40 amino acids.
  • CPPs may be derived from proteins or chimeric sequences or may be of completely artificial, synthetic or designed origin. They are occasionally amphipathic, but almost always possess a net positive (cationic) charge at physiological pH. CPPs are able to penetrate biological membranes, to trigger the movement of various biomolecules across cell membranes into the cytoplasm and to improve their intracellular routing, thereby facilitating interactions with the target. CPPs have been shown to be able to deliver oligonucleotide cargos into a wide variety of cell types. [0406] The features of the linker ⁇ oligonucleotide portion of the above modified oligonucleotide conjugate correspond to the features of the above ⁇ described modified oligonucleotides.
  • A is mono ⁇ or bicyclic heteroaryl group in which at least one carbon ring ⁇ atom is replaced with N;
  • X, Y, and Z are each independently selected from C 1 ⁇ 30 alkyl, C 1 ⁇ 30 alkenyl, (S) n , (O) n , NR 5 , (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, C 5 ⁇ 10 heteroaryl, or absent; wherein at least one of X, Y and Z is present; wherein each C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl or C 5 ⁇ 10 heteroaryl is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6
  • X ⁇ Y ⁇ Z is not a furanosyl group.
  • X ⁇ Y ⁇ Z does not comprise a furanosyl group.
  • A is able to undertake Watson ⁇ Crick base pairing.
  • A is a natural or artificial nucleobase.
  • a nucleobase, or nitrogenous base is a nitrogen ⁇ containing biological compound which are bonded to a five ⁇ carbon sugar (ribose or 2’ ⁇ deoxyribose) to form a nucleoside.
  • Nucleobases generally have ring structures which are derived from purine (purine bases) or pyrimidine (pyrimidine bases). There are five “primary” or “canonical” nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). These five primary nucleobases are natural nucleobases. Non ⁇ primary nucleobases may also be natural nucleobases, i.e. modified nucleobases which occur in nature, such as aminoadenine (Z).
  • nucleobases include the modified nucleobases 5 ⁇ methylcytosine (m 5 C), pseudouridine ( ⁇ ), dihydrouridine (D), inosine (I), and 7 ⁇ methylguanosine (m 7 G).
  • Artificial nucleobases are capable of forming hydrogen bonds and therefore are capable of taking part in base pairing via these hydrogen bonds.
  • Other artificial nucleobases include aminoallyl nucleobases such as aminoallyl uracil or aminoallyl cytosine which are used in post ⁇ labelling of nucleic acids by fluorescence detection, i.e. providing a non ⁇ isotopic tag.
  • both and X may be attached to A at any appropriate point of connection.
  • A is monocyclic, and X are attached in a meta or para configuration.
  • A is monocyclic, and X are attached in a meta configuration.
  • A is monocyclic, and X are attached in a para configuration.
  • A is selected from: be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration.
  • A is monocyclic, and X are attached in a meta configuration.
  • A is selected from: , . s, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is monocyclic, and X are attached in a meta or para configuration.
  • A is selected from: embodiments, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is monocyclic, and X are attached in a meta or para configuration.
  • A is monocyclic
  • X are attached in a meta configuration. In some embodiments where A is monocyclic, and X are attached in a para configuration.
  • A is selected from: embodiments, both and X may be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.
  • A is monocyclic
  • X are attached in a meta or para configuration. In some embodiments where A is monocyclic, and X are attached in a meta configuration. In some embodiments where A is monocyclic, and X are attached in a para configuration. .
  • R 5 is absent, or is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 5 is absent.
  • R 5 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 5 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 5 is trialkylsilyl.
  • R 5 is tert ⁇ butyldiphenylsilyl.
  • R 5 is acetyl.
  • R 5 is trihaloacetyl.
  • R 5 is phthalimidyl.
  • R 5 is H. In some embodiments, R 5 is C 1 ⁇ 10 alkyl. In some embodiments, R 5 is C 2 ⁇ 10 alkenyl. In some embodiments, R 5 is C 2 ⁇ 10 alkynyl. [0445] In some embodiments, R 6 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 6 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 6 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 6 is trialkylsilyl. In some embodiments, R 6 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 6 is acetyl. In some embodiments, R 6 is trihaloacetyl. In some embodiments, R 6 is phthalimidyl. In some embodiments, R 6 is H.
  • R 6 is C 1 ⁇ 10 alkyl. In some embodiments, R 6 is C 2 ⁇ 10 alkenyl. In some embodiments, R 6 is C 2 ⁇ 10 alkynyl. [0446] In some embodiments, R 7 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 7 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 7 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 7 is trialkylsilyl.
  • R 7 is tert ⁇ butyldiphenylsilyl.
  • R 7 is acetyl.
  • R 7 is trihaloacetyl.
  • R 7 is phthalimidyl.
  • R 7 is H.
  • R 7 is C 1 ⁇ 10 alkyl.
  • R 7 is C 2 ⁇ 10 alkenyl.
  • R 7 is C 2 ⁇ 10 alkynyl.
  • R 8 selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 8 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • R 8 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 8 is trialkylsilyl. In some embodiments, R 8 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 8 is acetyl. In some embodiments, R 8 is trihaloacetyl. In some embodiments, R 8 is phthalimidyl. In some embodiments, R 8 is H. In some embodiments, R 8 is C 1 ⁇ 10 alkyl. In some embodiments, R 8 is C 2 ⁇ 10 alkenyl. In some embodiments, R 8 is C 2 ⁇ 10 alkynyl.
  • R 12 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 12 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 12 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 12 is trialkylsilyl. In some embodiments, R 12 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 12 is acetyl. In some embodiments, R 12 is trihaloacetyl. In some embodiments, R 12 is phthalimidyl. In some embodiments, R 12 is H. In some embodiments, R 12 is C 1 ⁇ 10 alkyl. In some embodiments, R 12 is C 2 ⁇ 10 alkenyl. In some embodiments, R 12 is C 2 ⁇ 10 alkynyl.
  • R 13 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 13 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 13 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 13 is trialkylsilyl. In some embodiments, R 13 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 13 is acetyl. In some embodiments, R 13 is trihaloacetyl. In some embodiments, R 13 is phthalimidyl. In some embodiments, R 13 is H. In some embodiments, R 13 is C 1 ⁇ 10 alkyl. In some embodiments, R 13 is C 2 ⁇ 10 alkenyl. In some embodiments, R 13 is C 2 ⁇ 10 alkynyl.
  • R 14 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 14 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 14 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 14 is trialkylsilyl. In some embodiments, R 14 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 14 is acetyl. In some embodiments, R 14 is trihaloacetyl. In some embodiments, R 14 is phthalimidyl. In some embodiments, R 14 is H. In some embodiments, R 14 is C 1 ⁇ 10 alkyl. In some embodiments, R 14 is C 2 ⁇ 10 alkenyl. In some embodiments, R 14 is C 2 ⁇ 10 alkynyl.
  • R 18 when present, is selected from H, an optionally substituted C 1 ⁇ 10 alkyl group, trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 18 is selected from H and an optionally substituted C 1 ⁇ 10 alkyl group. In some embodiments, R 18 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 18 is H. In some embodiments, R 18 is an optionally substituted C 1 ⁇ 10 alkyl group.
  • R 18 is trialkylsilyl. In some embodiments, R 18 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 18 is acetyl. In some embodiments, R 18 is trihaloacetyl. In some embodiments, R 18 is phthalimidyl. [0452] In some embodiments, R 20 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 20 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 20 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 20 is trialkylsilyl. In some embodiments, R 20 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 20 is acetyl. In some embodiments, R 20 is trihaloacetyl. In some embodiments, R 20 is phthalimidyl. In some embodiments, R 20 is H.
  • R 20 is C 1 ⁇ 10 alkyl. In some embodiments, R 20 is C 2 ⁇ 10 alkenyl. In some embodiments, R 20 is C 2 ⁇ 10 alkynyl. [0453] In some embodiments, R 21 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 21 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 21 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 21 is trialkylsilyl. In some embodiments, R 21 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 21 is acetyl. In some embodiments, R 21 is trihaloacetyl. In some embodiments, R 21 is phthalimidyl. In some embodiments, R 21 is H.
  • R 21 is C 1 ⁇ 10 alkyl. In some embodiments, R 21 is C 2 ⁇ 10 alkenyl. In some embodiments, R 21 is C 2 ⁇ 10 alkynyl. [0454] In some embodiments, R 22 , when present, is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl.
  • R 22 is selected from trialkylsilyl, tert ⁇ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R 22 is selected from H, C 1 ⁇ 10 alkyl, C 2 ⁇ 10 alkenyl, and C 2 ⁇ 10 alkynyl. In some embodiments, R 22 is trialkylsilyl. In some embodiments, R 22 is tert ⁇ butyldiphenylsilyl. In some embodiments, R 22 is acetyl. In some embodiments, R 22 is trihaloacetyl. In some embodiments, R 22 is phthalimidyl. In some embodiments, R 22 is H.
  • R 22 is C 1 ⁇ 10 alkyl. In some embodiments, R 22 is C 2 ⁇ 10 alkenyl. In some embodiments, R 22 is C 2 ⁇ 10 alkynyl. [0455] In the preceding embodiments, the trialkylsilyl group, if present, may have the formula Si(C 1 ⁇ C 4 alkyl) 3 . In further embodiments, trialkylsilyl group, if present, is selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). In further embodiments, the trialkylsilyl group is trimethylsilyl (TMS).
  • the trialkylsilyl group is triethylsilyl (TES). In further embodiments, the trialkylsilyl group is tert ⁇ butyldimethylsilyl (TBS). In further embodiments, the trialkylsilyl group is triisopropylsilyl (TIPS).
  • the trihaloacetyl group if present, is trichloroacetyl or trifluoroacetyl. In some embodiments, the trihaloacetyl group is trichloroacetyl. In some embodiments, the trihaloacetyl group is trifluoroacetyl.
  • the base labile protecting group may be selected from trialkylsilyl, acetyl, trihaloacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trihalo acetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trichloroacetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from Si(C 1 ⁇ C 4 alkyl) 3 , acetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trihalo acetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trichloroacetyl, trifluoroacetyl, and phthalimidyl.
  • the base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl (TES), tert ⁇ butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trifluoroacetyl, and phthalimidyl.
  • R 9 is H.
  • X is selected from NR 5 , C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl; wherein each is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6 , C(O)NR 7 R 8 , SR 6 , S(O)R 6 , S(O) 2 R 6 , P(O)(OR 6 ) 2 , CN, NO 2 , and N 3 .
  • X is NR 5 .
  • X is C 3 ⁇ 10 cycloalkyl. In some embodiments, X is C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is C 5 ⁇ 10 cycloalkenyl. In some embodiments, X is C 6 ⁇ 10 aryl. In some embodiments, X is C 5 ⁇ 10 heteroaryl. [0465] In some embodiments, X is selected from NR 5 , C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 heterocycloalkyl, C 5 ⁇ 10 cycloalkenyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl. In some embodiments, X is NR 5 . In some embodiments, X is C 3 ⁇ 10 cycloalkyl.
  • X is C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is C 5 ⁇ 10 cycloalkenyl. In some embodiments, X is C 6 ⁇ 10 aryl. In some embodiments, X is C 5 ⁇ 10 heteroaryl. [0466] In some embodiments, X is selected from NR 5 and C 3 ⁇ 10 heterocycloalkyl. In some embodiments, X is NR 5 . In some embodiments, X is C 3 ⁇ 10 heterocycloalkyl. [0467] In some embodiments, X is selected from NR 5 , pyrrolidine, and piperidine. In some embodiments, X is NR 5 . In some embodiments, X is pyrrolidine.
  • X is piperidine.
  • Y is selected from C 1 ⁇ 30 alkyl, (CH 2 CH 2 O) m , C(O)n, C 3 ⁇ 10 cycloalkyl, and C 3 ⁇ 10 heterocycloalkyl; wherein each C 3 ⁇ 10 cycloalkyl or C 3 ⁇ 10 heterocycloalkyl is optionally substituted with at least one group selected from halo, C 1 ⁇ 10 alkyl, OR 6 , C(O)OR 6 , C(O)NR 7 R 8 , SR 6 , S(O)R 6 , S(O) 2 R 6 , P(O)(OR 6 ) 2 , CN, NO 2 , and N 3 .
  • Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is (CH 2 CH 2 O) m . In some embodiments, Y is C(O)n. In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. In some embodiments, Y is C 3 ⁇ 10 heterocycloalkyl. [0469] In some embodiments, Y is selected from C 1 ⁇ 30 alkyl, (CH 2 CH 2 O) m , C(O), C 3 ⁇ 10 cycloalkyl, and C 3 ⁇ 10 heterocycloalkyl. In some embodiments, Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is (CH 2 CH 2 O) m .
  • Y is C(O). In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. In some embodiments, Y is C 3 ⁇ 10 heterocycloalkyl. [0470] In some embodiments, Y is selected from C 1 ⁇ 30 alkyl, C(O), and C 3 ⁇ 10 cycloalkyl. In some embodiments Y is C 1 ⁇ 30 alkyl. In some embodiments, Y is C(O). In some embodiments, Y is C 3 ⁇ 10 cycloalkyl. [0471] In some embodiments, X ⁇ Y ⁇ Z comprises at least one tertiary amine.
  • X ⁇ Y ⁇ Z comprises at least one of NR 5 , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 N ⁇ heterocycloalkyl, C 6 ⁇ 10 aryl, and C 5 ⁇ 10 heteroaryl.
  • X ⁇ Y ⁇ Z comprises at least one of NR 5 , C(O), C 3 ⁇ 10 cycloalkyl, C 3 ⁇ 10 N ⁇ heterocycloalkyl, and C 6 ⁇ 10 aryl.
  • X ⁇ Y ⁇ Z comprises NR 5 .
  • X ⁇ Y ⁇ Z comprises C(O).
  • X ⁇ Y ⁇ Z comprises C 3 ⁇ 10 cycloalkyl. [0477] In some embodiments, X ⁇ Y ⁇ Z comprises C 3 ⁇ 10 N ⁇ heterocycloalkyl. [0478] In some embodiments, X ⁇ Y ⁇ Z comprises C 6 ⁇ 10 aryl. [0479] In some embodiments, X ⁇ Y ⁇ Z comprises C 5 ⁇ 10 heteroaryl.
  • X ⁇ Y ⁇ Z is selected from: [0481] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0482] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0483] In some embodiments, X ⁇ Y ⁇ Z is selected from: [0485] In some embodiments, . [0486] In some embodiments, . [0487] In some embodiments, . . [0490] In some embodiments, . [0491] In some embodiments, . [0492] In some embodiments, .
  • X ⁇ Y ⁇ Z is selected from: [0497] In some embodiments, X ⁇ Y ⁇ Z is selected from: , [0498] In some embodiments, X ⁇ Y ⁇ Z is selected from: , [ [ [ [ [ [ [ [0503] In some embodiments, [0504] In some embodiments, [0505] In some embodiments, [0506] In some embodiments, X [0507] Insomeembodiments,X [0508] In some embodiments, . [0509] In some embodiments, . [0510] In some embodiments, . [0513] In some embodiments, . [0514] In some embodiments, the modified oligonucleotide conjugate is selected from: , .
  • the modified oligonucleotide conjugate is selected from: . [0516] In some embodiments, the modified oligonucleotide conjugate is selected from: , , , , . [0517] In some embodiments, the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is: [0521] In some embodiments, the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is: [0528] In some embodiments, the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is selected from: [0532] In some embodiments, the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is: [0538] In some embodiments, the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is: [0544] In some embodiments, the modified oligonucleotide conjugate is:
  • the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is selected from:
  • the modified oligonucleotide conjugate is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the modified oligonucleotide conjugate is [0555] In some embodiments, the modified oligonucleotide conjugate [0557] In some embodiments, the modified oligonucleotide conjugate
  • the modified oligonucleotide conjugate is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the modified oligonucleotide conjugate is modified oligonucleotide conjugate [0564] In some embodiments, the modified oligonucleotide conjugate oligonucleotide conjugate is
  • the modified oligonucleotide conjugate modified oligonucleotide conjugate modified oligonucleotide conjugate
  • M-SH thiol-containing molecule
  • the oligonucleotide is an antisense oligonucleotide. In some embodiments, the oligonucleotide is an RNA interference (RNAi) oligonucleotide. In some embodiments, the oligonucleotide is an aptamer. In some embodiments, the oligonucleotide is a short interfering RNA (siRNA) oligonucleotide. In some embodiments, the oligonucleotide is a spliceswitching antisense oligonucleotide. In some embodiments, the oligonucleotide is an microRNA (miRNA) oligonucleotide.
  • miRNA microRNA
  • the oligonucleotide is a DNA oligonucleotide. In some embodiments, the oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is an XNA. In some embodiments, the oligonucleotide is an xRNA. In some embodiments, the oligonucleotide is an xDNA. In some embodiments, the oligonucleotide is an saRNA.
  • M is a small molecule.
  • M is a pharmacologically active small molecule.
  • M is a probe.
  • a probe is a molecule which is used to investigate biological mechanisms or validate targets for drug discovery. Probes may comprise a moiety which allows for their detection by an external source.
  • a probe may be radiolabelled, i.e. may comprise radioisotopes.
  • the probe may comprise a moiety which displays fluorescence or phosphorescence.
  • M is a fluorophore
  • M is fluorescein or a fluorescein derivative.
  • Fluorescein is a common diagnostic contrast agent with many derivatives which are well known in the art.
  • Exemplary derivatives include fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, mixtures of fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate, fluorescein succinimidyl esters, carboxyfluorescein, carboxyfluorescein succinimidyl ester, fluorescein pentafluorophenyl esters, fluorescein tetrafluorophenyl esters, and fluorescein amidites.
  • M is rhodamine or a rhodamine derivative.
  • Rhodamine derivatives are a well-known family of dyes which feature the rhodamine core structure. Examples of rhodamine derivatives include carboxytetramethylrhodamine, tetramethylrhodamine, tetramethylrhodamine isothiocyanate, sulforhodamine 101, sulforhodamine 101 acid chloride (Texas Red), and rhodamine red.
  • M is 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY).
  • M is a radiolabelled molecule.
  • Radiolabelled molecules are typically small molecules which are non-biologically active and which are isotopically enriched with a radioactive isotope such as 32P, 35S, 14C, and 3H.
  • M is a polypeptide. In some embodiments, M is a protein. In some embodiments, M is a polypeptide or protein connected to the modified oligonucleotide via a cysteine residue.
  • the polypeptide or protein of the invention may be a carrier protein.
  • a carrier protein is a protein or polypeptide which is capable of transporting an ion, small molecule, or macromolecule across a biological membrane, e.g. into a cell.
  • the carrier protein is selected to transport the modified oligonucleotide into the cell.
  • the oligonucleotide is an antisense oligonucleotide, RNAi oligonucleotide, aptamer, siRNA oligonucleotide, splice-switching antisense oligonucleotide, miRNA oligonucleotide, DNA oligonucleotide, gapmer, XNA oligonucleotide, xRNA oligonucleotide, xDNA oligonucleotide, or saRNA oligonucleotide which is intended to be transported into cells or a specific cell type.
  • the polypeptide or protein is an antigen binding fragment, a nanobody or an antibody.
  • Antibodies comprise antigen binding fragments.
  • Antigen binding fragments are polypeptide sequences which bind to antigens.
  • Antigen binding fragments are antigen- specific, i.e. can only react to and bind to one specific antigen, or can cross-react, i.e. can react to and bind more than one antigen.
  • Nanobodies, or single-domain antibodies are a type of antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies generally have a molecular weight around 12-15 kDa and so are significantly lighter than common antibodies which generally have a molecular weight around 150-160 kDa. Nanobodies.
  • Like antibodies are capable of selectively binding to a specific antigen.
  • an oligonucleotide By linking an oligonucleotide to an antigen binding fragment, a nanobody or an antibody, it is possible to provide a modified oligonucleotide conjugate which targets, for example, cells displaying a specific antigen. Delivery of a therapeutic oligonucleotide can, therefore, be targeted to only the cells in which the oligonucleotide is desired to have an effect.
  • the polypeptide or protein is a cell-penetrating peptide (CPP).
  • CPPs are usually short peptides, for example, of less than 30-40 amino acids. They may be derived from proteins or chimeric sequences or may be of completely artificial, synthetic or designed origin. They are occasionally amphipathic, but almost always possess a net positive (cationic) charge at physiological pH. CPPs are able to penetrate biological membranes, to trigger the movement of various biomolecules across cell membranes into the cytoplasm and to improve their intracellular routing, thereby facilitating interactions with the target. CPPs have been shown to be able to deliver oligonucleotide cargos into a wide variety of cell types.
  • the method comprises the step of M-SH being dissolved in a buffered solution to provide a M-SH buffered solution.
  • a buffered solution is a solution which contains a buffer which keeps the pH of the solution at a specific pH or within a specific pH range.
  • the buffered solution is a commercially available buffer.
  • the buffered solution comprises a buffer and water.
  • the buffered solution has a pH around physiological pH.
  • the buffered solution has a pH between 7.3 and 7.5.
  • the buffered solution has a pH of about 7.4.
  • the buffered solution comprises an aqueous buffer. In other embodiments, the buffered solution comprises a non-aqueous buffer. In some embodiments, the aqueous buffer is phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the method comprises the step of the modified oligonucleotide being dissolved in an aqueous solution to provide a modified oligonucleotide solution.
  • the method comprises the step of the M-SH buffered solution being mixed with the modified oligonucleotide solution.
  • the reaction between the modified oligonucleotide and M-SH is performed between 10-50 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed between 20-40 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed between 25-40 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed at 25 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed at physiological temperatures. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed at 37 °C.
  • the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has its temperature altered following the mixing. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has a temperature between 10-50 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has a temperature between 20-40 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution is heated to a temperature between 20-40 °C or 25-40 °C.
  • the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has a temperature of 25 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution is heated to a physiological temperature. In some embodiments, the solution resulting from the mixing of the M- SH buffered solution and the modified oligonucleotide solution is heated to 37 °C.
  • the thiol-ene reaction step is performed in the presence of a radical initiator.
  • a radical initiator is a substance or molecule which produces radical species under mild reaction conditions and therefor promotes radical reactions. These radical initiators will generally comprise a bond with a small bond dissociation energy, for example azo- or peroxide bonds.
  • the thiol-ene reaction step is performed in the presence of dithiothreitol.
  • the mixture was degassed for 5 mins. Then the catalyst Pd(dppf)Cl 2 (0.1 eq, 43 mg) was added to the mixture and further degassed for 5 mins. The reaction mixture was warmed upto 80 o C and stirred at this temperature for 6 hours. The reaction mixture was cooled to room temperature, diluted with DCM (10 mL) and filtered through a bed of celite washing several times with DCM (25 mL). Solvent was removed under vacuum.
  • reaction mixture was warmed upto 85 o C and stirred at this temperature for 18 hours.
  • the reaction mixture was cooled to room temperature, diluted with DCM and filtered through a bed of celite washing several times with DCM. Solvent was removed under vacuum. The residue was subjected to column chromatography (silica gel, 0 ⁇ 100% Heptane/Ethyl acetate) to afford the product 4 ⁇ (methyl(4 ⁇ vinylpyrimidin ⁇ 2 ⁇ yl)amino)cyclohexan ⁇ 1 ⁇ ol (130 mg, 96%) as a pale yellow gum.
  • Step 2 1 ⁇ (4 ⁇ vinylpyrimidin ⁇ 2 ⁇ yl)piperidin ⁇ 4 ⁇ ol [0611]
  • a solution of 2 ⁇ chloro ⁇ 4 ⁇ vinylpyrimidine (100 mg, 0.711 mmol), piperidin ⁇ 4 ⁇ ol (1.2 eq, 0.854 mmol) and anhydrous DIPEA (2.5 eq, 0.35 mL) in anhydrous DMF (1.5 mL) was stirred at 100 o C in a sealed tube for 18 hours. The reaction mixture was cooled to room temperature and solvent removed under vacuum.
  • Step 2 Procedure 2 was followed to synthesise 1 ⁇ (4 ⁇ vinylpyrimidin ⁇ 2 ⁇ yl)azetidin ⁇ 3 ⁇ ol from 1 ⁇ (2 ⁇ chloropyrimidin ⁇ 4 ⁇ yl)azetidin ⁇ 3 ⁇ ol in 70% yield as pale brown gum. Flash column chromatography (silica gel) was performed for purification using (0 ⁇ 50%) DCM ⁇ MeOH solvent system.
  • Step 2 was followed to synthesise 3 ⁇ (methyl(4 ⁇ vinylpyrimidin ⁇ 2 ⁇ yl)amino)cyclobutan ⁇ 1 ⁇ ol from 2 ⁇ chloro ⁇ 4 ⁇ vinylpyrimidine and commercially available 3 ⁇ (methylamino)cyclobutan ⁇ 1 ⁇ ol in 44% yield as yellow gum.
  • Step 2 Procedure 2 was followed to synthesise 2 ⁇ (methyl(4 ⁇ vinylpyrimidin ⁇ 2 ⁇ yl)amino)ethan ⁇ 1 ⁇ ol from commercially available 2 ⁇ ((4 ⁇ chloropyrimidin ⁇ 2 ⁇ yl)(methyl)amino)ethan ⁇ 1 ⁇ ol in 79% yield as pale brown viscous oil.
  • Step 1 was followed to synthesise 1 ⁇ (2 ⁇ chloropyrimidin ⁇ 4 ⁇ yl)piperidin ⁇ 4 ⁇ ol starting from commercially available compounds 2,4 ⁇ dichloropyrimidine and piperidin ⁇ 4 ⁇ ol.
  • Step 1 was followed to synthesise 3 ⁇ ((2 ⁇ chloropyrimidin ⁇ 4 ⁇ l)( th l) i ) l t 1 l t ti f i ll il bl d 24 dichloropyrimidine and 3 ⁇ (methylamino)cyclopentan ⁇ 1 ⁇ ol hydrochloride.
  • Method A [0750] Representative example: Deprotection of Compound 5 ⁇ Oligo1 conjugate [0751] 5 mg of Compound 5 ⁇ Oligo1 conjugate on CPG solid support was placed in a vial, treated with 100 ⁇ L of 0.4 M NaOH in MeOH/water (4:1) solution and shaken (Eppendorf ThermoMixerC) for 20 min at 80 °C. The sample was cooled down to room temperature, spun down and filtered. The filtrate was frozen and freeze ⁇ dried.
  • the sample was desalted on Glen Gel ⁇ PakTM 0.2 Desalting Column (catalogue no 61 ⁇ 5002 ⁇ 50) following the manufacturer’s protocol.
  • the collected sample was frozen, LCMS (Waters LCMS system with Acquity QDa detector, ACQUITY PREMIER Oligonucleotide BEH C18 column (130 ⁇ ; 2.1 x 50 mm, 1.7 ⁇ m; Waters) at 65 °C with a flow rate of 0.3 mL/min.
  • Eluent A 7 mM TEA, 80 mM HFIP in water
  • eluent B 3.5 mM TEA, 40 mM HFIP in 50% ACN; gradient 5 ⁇ 30% B in 8 min).
  • Sample was desalted on Glen Gel ⁇ PakTM 0.2 Desalting Column (catalogue no 61 ⁇ 5002 ⁇ 50) following manufacturer’s protocol. Collected sample was frozen, freeze ⁇ dried and analysed on LCMS. Sample was dissolved in 150 ⁇ L of water and analysed on LCMS (Waters LCMS system with Acquity QDa detector, ACQUITY PREMIER Oligonucleotide BEH C18 column (130 ⁇ ; 2.1 x 50 mm, 1.7 ⁇ m; Waters) at 65 °C with a flow rate of 0.3 mL/min.
  • LCMS Waters LCMS system with Acquity QDa detector, ACQUITY PREMIER Oligonucleotide BEH C18 column (130 ⁇ ; 2.1 x 50 mm, 1.7 ⁇ m; Waters) at 65 °C with a flow rate of 0.3 mL/min.
  • Compound 6_oligo 2 LCMS (ESI ⁇ ) m/z [M ⁇ H] ⁇ mass calculated: 4428.02, mass found: ⁇ [M ⁇ H] ⁇ +MeOH ⁇ 4460.03. [0762] Following Method A, Compound 6 – oligo 1 conjugate was deprotected and analysed. [0763] Compound 6_oligo 1: LCMS (ESI ⁇ ) m/z [M ⁇ H] ⁇ mass calculated: 4534.07, mass found: ⁇ [M ⁇ H] ⁇ +MeOH ⁇ 4566.12. [0764] Following Method B, Compound 9 ⁇ oligo 1 conjugate was deprotected and analysed.
  • a solution of 100 mM P-mercaptoethanol (P-ME) in the conjugation buffer was freshly prepared: 1.4 pL of P-mercaptoethanol was added to 200 pL of 5x PBS (phosphate-buffered saline) buffer. Then, the oligo sample (dissolved in around 50 pL of water) was mixed with 50 pL of water and 25 pL of conjugation buffer (with P-mercaptoethanol) and incubated for 2 h at 37 °C. LCMS (conditions as described previously) analysis was performed after 1 h (by mixing 10 pL of sample with 10 pL of water).
  • PBS phosphate-buffered saline
  • the glutathione (GSH) reaction was performed by mixing 50 pM in IX PBS with 250 pM of the freshly prepared glutathione solution in water.
  • the reaction was prepared in a vial by mixing 24 pL of the linker containing oligo (83.4 pM in water), 8 pL of 5X PBS (prepared from tablets), and 8 pL of the freshly prepared glutathione solution (1.25 mM in water) at 37 - 55 °C for 6 - 24 hours.
  • the glutathione (GSH) reaction was performed, according to Method B, by mixing 50 pM in IX PBS with 250 pM of the freshly prepared glutathione solution in water.
  • the reaction was prepared in a vial by mixing 24 pL of Compound 2_oligo 2 (83.4 pM in water), 8 pL of 5X PBS (prepared from tablets), and 8 pL of the freshly prepared glutathione solution (1.25 mM in water).
  • Electrophoresis was performed using a NuPAGETM 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well with IX NuPAGE MES SDS running buffer.
  • the gel was stained using 20 mL of SimpleBlueTM SafeStain, with gentle rocking for 2 h and washed three times with 15 mL water for 15 min.

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Abstract

The present invention discloses phosphoramidite linkers according to Formula (I), oligonucleotides bound to these linkers, and to oligonucleotide conjugates connected via these linkers.

Description

  LARC‐038/01WO 336450‐2421  Phosphoramidite Linkers  FIELD OF THE INVENTION  [0001] The invention relates to phosphoramidite linkers, oligonucleotides bound to these linkers, and  to  oligonucleotide  conjugates  connected  via  the  linkers  of  the  invention. Methods  of  preparing  5  oligonucleotide conjugates are also provided.  BACKGROUND  [0002] Oligonucleotide conjugates are an emerging technology, and it has been demonstrated that  conjugated moieties can be used in therapeutics to successfully treat disease. In addition, conjugated  oligonucleotides represent an important class of laboratory tools, including labelled oligonucleotides 10  for use in understanding intracellular trafficking. Conjugated oligonucleotides can also be broadly used  to modulate prokaryotic and eukaryotic gene expression.  [0003] Peptide‐oligonucleotide  conjugates  are,  in  particular,  a  field  of  increasing  interest.  The  conjugation of an oligonucleotide to a carrier protein, for example, provides a method of increasing  cellular uptake, tissue delivery, and bioavailability of therapeutic oligonucleotides such as antisense 15  oligonucleotides or small  interfering RNAs  (described  in Klabenkova et al. Molecules 2021, 26(17),  5420). Accordingly, this conjugation improves the overall efficiency of these oligonucleotides in vivo.  Likewise,  the  conjugation  of  these  therapeutic  oligonucleotides  to  an  antigen  binding  fragment,  nanobody, or antibody allows for greatly  increased specificity of action for the oligonucleotide. For  these advantages to be realised it is necessary for the linker joining the peptide and oligonucleotide 20  to be both biologically stable and possible to synthesise/attach in conditions suitable for both peptides  and oligonucleotides.  [0004] It is common for peptide linkers to make use of sulfide groups. A traditional method of forming  links is via disulfide bonds, for example between cysteine residues. However, these disulfides are not  stable in vivo and are known to undergo thiol exchange. As such, other linkers have been developed 25  for use in oligonucleotides and peptides.  [0005] One standard type of linker is the triazole linker. These linkers, however, require additional  modification of the protein with either an azide or alkyne group, and  therefore  frequently require  post‐synthetic oligonucleotide modification. These  linkers  also  typically  require  the presence of  a  metal catalyst, such as copper, in order to provide the desired linked molecule. These metals can be 30  detrimental to drug molecules and can lead to the degradation of oligonucleotides. Metal‐free triazole  formation reactions also have to deal with the bulky size of the reagents and the compatibility of the  necessary strained alkyne with the conditions necessary for oligonucleotide synthesis. Triazole linkers  1 11459075 v1   are also frequently not stable during extended storage periods, and triazole derivatives can display  biological activity, which may  lead  to unintended effects when  the oligonucleotide conjugates are  provided in vivo.  [0006] Maleimides are the most frequently used reagent  in oligonucleotide conjugation reactions.  However,  maleimide  linkers  have  a  number  of  significant  disadvantages.  Maleimide  modified  oligonucleotides can be challenging to synthesise as the maleimide group is generally not stable to the  conditions used  in oligonucleotide synthesis. At present, two approaches are used to mitigate this,  often  the oligonucleotide must be synthesised with an amine modification and  it  is only after  the  oligonucleotide has been cleaved from the solid support and purified that this amine is reacted with  a bifunctional small molecule  linker with both an N‐hydroxysuccinimide (NHS) ester and maleimide  group. The alternative approach is to make use of commercially available maleimide modifiers, which  are both expensive and require additional processing steps.   [0007] Aside from the difficulties in their preparation, maleimide functionalised oligonucleotides also  generally display a relatively short half‐life  in pH neutral conditions. They also display a narrow pH  range  for  selective  reaction with  thiols over  amines,  and  a  lack of  selectivity between  thiols  and  disulfide bonds.  [0008] Accordingly, there is a need to provide new linkers for oligonucleotides.  SUMMARY OF INVENTION  [0009] In accordance with a first aspect of the invention, there is provided a compound of formula  (I): 
Figure imgf000003_0001
, wherein:  A is mono‐ or bicyclic heteroaryl group in which at least one carbon ring‐atom is replaced with N;  X, Y, and Z are each independently selected from C1‐30 alkyl, C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m,  C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, C5‐10 heteroaryl, or absent;  wherein at least one of X, Y and Z is present;   wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl or C5‐10 heteroaryl  is  optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8,  SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3;   n is 1 or 2;  m is an integer from 1‐30;     each   is either a single bond or double bond;  W is OR2 or NR3R4;  R1 and R2 are each independently selected from H and a base labile protecting group;  R3 and R4 are each independently selected from H and an optionally substituted C1‐10 alkyl;  R5 is absent, is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl;  R6 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R7 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R8 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R9, is selected from H, C1‐10 alkyl, halide, C(O)C1‐10 alkyl, C1‐10 haloalkyl, C(O)OR12, C(O)SR12, C(O)NR13R14,  CN, or NO2;   R10, and R11 are each independently selected from H, C1‐10 alkyl, halide, or C1‐10 haloalkyl;  R12 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R13 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  and  R14 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  [0010] In  accordance  with  a  second  aspect  of  the  invention,  there  is  provided  a  modified  oligonucleotide, wherein the 5’ end of the oligonucleotide  is attached to a compound as described  above via a covalent bond which replaces the W moiety to give a modified oligonucleotide of formula  (II): 
Figure imgf000004_0001
  wherein Q is S or O.  [0011] In  accordance  with  a  third  aspect  of  the  invention,  there  is  provided  a  modified  oligonucleotide conjugate, wherein the modified oligonucleotide described above is conjugated to a  thiol‐containing moiety (M‐SH) via the vinyl group to provide a conjugate of formula (II):   
Figure imgf000005_0001
  wherein Q is S or O.  [0012] In accordance with a fourth aspect of the invention, there is provided a method of conjugating  the modified oligonucleotide described  in  the second aspect of  the  invention  to a  thiol‐containing  molecule (M‐SH) to form the modified oligonucleotide conjugate described in the third aspect of the  invention, wherein the method comprises a thiol‐ene reaction between the thiol and the vinyl group  of the modified oligonucleotide the second aspect of the invention.  [0013] Further embodiments of the invention are described in the appended claims.    BRIEF DESCRIPTION OF THE DRAWINGS  [0014] Figure 1a shows hours 0‐3  for  the  time course monitoring via HPLC  for  the  formation of a  glutathione‐linker‐oligonucleotide conjugate of the invention.  [0015] Figure 1b shows hours 3.5‐6 for the time course monitoring via HPLC for the formation of a  glutathione‐linker‐oligonucleotide conjugate of the invention.  [0016] Figure 2 is a graph showing the area under the curve for the formation of a glutathione‐linker‐ oligonucleotide conjugate of the invention.  [0017] Figure 3  shows  the SDS‐PAGE  for  the  formation of a human  serum albumen  (HSA)‐linker‐ oligonucleotide conjugate of the invention.    DETAILED DESCRIPTION OF THE INVENTION  Definitions  [0018] While  the  following  terms  are  believed  to  be well  understood  by  the  skilled  person,  the  following definitions are set forth to facilitate explanation of the presently disclosed subject matter.  [0019] The terms below, as used herein, have the following meanings, unless indicated otherwise:  [0020] “Amino” refers to the ‐NH2 radical.  [0021] “Cyano” refers to the ‐CN radical.  [0022] “Halo” or “halogen” refers to bromo, chloro, fluoro or iodo radical.  [0023] “Hydroxy” or “hydroxyl” refers to the ‐OH radical.    [0024] “Imino” refers to the =NH substituent.  [0025] “Nitro” refers to the ‐NO2 radical.  [0026] “Oxo” refers to the =O substituent.  [0027] “Alkyl” or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain  radical having from one to thirty carbon atoms unless otherwise specified, and which is attached to  the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 30  are included. An alkyl comprising up to 10 carbon atoms is a C1‐C10 alkyl, an alkyl comprising up to 6  carbon atoms is a C1‐C6 alkyl and an alkyl comprising up to 5 carbon atoms is a C1‐C5 alkyl. A C1‐C5 alkyl  includes C5 alkyls, C4 alkyls, C3 alkyls, C2 alkyls and C1 alkyl  (i.e., methyl). A C1‐C6 alkyl  includes all  moieties described above for C1‐C5 alkyls but also includes C6 alkyls. A C1‐C10 alkyl includes all moieties  described above for C1‐C5 alkyls and C1‐C6 alkyls, but also includes C7, C8, C9 and C10 alkyls. Non‐limiting  examples of C1‐C10 alkyl include methyl, ethyl, n‐propyl, i‐propyl, sec‐propyl, n‐butyl, i‐butyl, sec‐butyl,  t‐butyl, n‐pentyl, t‐amyl, n‐hexyl, n‐heptyl, n‐octyl, n‐nonyl, and n‐decyl.  [0028] “Alkenyl” or “alkenyl group” refers to a straight or branched hydrocarbon chain radical having  from two to thirty carbon atoms unless otherwise specified, and having one or more carbon‐carbon  double bonds. For the avoidance of doubt, the terms “alkenyl” or “alkenyl group” refer to straight or  branched hydrocarbon chain radicals comprising E and/or Z (trans and/or cis) alkenes. Each alkenyl  group is attached to the rest of the molecule by a single bond. Alkenyl groups comprising any number  of carbon atoms from 2 to 30 are included. An alkenyl group comprising up to 10 carbon atoms is a  C2‐C10 alkenyl, an alkenyl group comprising up to 6 carbon atoms is a C2‐C6 alkenyl and an alkenyl group  comprising up to 5 carbon atoms is a C2‐C5 alkenyl. A C2‐C5 alkenyl includes C5 alkenyls, C4 alkenyls, C3  alkenyls, and C2 alkenyls. A C2‐C6 alkenyl includes all moieties described above for C2‐C5 alkenyls but  also includes C6 alkenyls. A C2‐C10 alkenyl includes all moieties described above for C2‐C5 alkenyls and  C2‐C6 alkenyls, but also  includes C7, C8, C9 and C10 alkenyls. Non‐limiting examples of C2‐C10 alkenyl  include ethenyl (vinyl), 1‐propenyl, 2‐propenyl (allyl), iso‐propenyl, 2‐methyl‐1‐propenyl, 1‐butenyl, 2‐ butenyl, 3‐butenyl, 1‐pentenyl, 2‐pentenyl, 3‐pentenyl, 4‐pentenyl, 1‐hexenyl, 2‐hexenyl, 3‐hexenyl,  4‐hexenyl,  5‐hexenyl,  1‐heptenyl,  2‐heptenyl,  3‐heptenyl,  4‐heptenyl,  5‐heptenyl,  6‐heptenyl,  1‐ octenyl,  2‐octenyl,  3‐octenyl,  4‐octenyl,  5‐octenyl,  6‐octenyl,  7‐octenyl,  1‐nonenyl,  2‐nonenyl,  3‐ nonenyl, 4‐nonenyl, 5‐nonenyl, 6‐nonenyl, 7‐nonenyl, 8‐nonenyl, 1‐decenyl, 2‐decenyl, 3‐decenyl, 4‐ decenyl, 5‐decenyl, 6‐decenyl, 7‐decenyl, 8‐decenyl, and 9‐decenyl.  [0029] “Alkynyl” or “alkynyl group” refers to a straight or branched hydrocarbon chain radical having  from two to thirty carbon atoms unless otherwise specified, and having one or more carbon‐carbon  triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl groups  comprising any number of carbon atoms from 2 to 30 are included. An alkynyl group comprising up to    10 carbon atoms is a C2‐C10 alkynyl, an alkynyl group comprising up to 6 carbon atoms is a C2‐C6 alkynyl  and an alkynyl group comprising up to 5 carbon atoms is a C2‐C5 alkynyl. A C2‐C5 alkynyl includes C5  alkynyls, C4 alkynyls, C3 alkynyls, and C2 alkynyls. A C2‐C6 alkynyl includes all moieties described above  for C2‐C5 alkynyls but also includes C6 alkynyls. A C2‐C10 alkynyl includes all moieties described above  for C2‐C5 alkynyls and C2‐C6 alkynyls, but also includes C7, C8, C9 and C10 alkynyls. Non‐limiting examples  of C2‐C10 alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like.  [0030] “Aryl” refers to a hydrocarbon ring system radical comprising 6 to 18 carbon atoms (C6‐C18)  and at least one aromatic ring. For purposes of this  invention, the aryl radical can be a monocyclic,  bicyclic,  tricyclic or  tetracyclic  ring  system, which  can  include  fused or bridged  ring  systems. Aryl  radicals  include, but are not  limited  to, aryl  radicals derived  from aceanthrylene, acenaphthylene,  acephenanthrylene, anthracene, azulene, benzene,  chrysene,  fluoranthene,  fluorene, as‐indacene,  sindacene,  indane,  indene,  naphthalene,  phenalene,  phenanthrene,  pleiadene,  pyrene,  and  triphenylene. Unless  stated otherwise  specifically  in  the  specification,  the  term “aryl”  is meant  to  include aryl radicals that are optionally substituted.  [0031] “Cycloalkyl”  refers  to  a  stable  non‐aromatic  monocyclic  or  polycyclic  fully  saturated  hydrocarbon  radical  consisting  solely of  carbon  and hydrogen  atoms, which  can  include  fused or  bridged ring systems, having from three to ten carbon atoms, and which is attached to the rest of the  molecule by a single bond. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl,  cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example,  adamantyl, norbornyl, decalinyl, 7,7‐dimethyl‐bicyclo[2.2.1]heptanyl, and the like.  [0032] “Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo  radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2‐trifluoroethyl,  1,2‐difluoroethyl, 3‐bromo‐2‐fluoropropyl, 1,2‐dibromoethyl, and the like.  [0033] “Heterocyclyl,” “heterocyclic ring” or “heterocycle” refers to a stable 3‐ to 20‐membered (also  denoted as “C3‐C20”) non‐aromatic, partially aromatic, or aromatic ring radical which may consist of  two to fourteen carbon atom and from one to six heteroatoms. A heterocyclyl, heterocyclic ring, or  heterocycle can also refer to a non‐aromatic, partially aromatic, or aromatic ring radical which consists  of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting  of nitrogen, oxygen,  sulfur and  selenium. Heterocyclyl or heterocyclic  rings  include heteroaryls as  defined below. Unless stated otherwise specifically in the specification, the heterocyclyl radical can be  a monocyclic, bicyclic,  tricyclic or  tetracyclic  ring  system, which  can  include  fused or bridged  ring  systems;  and  the  nitrogen,  carbon,  sulfur  or  selenium  atoms  in  the  heterocyclyl  radical  can  be  optionally oxidised; the nitrogen atom can be optionally quaternised; and the heterocyclyl radical can  be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to,    dioxolanyl,  thienyl[1,3]dithianyl,  decahydroisoquinolyl,  imidazolinyl,  imidazolidinyl,  isothiazolidinyl,  isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2‐oxopiperazinyl, 2‐oxopiperidinyl,  2‐oxopyrrolidinyl,  oxazolidinyl,  piperidinyl,  piperazinyl,  4‐piperidonyl,  pyrrolidinyl,  pyrazolidinyl,  quinuclidinyl,  thiazolidinyl,  tetrahydrofuryl,  trithianyl,  tetrahydropyranyl,  thiomorpholinyl,  thiamorpholinyl,  1‐oxo‐thiomorpholinyl,  1,1‐dioxo‐thiomorpholinyl,  indolinyl,  isoindolinyl,  tetrahydroquinolyl,  tetrahydroisoquinolyl,  coumaranyl,  phthalanyl,  chromanyl,  isochromanyl,  chromenyl.  [0034] “Heteroaryl”  refers  to a 5‐  to 20‐membered  (also denoted as “C5‐C20”)  ring  system  radical  comprising hydrogen atoms, one to fourteen carbon atom and from one to six heteroatoms selected  from the group consisting of nitrogen, oxygen, sulfur and selenium, and at least one aromatic ring. A  heteroaryl can also refer to a ring system radical comprising one to thirteen carbon atoms, one to six  heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and selenium, and at least  one aromatic ring. For purposes of this invention, the heteroaryl radical can be a monocyclic, bicyclic,  tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen,  carbon, sulfur or selenium atoms  in the heteroaryl radical can be optionally oxidised; the nitrogen  atom  can be optionally quaternised.  Examples  include, but  are not  limited  to,  azepinyl,  acridinyl,  benzimidazolyl,  benzothiazolyl,  benzindolyl,  benzodioxolyl,  benzofuranyl,  benzooxazolyl,  benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4‐benzodioxanyl, benzonaphthofuranyl,  benzoxazolyl,  benzodioxolyl,  benzodioxinyl,  benzopyranyl,  benzopyranonyl,  benzofuranyl,  benzofuranonyl, benzothienyl  (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2‐a]pyridinyl,  carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl,  indazolyl,  indolyl,  indazolyl,  isoindolyl,  indolinyl,  isoindolinyl,  isoquinolyl,  indolizinyl,  isoxazolyl,  naphthyridinyl, oxadiazolyl, 2‐oxoazepinyl, oxazolyl, oxiranyl, 1‐oxidopyridinyl, 1‐oxidopyrimidinyl, 1‐ oxidopyrazinyl,  1‐oxidopyridazinyl,  1‐phenyl‐1H‐pyrrolyl,  phenazinyl,  phenothiazinyl,  phenoxazinyl,  phthalazinyl,  pteridinyl,  purinyl,  pyrrolyl,  pyrazolyl,  pyridinyl,  pyrazinyl,  pyrimidinyl,  pyridazinyl,  quinazolinyl,  quinoxalinyl,  quinolinyl,  quinuclidinyl,  isoquinolinyl,  tetrahydroquinolinyl,  thiazolyl,  thiadiazolyl,  triazolyl,  tetrazolyl,  triazinyl,  and  thiophenyl  (i.e.  thienyl).  A  5‐  to  20‐membered  heteroaryl may be denoted herein as “C5‐C20 heteroaryl”, meaning 5‐ to 20‐membered ring system  radical comprising hydrogen atoms, one to fourteen carbon atoms, one to six heteroatoms selected  from the group consisting of nitrogen, oxygen, sulfur and selenium, and at least one aromatic ring. For  example, a 5‐ to 10‐ membered heteroaryl radical may be denoted herein as “C5‐C10 heteroaryl”.  [0035] “Heterocycloalkyl” refers to a stable non‐aromatic monocyclic or polycyclic fully saturated or  unsaturated hydrocarbon radical which consists of carbon and hydrogen atoms and from one to six  heteroatoms  selected  from  the group  consisting of nitrogen, oxygen,  sulfur and  selenium. An  “N‐   heterocycloalkyl” is a heterocycloalkyl comprising at least one ring nitrogen. Unless stated otherwise  specifically in the specification, the heterocycloalkyl radical can be a monocyclic, bicyclic, tricyclic or  tetracyclic ring system, which can  include fused or bridged ring systems; and the nitrogen, carbon,  sulfur or selenium atoms in the heterocycloalkyl radical can be optionally oxidised; the nitrogen atom  can be optionally quaternised; and  the heterocycloalkyl  radical  can be partially or  fully  saturated.  Examples  of  such  heterocycloalkyl  radicals  include,  but  are  not  limited  to,  dioxolanyl,  thienyl[1,3]dithianyl, decahydroisoquinolyl,  imidazolinyl,  imidazolidinyl,  isoxazolidinyl, morpholinyl,  octahydroindolyl,  octahydroisoindolyl,  2‐oxopiperazinyl,  2‐oxopiperidinyl,  2‐oxopyrrolidinyl,  oxazolidinyl,  piperidinyl,  piperazinyl,  4‐piperidonyl,  pyrrolidinyl,  pyrazolidinyl,  quinuclidinyl,  thiazolidinyl,  tetrahydrofuryl,  trithianyl,  tetrahydropyranyl,  thiomorpholinyl,  thiamorpholinyl,  1‐oxo‐thiomorpholinyl,  1,1‐dioxo‐thiomorpholinyl,  indolinyl,  isoindolinyl,  tetrahydroquinolyl,  tetrahydroisoquinolyl, coumaranyl, chromanyl, isochromanyl. A 3‐ to 10‐ membered heterocycloalkyl  may  be  denoted  herein  as  “C3‐C10  heterocycloalkyl”,  meaning  a  stable  3‐  to  10‐  membered  non‐aromatic monocyclic  or  polycyclic  fully  saturated  or  unsaturated  hydrocarbon  radical which  consists of carbon and hydrogen atoms and  from one to six heteroatoms selected  from  the group  consisting of nitrogen, oxygen, sulfur and selenium.  [0036] The term “substituted” used herein means any of the above groups (e.g. alkyl, alkenyl, alkynyl,  aryl,  cycloalkyl,  haloalkyl,  heterocyclyl,  heterocycloalkyl  and/or  heteroaryl)  wherein  at  least  one  hydrogen atom is replaced by a bond to a non‐hydrogen atom. “Substituted” also means any of the  above groups  in which one or more hydrogen atoms are  replaced by a higher‐order bond  (e.g., a  double‐ or triple‐bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups;  and nitrogen in groups such as imines, oximes, hydrazones, and nitriles.   [0037] As used herein,  the symbol 
Figure imgf000009_0001
  (hereinafter can be  referred  to as “a point of attachment  bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is  depicted as being attached to the point of attachment bond and the other of which is not depicted as  being attached to the point of attachment bond.   [0038] As used herein,  the  term “protecting group” refers  to a reversibly  formed derivative of an  existing functional group  in a molecule, wherein the functional group  is derivatised to decrease  its  reactivity (i.e. the functional group is “protected”). This is done to prevent the protected functional  group from reacting under the synthetic conditions to which the molecule comprising the protected  functional group is subjected in one or more subsequent steps. The protecting group can subsequently  be removed under orthogonal reaction conditions to return the functional group to its non‐derivatised  state. A commonly protected functional group  is an alcohol, with common protecting groups being  acetyl, trihaloacetyl, benzoyl, benzyl, methoxyethoxymethyl ether, dimethoxytrityl, methoxymethyl    ether,  p‐methoxybenxyl  ether,  p‐methoxyphenyl  ether, methylthiomethyl  ether,  pivaloyl,  t‐butyl  ethers,  tetrahydropyranyl,  tetrahydrofuran,  trityl,  silyl  ethers  such  as  trimethylsilyl (TMS), t‐ butyldimethylsilyl (TBDMS or TBS), tri‐i‐propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers,  methyl ethers, and ethoxyethyl ethers. Amines are another commonly protected functional group,  with  common  protecting  groups  being  carbobenxyloxy,  p‐methoxybenzyl  carbonyl,  t‐ butyloxycarbonyl,  9‐fluorenylmethyloxycarbonyl  (Fmoc),  acetyl,  trihaloacetyl,  benzoyl,  benzyl,  carbamate,  p‐methoxybenzyl,  3,4‐dimethoxybenzyl,  p‐methoxyphenyl,  tosyl,  troc  (tricholoroethyl  chloroformate), nosyl, and phthalimidyl.  [0039] As used herein, the term “base labile” refers to a group which is removable from a molecule  when  subjected  to  basic  reaction  conditions.  Accordingly,  a  “base  labile  protecting  group”  is  a  protecting group as defined above which can be removed from the relevant functional group via the  application  of  a  base.  Examples  of  base  labile  protecting  groups  include  acetyl,  benzoyl,  Fmoc,  trialkylsilyl, tert‐butyldiphenylsilyl, acetyl, trihaloacetyl, cyanoethyl, and phthalimidyl groups.  [0040] As  used  herein,  “oligonucleotide”  refers  to  an  oligomer  comprising  multiple  nucleotide  monomeric units and may be a nucleic acid or nucleic acid analogue.  [0041] A “nucleic acid analogue” is a compound that has an arrangement of nucleobases that mimics  the arrangement of nucleobases  in nucleic acids containing a 2’ deoxyribose 5’ monophosphate or  ribose 5’ monophosphate backbone, wherein the nucleic acid analogue is capable of base pairing with  a  complementary nucleic  acid. Examples of backbone moieties  include  amino  acids  as  in peptide  nucleic acids, glycol molecules as in glycol nucleic acids, threofuranosyl sugar molecules as in threose  nucleic acids, morpholine rings and phosphorodiamidate groups as in morpholinos, and cyclohexenyl  molecules as in cyclohexenyl nucleic acids.  [0042] A “furanose” or “furanosyl” is a carbohydrate containing a 5‐membered ring system consisting  of four carbon atoms and one oxygen atom,  i.e. a tetrahydrofuran derivative. Exemplary furanoses  include  ribose,  deoxyribose  and  dideoxyribose.  Further  furanoses  include  arabinofuranose  (arabinose),  xylofuranose  (xylose),  lyxofuranose  (lyxose),  xylulofuranose  (xylulose),  ribulofuranose  (ribulose),  erythrofuranose  (erythrose),  and  threofuranose  (threose).  Also  contained  within  the  definition of a furanose are furanoses where the tetrahydrofuran is substituted with groups other than  OH and CH2OH. For example, the furanose may comprise OH protecting groups such as those defined  above, e.g. a methyl ether, acetyl, or benzyl group, such that a 5’, 4’, 3’, or 2’ furanose OH may be  protected to provide an OMe, OAc, or OBn group. Other substitutions may be any of those described  herein. For example, the furanose may be substituted with one or more halo, alkyl, alkenyl, alkynyl,  heteroalkyl, haloalkyl, cyano, or nitro groups. Another possible substitution would be a furanose OH  being replaced with an ‐O‐CH2‐CH2‐O‐CH3 group.    [0043] The oligonucleotides of  the  invention may comprise a nucleoside or nucleosides  that have  been modified, for instance the modifications may be to the sugar moiety. In some examples, the 2’‐ position of the sugar moiety may be modified. A modification may be to any moiety that is not “‐H”  for DNA or not “‐OH” for RNA. Examples of such 2’ modifications are ‐O‐CH3 or ‐O‐CH2‐CH2‐O‐CH3, and  further examples are provided herein. In some examples, the 4’ position of the sugar moiety may be  modified,  for  instance  to  result  in  a  bridge  between  the  2’  position  and  the  4’  position.  The  oligonucleotide may comprise one, two, three, four, or more types of nucleosides. The oligonucleotide  may  comprise  a  combination  of  modified  nucleosides  and  unmodified  nucleosides.  The  oligonucleotide may comprise only modified nucleosides. The nucleotides of the oligonucleotide may  all be modified in the same manner or may be modified in two or more different manners.  [0044] Oligonucleotides display directionality in the internucleoside linkages, and as such, the ends  of the oligonucleotide are designated the 5’ (five prime) and 3’ (three prime) end. The 5’ end of an  oligonucleotide has the fifth carbon of the sugar ring at its terminus, and the 3’ end terminates in the  hydroxyl group of the third carbon in the sugar ring. An exemplary nucleoside is provided below, with  the 5’ and 3’ carbons labelled: 
Figure imgf000011_0001
.  [0045] Generally,  oligonucleotides  are  prepared  from  the  3’  end  to  the  5’  end,  i.e.  with  each  additional  nucleotide  attached  to  the  5’  end  of  the  oligonucleotide.  Often,  oligonucleotides  are  prepared making use of solid phase synthesis. Under this approach, the 3’ end of the first nucleotide  is bonded to a solid support and each additional nucleotide is reacted in turn to provide the desired  oligonucleotide. Once  the oligonucleotide has been prepared,  the 3’ end of  the oligonucleotide  is  cleaved from the solid support so that the oligonucleotide may be isolated.  [0046] The oligonucleotide may comprise a modification to one or more internucleoside linkages. For  instance,  the  oligonucleotide  may  comprise  one  or  more  phosphorothioate  linkages.  In  some  embodiments, all of  the  internucleotide  linkages within  the oligonucleotide are phosphorothioate  linkages.  The oligonucleotide may be or may  comprise  an oligonucleotide phosphorothioate.  The  oligonucleotide may comprise one or more phosphorodiamidate linkage. In some embodiments, all  of the intermonomer linkages within the oligonucleotide are phosphorodiamidate linkages.  [0047] The oligonucleotide may comprise one or more of a deoxyribonucleotide, a ribonucleotide,  an  arabinonucleotide,  a  2′‐Fluoroarabinonucleotide  (FANA),  a  2′‐O‐methyl  (2’OMe)  nucleotide,  a  phosphorothioate  2′‐O‐methyl  (PS‐2’OMe)  nucleotide,  a  2'‐O‐methoxyethyl  (MOE)  nucleotide,  a    phosphorothioate  2’‐O‐methoxyethyl  (PS‐MOE)  nucleotide,  a  phosphorodiamidate  morpholino  monomer,  a  locked nucleotide,  a P‐alkyl phosphonate nucleotide,  a  threose nucleotide,  a hexitol  nucleotide,  a  2’  hydroxy‐hexitol  nucleotide,  a  cyclohexene  nucleotide,  a  3’  deoxi‐DNA  (2’‐5’)  nucleotide,  a  peptide  nucleic  acid  (PNA)  residue,  a  2’‐O,4’‐C‐ethylene‐bridged  nucleotide,  or  any  combination thereof.  [0048] The  oligonucleotide  may  be  or  may  comprise  a  DNA  oligomer,  an  RNA  oligomer,  an  arabinonucleic acid  (ANA) oligomer, a 2′‐Fluoroarabinonucleic acid  (FANA) oligomer, a 2′‐O‐methyl  ribonucleic  acid  (2’OMe)  oligomer,  a  phosphorothioate  2′‐O‐methyl  ribonucleic  acid  (PS‐2’OMe)  oligomer, a 2'‐O‐methoxyethyl (MOE) nucleic acid oligomer, a phosphorothioate 2’‐O‐methoxyethyl  (PS‐MOE) nucleic acid oligomer, a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic  acid (LNA) oligomer, a P‐alkyl phosphonate nucleic acid (phNA) oligomer, a threose nucleic acid (TNA)  oligomer, a hexitol nucleic acid (HNA) oligomer, a 2’ hydroxy‐hexitol (AtNA) oligomer, a cyclohexene  nucleic acid (CeNA) oligomer, a 3’ deoxi‐DNA (2’‐5’) oligomer, a peptide nucleic acid (PNA) oligomer,  a 2’‐O,4’‐C‐ethylene‐bridged nucleic acid (ENA) oligomer, or any combination thereof.  [0049] Any of the oligonucleotides of the present disclosure may be present as a pharmaceutically  acceptable  salt, ester,  salt of  said ester, or hydrate of  said oligonucleotide,  and  references  to  an  oligonucleotide encompass such compounds. The oligonucleotide of the present disclosure may be  present as a prodrug.  [0050] In  some  embodiments,  the  oligonucleotide  is  an  antisense  oligonucleotide.  An  antisense  oligonucleotide,  in the context of the present disclosure,  is an oligonucleotide comprising subunits  that comprise moieties capable of binding to nucleobases. Hence, antisense oligonucleotides can be  designed  to  be  capable  of  hybridising  to  specific  nucleic  acid  sequences.  The  subunits may  be  monomers, each monomer comprising a moiety capable of binding  to a nucleobase. The moieties  capable of binding to a nucleobase may bind by base‐specific hydrogen bonding, such as Watson‐Crick  base pairing.  [0051] The term “antisense” refers to oligonucleotides that are at least partially complementary to a  region of a sense strand of a nucleic acid. The degree of complementarity may not be exact, as long  as the antisense oligonucleotide and the pre‐mRNA can hybridise under physiological conditions. In  some examples, the antisense oligonucleotide and the pre‐mRNA may be complementary apart from  5,  4,  3,  2,  or  1  mismatches.  In  some  examples,  the  antisense  oligonucleotide  is  perfectly  complementary  to  a  region  of  the  pre‐mRNA.  In  other  examples,  the  antisense  oligonucleotide  comprises  a  region  that  is  perfectly  complementary  to  a  region  of  the  pre‐mRNA, wherein  the  complementary regions are of a sufficient length to allow binding by hybridisation.    [0052] The term “aptamer” refers to oligonucleotides that are short, single stranded nucleic acids or  nucleic acid analogues, such as single stranded DNA (ssDNA) or single stranded RNA (ssRNA), which  selectively  bind  to  a  specific  target.  This  target may  be  a  protein,  peptide,  carbohydrate,  small  molecule, toxin, or potentially a cell. Aptamers are highly versatile as their binding is determined by  their tertiary structure. Target recognition and binding results from the aptamer “fitting” the desired  target through base stacking, intercalation, hydrophobic interactions and sterics.  [0053] As used in this disclosure, the term “short interfering RNA” (siRNA), also called silencing RNA,  refers  to  oligonucleotides  that  are  short,  double  stranded  RNA molecules which  are  non‐coding.  siRNAs may be synthetic or naturally occurring. Typically, siRNAs are 20‐24 base pairs long. Typically,  siRNAs include a number of overhanging nucleotides (for example, 1, 2, or 3) at each end. These siRNAs  are therefore typically 22‐30 nucleotides long. siRNAs are used in gene silencing as these siRNAs will  bind to their complementary sequence and prevent gene transcription.  [0054] The  term  “splice‐switching  antisense  oligonucleotide”,  as  used  herein,  refers  to  synthetic  antisense nucleic acid oligomers which are configured to inhibit or prevent protein production relating  to a  specific gene.  Splice‐switching antisense oligonucleotides bind  to pre‐mRNA via base pairing,  preventing the pre‐mRNA from interacting with the splicing machinery of the cell. This disrupts the  normal splicing process of gene transcription by blocking either RNA‐RNA base pairing or protein‐RNA  binding resulting from the pre‐mRNA/splicing machinery interactions.   [0055] The term “microRNA” (miRNA), as used herein, refers to oligonucleotides that are short, single  stranded RNA molecules which are non‐coding. Typically miRNAs are 21‐23 nucleotides long. miRNAs  may be  synthetic or naturally occurring. These miRNAs are used  to base pair  to mRNA molecules  comprising the complementary nucleotide sequence. These mRNA molecules are then gene silenced.  [0056] As  used  in  this  disclosure,  xeno  nucleic  acid  (XNA)  oligomers  are  synthetic  nucleic  acid  analogues where the naturally occurring sugar backbone (2’ deoxyribose 5’ monophosphate or ribose  5’ monophosphate)  of  an  RNA  or DNA  sequence  has  been  replaced with  a  synthetic  alternative  resulting  in an arrangement of nucleobases that mimics the arrangement of nucleobases  in nucleic  acids containing 2’ deoxyribose 5’ monophosphate or ribose 5’ monophosphate. These nucleic acid  analogues are generally capable of base pairing with a complementary nucleic acids. This synthetic  alternative  may  be  an  alternate  sugar  or  may  be  a  non‐sugar  moiety.  Exemplary  sugar  replacing/backbone moieties  include amino acids as  in peptide nucleic acids, glycol molecules as  in  glycol nucleic acids, threofuranosyl sugar molecules as in threose nucleic acids, morpholine rings and  phosphorodiamidate groups as  in morpholinos,  cyclohexenyl molecules as  in  cyclohexenyl nucleic  acids, 1,5‐anhydrohexitol nucleic acids, locked nucleic acids, and fluoro arabino nucleic acids. Locked  nucleic acids are nucleic acids where a carbon bridge has been introduced to ribose to connect the 2’    oxygen and the 4’ carbon, “locking” the sugar conformation. PNAs are nucleic acids where the sugar‐ phosphate backbone has been replaced with a synthetic peptide, i.e. a polymer prepared from amino  acid monomers.  [0057] The term “small activating ribonucleic acid” (saRNA), as used herein, refers to small double‐ stranded RNAs that target gene promoters to cause RNA activation. These saRNAs therefore induce  transcriptional gene activation. These saRNAs are typically around 21 nucleotides in length with the 3’  end of each strand having a two nucleotide overhang.  [0058] Gapmers, as used herein, are short antisense oligonucleotide structures comprising a DNA  sequence with RNA‐like sequences on both sides of the DNA sequence. These gapmers are designed  to take part in gene silencing and are designed to hybridise to a target piece of RNA. Once hybridised  to the target RNA, RNase H cleavage is induced.  [0059] As used  in  this disclosure  the  terms “expanded RNA”  (xRNA) and “expanded DNA”  (xDNA)  refer to nucleotide systems which comprise size‐expanded nucleotides. Size‐expanded nucleotides are  nucleotides where the naturally occurring bases are fused with a benzene ring to provide the four  expanded bases xA, xG, xT, and xC, which have the structures shown below. These expanded bases  generally form base pairs with the associated natural base, i.e. xA‐T, xG‐C, xT‐A, and xC‐G. 
Figure imgf000014_0001
  [0060] Physiological conditions are the conditions (such as temperature, pH, concentration of various  ions, etc)  found  in natural,  in  vivo,  situations, or  conditions  that  correspond  such a  situation. For  instance, the antisense oligonucleotide may bind by hybridisation in intracellular conditions, such as  the  intracellular conditions of human cells. The physiological conditions may be  those during pre‐ mRNA  splicing  in  cells  found  in  the  liver,  for  instance  the  intracellular  conditions  of  human  hepatocytes. Confirmation of hybridisation in physiological conditions may be performed in vitro, for  instance at a temperature, pH, and salt concentration that approximates intracellular conditions.    Linkers    [0061] In an embodiment of the invention, there is provided a compound of formula (I): 
Figure imgf000014_0002
    A is mono‐ or bicyclic heteroaryl group in which at least one carbon ring‐atom is replaced with N;  X, Y, and Z are each independently selected from C1‐30 alkyl, C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m,  C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, C5‐10 heteroaryl, or absent;  wherein at least one of X, Y and Z is present;   wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl or C5‐10 heteroaryl  is  optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8,  SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3;   n is 1 or 2;  m is an integer from 1‐30;   each   is either a single bond or double bond;  W is OR2 or NR3R4;  R1 and R2 are each independently selected from H and a base labile protecting group;  R3 and R4 are each independently selected from H and an optionally substituted C1‐10 alkyl;  R5 is absent, is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl;  R6 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R7 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R8 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R9, is selected from H, C1‐10 alkyl, halide, C(O)C1‐10 alkyl, C1‐10 haloalkyl, C(O)OR12, C(O)SR12, C(O)NR13R14,  CN, or NO2;   R10, and R11 are each independently selected from H, C1‐10 alkyl, halide, or C1‐10 haloalkyl;  R12 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R13 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  and  R14 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  [0062] In embodiments of the invention, X‐Y‐Z is not a furanosyl group.  [0063] In embodiments of the invention, X‐Y‐Z does not comprise a furanosyl group.  [0064] In embodiments of the invention, A is able to undertake Watson‐Crick base pairing.  [0065] In embodiments of  the  invention, A  is a natural or artificial nucleobase. A nucleobase, or  nitrogenous base, is a nitrogen‐containing biological compound which is bonded to a five‐carbon sugar  (ribose or 2’‐deoxyribose) to form a nucleoside. These nucleosides are bonded to phosphate groups  to form nucleotides, which are the monomeric units of nucleic acid polymers, for example RNA and  DNA. Nucleobases generally have  ring  structures which are derived  from purine  (purine bases) or  pyrimidine  (pyrimidine  bases).  There  are  five  “primary”  or  “canonical”  nucleobases,  adenine  (A),    cytosine  (C),  guanine  (G),  thymine  (T),  and uracil  (U). These  five primary nucleobases  are natural  nucleobases. Non‐primary nucleobases may also be natural nucleobases,  i.e. modified nucleobases  which occur in nature, such as aminoadenine (Z). Other natural non‐primary nucleobases include the  modified nucleobases 5‐methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and  7‐methylguanosine  (m7G). A  large  number  of  artificial  nucleobases  exist,  such  as  isoguanine  and  isocytosine. Artificial nucleobases are capable of forming hydrogen bonds and therefore are capable  of  taking part  in Watson‐Crick base pairing via  these hydrogen bonds. Other artificial nucleobases  include aminoallyl nucleobases such as aminoallyl uracil or aminoallyl cytosine which are used in post‐ labelling  of  nucleic  acids  by  fluorescence  detection,  i.e.  providing  a  non‐isotopic  tag.  Artificial  nucleobases are commonly used in oligonucleotides for use as anticancer or antiviral agents.   [0066] In embodiments of the invention A is selected from 
Figure imgf000016_0001
  ;  wherein:   each   is either a single bond or double bond;  each of A1, A2, A3 is selected from NR18 and CR19, where at least one of A1, A2, and A3 is NR18;  each of A4, A5, A6, and A7 is selected from NR18 and CR19 where at least one of A4, A5, A6, and A7 is NR18;  at  least one R15, R16, or R17 group  is present and  independently selected from H, OR20, SR20, =O, =S,  NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo, wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl is optionally substituted with at least one group selected from halo, OR5, C(O)OR5, C(O)NR6R7,  SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;   R18, if present, is H, an optionally substituted C1‐10 alkyl group, or a base labile protecting group;  R19  is  selected  from H, OR20, SR20, =O, =S, NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo,  wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl is optionally substituted with at least one group  selected from halo, OR5, C(O)OR5, C(O)NR6R7, SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;  R20 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R21 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl; and  R22 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In 
Figure imgf000016_0002
these  embodiments, both    and X may be  attached  to A  at  any  appropriate point of  connection. In some embodiments where A is monocyclic, 
Figure imgf000016_0003
 and X are attached in a meta    or para configuration. In some embodiments where A is monocyclic, 
Figure imgf000017_0001
 and X are attached  in  a meta  configuration.  In  some  embodiments where  A  is monocyclic, 
Figure imgf000017_0002
  and  X  are  attached in a para configuration.  [0067] In some embodiments A, is selected from: 
Figure imgf000017_0003
  be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen  within  the  structure of A.  In  some  embodiments where A  is monocyclic, 
Figure imgf000017_0004
  and X  are  attached in a meta or para configuration. In some embodiments where A is monocyclic, 
Figure imgf000017_0005
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments  where  A  is  monocyclic, 
Figure imgf000017_0006
 and X are attached in a para configuration.    [0068] In some embodiments, 
Figure imgf000017_0007
.  [0069] In some embodiments, 
Figure imgf000017_0008
.  [0070] In some embodiments, 
Figure imgf000017_0009
.   
Figure imgf000018_0001
     
Figure imgf000018_0002
[0076] In some embodiments, 
Figure imgf000018_0003
.  [0077] In some embodiments, A is selected from: 
Figure imgf000018_0004
  appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A.  In some embodiments where A  is monocyclic, 
Figure imgf000018_0005
 and X are attached  in a meta or para 
Figure imgf000018_0006
configuration.  In some embodiments where A  is monocyclic,   and X are attached  in a    meta configuration. In some embodiments where A is monocyclic, 
Figure imgf000019_0001
 and X are attached  in a para configuration.   
Figure imgf000019_0002
.  [0080] In some embodiments, 
Figure imgf000019_0003
.  [0081] In some embodiments, 
Figure imgf000019_0004
.  [0082] In some embodiments, 
Figure imgf000019_0005
.  [0083] In some embodiments, 
Figure imgf000019_0006
.  [0084] In some embodiments, 
Figure imgf000019_0007
.   [0085] In some embodiments, A is selected from:     
Figure imgf000020_0001
embodiments, both   and X may be attached to A at any appropriate point of connection,  e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is  monocyclic, 
Figure imgf000020_0002
 and X are attached in a meta or para configuration. In some embodiments  where  A  is  monocyclic, 
Figure imgf000020_0003
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments where A is monocyclic, 
Figure imgf000020_0004
 and X are attached in a para configuration.  [0086] In some embodiments, A is selected from:   
Figure imgf000020_0006
embodiments, both   and X may be attached to A at any appropriate point of connection,  e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is  monocyclic, 
Figure imgf000020_0005
 and X are attached in a meta or para configuration. In some embodiments    where  A  is  monocyclic, 
Figure imgf000021_0001
  and  X  are  attached  in  a  meta  configuration.  In  some 
Figure imgf000021_0002
embodiments where A is monocyclic,   and X are attached in a para configuration. 
Figure imgf000021_0003
[0089] In some embodiments, 
Figure imgf000021_0004
  [0090] In some embodiments, 
Figure imgf000021_0005
  [0091] In some embodiments, 
Figure imgf000021_0006
  [0092] In some embodiments, 
Figure imgf000021_0007
  [0093] In some embodiments, 
Figure imgf000021_0008
  [0094] In some embodiments, 
Figure imgf000021_0009
    [0095] In some embodiments, 
Figure imgf000022_0001
.  [0096] In some embodiments, 
Figure imgf000022_0002
.  [0097] In some embodiments, 
Figure imgf000022_0003
.  [0098] In some embodiments, 
Figure imgf000022_0004
.  [0099] In some embodiments, R1 is selected from H, cyanoethyl, trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, or phthalimidyl.  In  some embodiments, R1  is H.  In  some embodiments, R1  is  cyanoethyl.  In  some  embodiments,  R1  is  trialkylsilyl.  In  some  embodiments,  R1  is  tert‐ butyldiphenylsilyl.  In some embodiments, R1  is acetyl.  In some embodiments, R1  is trihaloacetyl.  In  some embodiments, R1 is phthalimidyl.   [0100] In some embodiments, R2 is selected from H, cyanoethyl, trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, or phthalimidyl.  In  some embodiments, R2  is H.  In  some embodiments, R2  is  cyanoethyl.  In  some  embodiments,  R2  is  trialkylsilyl.  In  some  embodiments,  R2  is  tert‐ butyldiphenylsilyl.  In some embodiments, R2  is acetyl.  In some embodiments, R2  is trihaloacetyl.  In  some embodiments, R1 is phthalimidyl.   [0101] In some embodiments, wherein R3 and R4 are each an optionally substituted C1‐10 alkyl. In some  embodiments, R3 and R4 are each an unsubstituted branched chain C1‐10 alkyl. In some embodiments,  R3 and R4 are each isopropyl.  [0102] In  some embodiments, R5  is absent, or  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R5  is absent.  In  some embodiments, R5  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl, acetyl,  trihaloacetyl, and phthalimidyl. In some embodiments, R5 is selected from H, C1‐10 alkyl, C2‐10 alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R5  is  trialkylsilyl.  In  some  embodiments  R5  is  tert‐ butyldiphenylsilyl.  In some embodiments, R5  is acetyl.  In some embodiments, R5  is trihaloacetyl.  In    some embodiments, R5 is phthalimidyl. In some embodiments, R5 is H. In some embodiments, R5 is C1‐ 10 alkyl. In some embodiments, R5 is C2‐10 alkenyl. In some embodiments, R5 is C2‐10 alkynyl.  [0103] In  some  embodiments,  R6  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R6  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R6 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R6 is trialkylsilyl. In some embodiments, R6 is tert‐butyldiphenylsilyl. In some embodiments, R6 is acetyl.  In  some  embodiments,  R6  is  trihaloacetyl.  In  some  embodiments,  R6  is  phthalimidyl.  In  some  embodiments, R6 is H. In some embodiments, R6 is C1‐10 alkyl. In some embodiments, R6 is C2‐10 alkenyl.  In some embodiments, R6 is C2‐10 alkynyl.  [0104] In  some  embodiments,  R7  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R7  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R7 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R7 is trialkylsilyl. In some embodiments, R7 is tert‐butyldiphenylsilyl. In some embodiments, R7 is acetyl.  In  some  embodiments,  R7  is  trihaloacetyl.  In  some  embodiments,  R7  is  phthalimidyl.  In  some  embodiments, R7 is H. In some embodiments, R7 is C1‐10 alkyl. In some embodiments, R7 is C2‐10 alkenyl.  In some embodiments, R7 is C2‐10 alkynyl.  [0105] In  some  embodiments,  R8  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R8  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R8 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R8 is trialkylsilyl. In some embodiments, R8 is tert‐butyldiphenylsilyl. In some embodiments, R8 is acetyl.  In  some  embodiments,  R8  is  trihaloacetyl.  In  some  embodiments,  R8  is  phthalimidyl.  In  some  embodiments, R8 is H. In some embodiments, R8 is C1‐10 alkyl. In some embodiments, R8 is C2‐10 alkenyl.  In some embodiments, R8 is C2‐10 alkynyl.  [0106] In  some  embodiments,  R12  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R12 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R12 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R12  is trialkylsilyl.  In some embodiments, R12  is tert‐butyldiphenylsilyl.  In some embodiments, R12  is  acetyl. In some embodiments, R12 is trihaloacetyl. In some embodiments, R12 is phthalimidyl. In some  embodiments, R12  is H.  In  some embodiments, R12  is C1‐10 alkyl.  In  some embodiments, R12  is C2‐10  alkenyl. In some embodiments, R12 is C2‐10 alkynyl.    [0107] In  some  embodiments,  R13  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R13 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R13 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R13  is trialkylsilyl.  In some embodiments, R12  is tert‐butyldiphenylsilyl.  In some embodiments, R13  is  acetyl. In some embodiments, R13 is trihaloacetyl. In some embodiments, R13 is phthalimidyl. In some  embodiments, R13  is H.  In  some embodiments, R13  is C1‐10 alkyl.  In  some embodiments, R13  is C2‐10  alkenyl. In some embodiments, R13 is C2‐10 alkynyl.  [0108] In  some  embodiments,  R14  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R14 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R14 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R14  is trialkylsilyl.  In some embodiments, R14  is tert‐butyldiphenylsilyl.  In some embodiments, R14  is  acetyl. In some embodiments, R14 is trihaloacetyl. In some embodiments, R14 is phthalimidyl. In some  embodiments, R14  is H.  In  some embodiments, R14  is C1‐10 alkyl.  In  some embodiments, R14  is C2‐10  alkenyl. In some embodiments, R14 is C2‐10 alkynyl.  [0109] In some embodiments, R18, when present, is selected from H, an optionally substituted C1‐10  alkyl  group,  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments,  R18  is  selected  from  H  and  an  optionally  substituted  C1‐10  alkyl  group.  In  some  embodiments,  R18  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R18 is H. In some embodiments, R18 is an optionally substituted  C1‐10  alkyl  group.  In  some  embodiments,  R18  is  trialkylsilyl.  In  some  embodiments,  R18  is  tert‐ butyldiphenylsilyl. In some embodiments, R18 is acetyl. In some embodiments, R18 is trihaloacetyl. In  some embodiments, R18 is phthalimidyl.  [0110] In some embodiments, R20, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R20  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R20 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R20 is trialkylsilyl. In some embodiments, R20 is tert‐butyldiphenylsilyl. In some  embodiments, R20 is acetyl. In some embodiments, R20 is trihaloacetyl. In some embodiments, R20 is  phthalimidyl.  In  some  embodiments,  R20  is  H.  In  some  embodiments,  R20  is  C1‐10  alkyl.  In  some  embodiments, R20 is C2‐10 alkenyl. In some embodiments, R20 is C2‐10 alkynyl.  [0111] In some embodiments, R21, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some    embodiments,  R21  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R21 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R21 is trialkylsilyl. In some embodiments, R21 is tert‐butyldiphenylsilyl. In some  embodiments, R21 is acetyl. In some embodiments, R21 is trihaloacetyl. In some embodiments, R21 is  phthalimidyl.  In  some  embodiments,  R21  is  H.  In  some  embodiments,  R21  is  C1‐10  alkyl.  In  some  embodiments, R21 is C2‐10 alkenyl. In some embodiments, R21 is C2‐10 alkynyl.  [0112] In some embodiments, R22, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R22  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R22 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R22 is trialkylsilyl. In some embodiments, R22 is tert‐butyldiphenylsilyl. In some  embodiments, R22 is acetyl. In some embodiments, R22 is trihaloacetyl. In some embodiments, R22 is  phthalimidyl.  In  some  embodiments,  R22  is  H.  In  some  embodiments,  R22  is  C1‐10  alkyl.  In  some  embodiments, R22 is C2‐10 alkenyl. In some embodiments, R22 is C2‐10 alkynyl.  [0113] In  the  preceding  embodiments,  the  trialkylsilyl  group,  if  present, may  have  the  formula  Si(C1‐C4 alkyl)3.  In further embodiments, trialkylsilyl group,  if present,  is selected from trimethylsilyl  (TMS),  triethylsilyl  (TES),  tert‐butyldimethylsilyl  (TBS),  and  triisopropylsilyl  (TIPS).  In  further  embodiments, the trialkylsilyl group  is trimethylsilyl (TMS). In further embodiments, the trialkylsilyl  group  is  triethylsilyl  (TES).  In  further embodiments,  the  trialkylsilyl group  is  tert‐butyldimethylsilyl  (TBS). In further embodiments, the trialkylsilyl group is triisopropylsilyl (TIPS).  [0114] In  the  preceding  embodiments,  the  trihaloacetyl  group,  if  present,  is  trichloroacetyl  or  trifluoroacetyl.  In  some  embodiments,  the  trihaloacetyl  group  is  trichloroacetyl.  In  some  embodiments, the trihaloacetyl group is trifluoroacetyl.  [0115] In the preceding embodiments, therefore, the base labile protecting group may be selected  from  trialkylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  The  base  labile  protecting  group may  be  selected from Si(C1‐C4 alkyl)3, acetyl, trihalo acetyl, and phthalimidyl. The base labile protecting group  may be  selected  from  Si(C1‐C4 alkyl)3,  acetyl,  trichloroacetyl,  trifluoroacetyl,  and phthalimidyl. The  base  labile  protecting  group  may  be  selected  from  Si(C1‐C4 alkyl)3,  acetyl,  trifluoroacetyl,  and  phthalimidyl. The base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl  (TES), tert‐butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trihalo acetyl, and phthalimidyl. The  base  labile  protecting  group  may  be  selected  from  trimethylsilyl  (TMS),  triethylsilyl  (TES),  tert‐ butyldimethylsilyl  (TBS),  triisopropylsilyl  (TIPS),  acetyl,  trichloroacetyl,  trifluoroacetyl,  and  phthalimidyl. The base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl  (TES), tert‐butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trifluoroacetyl, and phthalimidyl.    [0116] In some embodiments, R9 is H. In some embodiments, R10 is H. In some embodiments R11 is H.  In some embodiments, R9 and R10 are both H. In some embodiments, R9 and R11 are both H. In some  embodiments, R10 and R11 are both H. In some embodiments, R9, R10, and R11 are each H.  [0117] In some embodiments, R10 is F. In some embodiments, R11 is F. In some embodiments, R10 and  R11 are both F.  [0118] In some embodiments, R15  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R15 is selected from H, =O, and NR21R22. In some embodiments, R15 is H. In some  embodiments, R15 is =O. In some embodiments, R15 is NR21R22.  [0119] In some embodiments, R16  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R16 is selected from H, =O, and NR21R22. In some embodiments, R16 is H. In some  embodiments, R16 is =O. In some embodiments, R16 is NR21R22.  [0120] In some embodiments, R17  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R17 is selected from H, =O, and NR21R22. In some embodiments, R17 is H. In some  embodiments, R17 is =O. In some embodiments, R17 is NR21R22.  [0121] In some embodiments Z is absent and X and Y are each independently selected from C1‐30 alkyl,  C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m, C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl,  C6‐10 aryl, or C5‐10 heteroaryl; wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐ 10 aryl or C5‐10 heteroaryl is optionally substituted with at least one group selected from halo, C1‐10 alkyl,  OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3.  [0122] In  some embodiments, X  is  selected  from NR5, C3‐10  cycloalkyl, C3‐10 heterocycloalkyl, C5‐10  cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl; wherein each is optionally substituted with at least one  group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN,  NO2, and N3.  In some embodiments, X  is NR5.  In some embodiments, X  is C3‐10 cycloalkyl.  In some  embodiments,  X  is  C3‐10  heterocycloalkyl.  In  some  embodiments,  X  is  C5‐10  cycloalkenyl.  In  some  embodiments, X is C6‐10 aryl. In some embodiments, X is C5‐10 heteroaryl.  [0123] In  some embodiments, X  is  selected  from NR5, C3‐10  cycloalkyl, C3‐10 heterocycloalkyl, C5‐10  cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl. In some embodiments, X is NR5. In some embodiments, X  is C3‐10 cycloalkyl. In some embodiments, X is C3‐10 heterocycloalkyl. In some embodiments, X is C5‐10  cycloalkenyl. In some embodiments, X is C6‐10 aryl. In some embodiments, X is C5‐10 heteroaryl.  [0124] In  some  embodiments,  X  is  selected  from  NR5  and  C3‐10  heterocycloalkyl.  In  some  embodiments, X is NR5. In some embodiments, X is C3‐10 heterocycloalkyl.  [0125] In  some  embodiments,  X  is  selected  from  NR5,  pyrrolidine,  and  piperidine.  In  some  embodiments, X is NR5. In some embodiments, X is pyrrolidine. In some embodiments, X is piperidine.    [0126] In some embodiments, Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O)n, C3‐10 cycloalkyl, and  C3‐10 heterocycloalkyl; wherein each C3‐10 cycloalkyl or C3‐10 heterocycloalkyl is optionally substituted  with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6,  P(O)(OR6)2,  CN, NO2,  and  N3.  In  some  embodiments,  Y  is  C1‐30  alkyl.  In  some  embodiments,  Y  is  (CH2CH2O)m.  In some embodiments, Y  is C(O)n.  In some embodiments, Y  is C3‐10 cycloalkyl.  In some  embodiments, Y is C3‐10 heterocycloalkyl.  [0127] In some embodiments, Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O), C3‐10 cycloalkyl, and C3‐ 10 heterocycloalkyl. In some embodiments, Y is C1‐30 alkyl. In some embodiments, Y is (CH2CH2O)m. In  some embodiments, Y is C(O). In some embodiments, Y is C3‐10 cycloalkyl. In some embodiments, Y is  C3‐10 heterocycloalkyl.  [0128] In  some  embodiments,  Y  is  selected  from  C1‐30  alkyl,  C(O),  and  C3‐10  cycloalkyl.  In  some  embodiments  Y  is  C1‐30  alkyl.  In  some  embodiments,  Y  is  C(O).  In  some  embodiments,  Y  is  C3‐10  cycloalkyl.  [0129] In some embodiments, X‐Y‐Z comprises at least one tertiary amine.  [0130] In  some embodiments, X‐Y‐Z  comprises  at  least one of NR5, C(O), C3‐10  cycloalkyl, C3‐10 N‐ heterocycloalkyl, C6‐10 aryl, and C5‐10 heteroaryl.  [0131] In  some embodiments, X‐Y‐Z  comprises  at  least one of NR5, C(O), C3‐10  cycloalkyl, C3‐10 N‐ heterocycloalkyl, and C6‐10 aryl.  [0132] In some embodiments, X‐Y‐Z comprises NR5.  [0133] In some embodiments, X‐Y‐Z comprises C(O).  [0134] In some embodiments, X‐Y‐Z comprises C3‐10 cycloalkyl.  [0135] In some embodiments, X‐Y‐Z comprises C3‐10 N‐heterocycloalkyl.  [0136] In some embodiments, X‐Y‐Z comprises C6‐10 aryl.  [0137] In some embodiments, X‐Y‐Z comprises C5‐10 heteroaryl.  [0138] In some embodiments, X‐Y‐Z is selected from:  
Figure imgf000027_0001
[0139] In some embodiments, X‐Y‐Z is selected from:        
Figure imgf000028_0001
[0144] In some embodiments, 
Figure imgf000028_0002
.   [0145] In some 
Figure imgf000028_0003
   [0146] In some 
Figure imgf000028_0004
  [0147] In some embodiments, 
Figure imgf000028_0005
.  [0148] In some embodiments, 
Figure imgf000028_0006
.  [0149] In some embodiments, 
Figure imgf000028_0007
.     
Figure imgf000029_0001
[0153] In some embodiments, X‐Y‐Z is selected from:   ,
Figure imgf000029_0002
[0154] In some embodiments, X‐Y‐Z is selected from:   ,
Figure imgf000029_0003
[0155] In some embodiments, X‐Y‐Z is selected from:  
Figure imgf000029_0004
[0156] In some embodiments, X‐Y‐Z is selected from:    
Figure imgf000030_0001
  
Figure imgf000030_0002
   [0161] In some embodiments, 
Figure imgf000030_0003
.   [0162] In some embodiments, 
Figure imgf000030_0004
.  [0163] In some embodiments, 
Figure imgf000030_0005
.  [0164] In some embodiments, 
Figure imgf000030_0006
.  [0165] In some embodiments, 
Figure imgf000030_0007
.  [0166] In some embodiments, 
Figure imgf000030_0008
.  [0167] In some embodiments, 
Figure imgf000030_0009
.    [0168] In some embodiments, 
Figure imgf000031_0001
.   
Figure imgf000031_0002
[0172] In some embodiments, the compound is selected from:  ,
Figure imgf000031_0003
[0173] In some embodiments, the compound is selected from:     
Figure imgf000032_0001
.  [0174] In some embodiments, the compound is selected from: 
Figure imgf000032_0002
 
Figure imgf000033_0001
  [0175] In some embodiments, the compound is selected from:   
Figure imgf000033_0002
.  [0176] In some embodiments, the compound is:
Figure imgf000033_0003
.  [0177] In some embodiments, the compound 
Figure imgf000033_0004
.   
Figure imgf000034_0001
[0178] In some embodiments, the compound is: .  [0179] In some embodiments, the compound is:
Figure imgf000034_0002
.  [0180] In some embodiments, the compound is: 
Figure imgf000034_0003
.  [0181] In some embodiments, the compound is: 
Figure imgf000034_0004
.  [0182] In some embodiments, the compound is: 
Figure imgf000034_0005
.  [0183] In some embodiments, the compound is: 
Figure imgf000034_0006
.  [0184] In some embodiments, the compound is: 
Figure imgf000034_0007
.  [0185] In some embodiments, the compound is: 
Figure imgf000034_0008
.  [0186] In some embodiments, the compound is selected from:   
Figure imgf000035_0001
  [0187] In some embodiments, the compound is selected from:   
Figure imgf000035_0002
   
Figure imgf000036_0001
.  [0189] In some embodiments, the compound is selected from:     
Figure imgf000037_0001
[0191] In some embodiments, the compound is:
Figure imgf000037_0002
. 
Figure imgf000037_0003
  [0193] In some embodiments, the compound is: 
Figure imgf000037_0004
.  [0194] In some embodiments, the compound is:
Figure imgf000037_0005
.    [0195] In some embodiments, the compound is:
Figure imgf000038_0001
.  [0196] In some embodiments, the compound is:
Figure imgf000038_0002
.  [0197] In some embodiments, the compound is:
Figure imgf000038_0003
.  [0198] In some embodiments, the compound is:
Figure imgf000038_0004
. 
Figure imgf000038_0005
  [0200] In some embodiments, the compound is:
Figure imgf000038_0006
.  [0201] In some embodiments, the compound is:
Figure imgf000038_0007
.  [0202] In some embodiments, the compound is:
Figure imgf000038_0008
.  [0203] In some embodiments, the compound 
Figure imgf000038_0009
.   
Figure imgf000039_0001
  [0205] In some embodiments, the compound is selected from: 
Figure imgf000039_0002
[0206] In some embodiments, the compound is selected from:     
Figure imgf000040_0001
[0207] In  some  embodiments,  the  compound  is  selected 
Figure imgf000040_0002
Figure imgf000040_0003
  [
Figure imgf000041_0001
[0209] In some embodiments, the compound i
Figure imgf000041_0002
[0210] In some embodiments, the compound i
Figure imgf000041_0003
  [0211] In some embodiments, the compound i
Figure imgf000041_0004
  [0212] In some embodiments, the compound i
Figure imgf000041_0005
    [0213] In some embodiments, the compound 
Figure imgf000042_0001
.  [0214] In some embodiments, the compound is:
Figure imgf000042_0002
. 
Figure imgf000042_0003
  [0216] In some embodiments, the compound is:
Figure imgf000042_0004
.  [0217] In some embodiments, the compound is:
Figure imgf000042_0005
.  [0218] In some embodiments, the compound is:
Figure imgf000042_0006
.   
Figure imgf000042_0007
[0221] In some embodiments, the compound is:
Figure imgf000042_0008
.    [0222] In some embodiments, the compound is:
Figure imgf000043_0001
.  [0223] In some embodiments, the compound is
Figure imgf000043_0002
.  [0224] In some embodiments, the compound is: 
Figure imgf000043_0003
.    Modified oligonucleotide    [0225] The  compounds  described  above  are  “building  blocks”  for  use  in  the  conjugation  of  oligonucleotides  to  other  structures  of  interest,  such  as  small  molecules  or  peptides.  The  phosphite/phosphoramidite portion of these compounds is suitable for reaction with the 5’ end of an  oligonucleotide (i.e. with the 5’ sugar hydroxyl group), such that the  linker  is  incorporated  into the  oligonucleotide  backbone.  The  phosphite/phosphoramidite  portion  of  the  compounds  of  the  invention may be reacted with an oligonucleotide under the same or similar synthetic conditions as  used  to  build  the  oligonucleotide  itself.  This  approach  minimises  the  risk  of  degrading  the  oligonucleotides when  introducing the  linker moiety. Generally, the  linker molecule will be reacted  with  the oligonucleotide  first  to provide a modified oligonucleotide. This modified oligonucleotide  may then be reacted with a molecule containing at least one thiol group.  [0226] Accordingly, there is provided, in an embodiment, a modified oligonucleotide, wherein the 5’  end of the oligonucleotide is attached to any of the compounds described above via a covalent bond  which replaces the W moiety to give a modified oligonucleotide of formula (II): 
Figure imgf000043_0004
;  wherein Q is S or O.  [0227] In  some  embodiments,  the  oligonucleotide  is  an  antisense  oligonucleotide.  In  some  embodiments,  the  oligonucleotide  is  an  RNA  interference  (RNAi)  oligonucleotide.  In  some  embodiments, the oligonucleotide is an aptamer. In some embodiments, the oligonucleotide is a short    interfering  RNA  (siRNA)  oligonucleotide.  In  some  embodiments,  the  oligonucleotide  is  a  splice‐ switching  antisense  oligonucleotide.  In  some  embodiments,  the  oligonucleotide  is  an microRNA  (miRNA) oligonucleotide. In some embodiments, the oligonucleotide is a DNA oligonucleotide. In some  embodiments, the oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is an XNA.  In some embodiments, the oligonucleotide is an xRNA. In some embodiments, the oligonucleotide is  an xDNA. In some embodiments, the oligonucleotide is an saRNA.  [0228] The features of the  linker portion of the above modified oligonucleotide correspond to the  features of the above‐described compounds. As such, in some embodiments:  A is mono‐ or bicyclic heteroaryl group in which at least one carbon ring‐atom is replaced with N;  X, Y, and Z are each independently selected from C1‐30 alkyl, C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m,  C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, C5‐10 heteroaryl, or absent;  wherein at least one of X, Y and Z is present;   wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl or C5‐10 heteroaryl  is  optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8,  SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3;   n is 1 or 2;  m is an integer from 1‐30;   each   is either a single bond or double bond;  R5 is absent, is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl;  R6 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R7 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R8 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R9, is selected from H, C1‐10 alkyl, halide, C(O)C1‐10 alkyl, C1‐10 haloalkyl, C(O)OR12, C(O)SR12, C(O)NR13R14,  CN, or NO2;   R10, and R11 are each independently selected from H, C1‐10 alkyl, halide, or C1‐10 haloalkyl;  R12 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R13 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  and  R14 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  [0229] In embodiments of the invention, X‐Y‐Z is not a furanosyl group.  [0230] In embodiments of the invention, X‐Y‐Z does not comprise a furanosyl group.  [0231] In embodiments of the invention, A is able to undertake Watson‐Crick base pairing.    [0232] In embodiments of  the  invention, A  is a natural or artificial nucleobase. A nucleobase, or  nitrogenous base,  is a nitrogen‐containing biological compound which are bonded to a five‐carbon  sugar (ribose or 2’‐deoxyribose) to form a nucleoside. These nucleosides are bonded to phosphate  groups to form nucleotides, which are the monomeric units of nucleic acid polymers, for example RNA  and DNA. Nucleobases generally have ring structures which are derived from purine (purine bases) or  pyrimidine  (pyrimidine  bases).  There  are  five  “primary”  or  “canonical”  nucleobases,  adenine  (A),  cytosine  (C),  guanine  (G),  thymine  (T),  and uracil  (U). These  five primary nucleobases  are natural  nucleobases. Non‐primary nucleobases may also be natural nucleobases,  i.e. modified nucleobases  which occur in nature, such as aminoadenine (Z). Other natural non‐primary nucleobases include the  modified nucleobases 5‐methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and  7‐methylguanosine  (m7G). A  large  number  of  artificial  nucleobases  exist,  such  as  isoguanine  and  isocytosine. Artificial nucleobases are capable of forming hydrogen bonds and therefore are capable  of taking part in base pairing via these hydrogen bonds. Other artificial nucleobases include aminoallyl  nucleobases such as aminoallyl uracil or aminoallyl cytosine which are used in post‐labelling of nucleic  acids by fluorescence detection, i.e. providing a non‐isotopic tag. Artificial nucleobases are commonly  used in oligonucleotides for use as anticancer or antiviral agents.   [0233] In embodiments of the invention A is selected from  and  ;  wherein:   each   is either a single bond or double bond;  each of A1, A2, A3 is selected from NR18 and CR19, where at least one of A1, A2, and A3 is NR18;  each of A4, A5, A6, and A7 is selected from NR18 and CR19 where at least one of A4, A5, A6, and A7 is NR18;  at  least one R15, R16, or R17 group  is present and  independently selected from H, OR20, SR20, =O, =S,  NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo, wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl is optionally substituted with at least one group selected from halo, OR5, C(O)OR5, C(O)NR6R7,  SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;   R18, if present, is H, an optionally substituted C1‐10 alkyl group, or a base labile protecting group;  R19  is  selected  from H, OR20, SR20, =O, =S, NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo,  wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl is optionally substituted with at least one group  selected from halo, OR5, C(O)OR5, C(O)NR6R7, SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;  R20 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R21 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl; and    R22 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In  these  embodiments, both 
Figure imgf000046_0001
  and X may be  attached  to A  at  any  appropriate point of 
Figure imgf000046_0002
connection. In some embodiments where A is monocyclic,   and X are attached in a meta  or para configuration. In some embodiments where A is monocyclic, 
Figure imgf000046_0003
 and X are attached 
Figure imgf000046_0004
in  a meta  configuration.  In  some  embodiments where  A  is monocyclic,    and  X  are  attached in a para configuration.  [0234] In some embodiments A, is selected from: 
Figure imgf000046_0005
  be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen  within  the  structure of A.  In  some  embodiments where A  is monocyclic, 
Figure imgf000046_0006
  and X  are  attached in a meta or para configuration. In some embodiments where A is monocyclic, 
Figure imgf000046_0007
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments  where  A  is  monocyclic, 
Figure imgf000046_0008
 and X are attached in a para configuration.      [0235] In some embodiments, 
Figure imgf000047_0001
  [0236] In some embodiments, 
Figure imgf000047_0002
  [0237] In some embodiments, 
Figure imgf000047_0003
  [0238] In some embodiments, 
Figure imgf000047_0004
  [0239] In some embodiments, 
Figure imgf000047_0005
  [0240] In some embodiments, 
Figure imgf000047_0006
  [0241] In some embodiments, 
Figure imgf000047_0007
  [0242] In some embodiments, 
Figure imgf000047_0008
  [0243] In some embodiments, 
Figure imgf000047_0009
  [0244] In some embodiments, A is selected from: 
Figure imgf000047_0010
Figure imgf000047_0011
  and  X may  be  attached  to  A  at  any    appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A. 
Figure imgf000048_0001
In some embodiments where A  is monocyclic,   and X are attached  in a meta or para 
Figure imgf000048_0002
configuration.  In some embodiments where A  is monocyclic,   and X are attached  in a  meta configuration. In some embodiments where A is monocyclic, 
Figure imgf000048_0003
 and X are attached  in a para configuration.   
Figure imgf000048_0004
.  [0247] In some embodiments, 
Figure imgf000048_0005
.  [0248] In some embodiments, 
Figure imgf000048_0006
.  [0249] In some embodiments, 
Figure imgf000048_0007
.  [0250] In some embodiments, 
Figure imgf000048_0008
.  [0251] In some embodiments, 
Figure imgf000048_0009
.     [0252] In some embodiments, A is selected from: 
Figure imgf000049_0001
  embodiments, both 
Figure imgf000049_0002
 and X may be attached to A at any appropriate point of connection,  e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is  monocyclic, 
Figure imgf000049_0003
 and X are attached in a meta or para configuration. In some embodiments  where  A  is  monocyclic, 
Figure imgf000049_0004
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments where A is monocyclic, 
Figure imgf000049_0005
 and X are attached in a para configuration.  [0253] In some embodiments, A is selected from:   
Figure imgf000049_0006
embodiments, both   and X may be attached to A at any appropriate point of connection,  e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is  monocyclic, 
Figure imgf000049_0007
 and X are attached in a meta or para configuration. In some embodiments    where  A  is  monocyclic, 
Figure imgf000050_0001
  and  X  are  attached  in  a  meta  configuration.  In  some 
Figure imgf000050_0002
embodiments where A is monocyclic,   and X are attached in a para configuration.    [
Figure imgf000050_0003
  [0256] In some embodiments, 
Figure imgf000050_0004
  [0257] In some embodiments, 
Figure imgf000050_0005
  [0258] In some embodiments, 
Figure imgf000050_0006
  [0259] In some embodiments, 
Figure imgf000050_0007
  [0260] In some embodiments, 
Figure imgf000050_0008
  [0261] In some embodiments, 
Figure imgf000050_0009
  [0262] In some embodiments, 
Figure imgf000051_0001
.  [0263] In some embodiments, 
Figure imgf000051_0002
.  [0264] In some embodiments, 
Figure imgf000051_0003
.  [0265] In some embodiments, 
Figure imgf000051_0004
.  [0266] In  some embodiments, R5  is absent, or  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  wherein R5  is absent.  In some embodiments, R5  is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R5 is selected from H, C1‐10 alkyl, C2‐10  alkenyl, and C2‐10 alkynyl.  In some embodiments, R5  is trialkylsilyl. In some embodiments, R5  is tert‐ butyldiphenylsilyl.  In some embodiments, R5  is acetyl.  In some embodiments, R5  is trihaloacetyl.  In  some embodiments, R5 is phthalimidyl. In some embodiments, R5 is H. In some embodiments, R5 is C1‐ 10 alkyl. In some embodiments, R5 is C2‐10 alkenyl. In some embodiments, R5 is C2‐10 alkynyl.  [0267] In  some  embodiments,  R6  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R6  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R6 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R6 is trialkylsilyl. In some embodiments, R6is tert‐butyldiphenylsilyl. In some embodiments, R6 is acetyl.  In  some  embodiments,  R6  is  trihaloacetyl.  In  some  embodiments,  R6  is  phthalimidyl.  In  some  embodiments, R6 is H. In some embodiments, R6 is C1‐10 alkyl. In some embodiments, R6 is C2‐10 alkenyl.  In some embodiments, R6 is C2‐10 alkynyl.  [0268] In  some  embodiments,  R7  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R7  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R7 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,    R7 is trialkylsilyl. In some embodiments, R7 is tert‐butyldiphenylsilyl. In some embodiments, R7 is acetyl.  In  some  embodiments,  R7  is  trihaloacetyl.  In  some  embodiments,  R7  is  phthalimidyl.  In  some  embodiments, R7 is H. In some embodiments, R7 is C1‐10 alkyl. In some embodiments, R7 is C2‐10 alkenyl.  In some embodiments, R7 is C2‐10 alkynyl.  [0269] In  some  embodiments,  R8  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R8  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R8 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R8 is trialkylsilyl. In some embodiments, R8 is tert‐butyldiphenylsilyl. In some embodiments, R8 is acetyl.  In  some  embodiments,  R8  is  trihaloacetyl.  In  some  embodiments,  R8  is  phthalimidyl.  In  some  embodiments, R8 is H. In some embodiments, R8 is C1‐10 alkyl. In some embodiments, R8 is C2‐10 alkenyl.  In some embodiments, R8 is C2‐10 alkynyl.  [0270] In  some  embodiments,  R12  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R12 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R12 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R12  is trialkylsilyl.  In some embodiments, R12  is tert‐butyldiphenylsilyl.  In some embodiments, R12  is  acetyl. In some embodiments, R12 is trihaloacetyl. In some embodiments, R12 is phthalimidyl. In some  embodiments, R12  is H.  In  some embodiments, R12  is C1‐10 alkyl.  In  some embodiments, R12  is C2‐10  alkenyl. In some embodiments, R12 is C2‐10 alkynyl.  [0271] In  some  embodiments,  R13  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R13 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R13 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R13  is trialkylsilyl.  In some embodiments, R13  is tert‐butyldiphenylsilyl.  In some embodiments, R13  is  acetyl. In some embodiments, R13 is trihaloacetyl. In some embodiments, R13 is phthalimidyl. In some  embodiments, R13  is H.  In  some embodiments, R13  is C1‐10 alkyl.  In  some embodiments, R13  is C2‐10  alkenyl. In some embodiments, R13 is C2‐10 alkynyl.  [0272] In  some  embodiments,  R14  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R14 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R14 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R14  is trialkylsilyl.  In some embodiments, R14  is tert‐butyldiphenylsilyl.  In some embodiments, R14  is  acetyl. In some embodiments, R14 is trihaloacetyl. In some embodiments, R14 is phthalimidyl. In some    embodiments, R14  is H.  In  some embodiments, R14  is C1‐10 alkyl.  In  some embodiments, R14  is C2‐10  alkenyl. In some embodiments, R14 is C2‐10 alkynyl.  [0273] In some embodiments, R18, when present, is selected from H, an optionally substituted C1‐10  alkyl  group,  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments,  R18  is  selected  from  H  and  an  optionally  substituted  C1‐10  alkyl  group.  In  some  embodiments,  R18  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R18 is H. In some embodiments, R18 is an optionally substituted  C1‐10  alkyl  group.  In  some  embodiments,  R18  is  trialkylsilyl.  In  some  embodiments,  R18  is  tert‐ butyldiphenylsilyl. In some embodiments, R18 is acetyl. In some embodiments, R18 is trihaloacetyl. In  some embodiments, R18 is phthalimidyl.  [0274] In some embodiments, R20, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R20  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R20 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R20 is trialkylsilyl. In some embodiments, R20 is tert‐butyldiphenylsilyl. In some  embodiments, R20 is acetyl. In some embodiments, R20 is trihaloacetyl. In some embodiments, R20 is  phthalimidyl.  In  some  embodiments,  R20  is  H.  In  some  embodiments,  R20  is  C1‐10  alkyl.  In  some  embodiments, R20 is C2‐10 alkenyl. In some embodiments, R20 is C2‐10 alkynyl.  [0275] In some embodiments, R21, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R21  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R21 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R21 is trialkylsilyl. In some embodiments, R21 is tert‐butyldiphenylsilyl. In some  embodiments, R21 is acetyl. In some embodiments, R21 is trihaloacetyl. In some embodiments, R21 is  phthalimidyl.  In  some  embodiments,  R21  is  H.  In  some  embodiments,  R21  is  C1‐10  alkyl.  In  some  embodiments, R21 is C2‐10 alkenyl. In some embodiments, R21 is C2‐10 alkynyl.  [0276] In some embodiments, R22, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R22  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R22 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R22 is trialkylsilyl. In some embodiments, R22 is tert‐butyldiphenylsilyl. In some  embodiments, R22 is acetyl. In some embodiments, R22 is trihaloacetyl. In some embodiments, R22 is  phthalimidyl.  In  some  embodiments,  R22  is  H.  In  some  embodiments,  R22  is  C1‐10  alkyl.  In  some  embodiments, R22 is C2‐10 alkenyl. In some embodiments, R22 is C2‐10 alkynyl.    [0277] In  the  preceding  embodiments,  the  trialkylsilyl  group,  if  present, may  have  the  formula  Si(C1‐C4 alkyl)3.  In further embodiments, trialkylsilyl group,  if present,  is selected from trimethylsilyl  (TMS),  triethylsilyl  (TES),  tert‐butyldimethylsilyl  (TBS),  and  triisopropylsilyl  (TIPS).  In  further  embodiments, the trialkylsilyl group  is trimethylsilyl (TMS). In further embodiments, the trialkylsilyl  group  is  triethylsilyl  (TES).  In  further embodiments,  the  trialkylsilyl group  is  tert‐butyldimethylsilyl  (TBS). In further embodiments, the trialkylsilyl group is triisopropylsilyl (TIPS).  [0278] In  the  preceding  embodiments,  the  trihaloacetyl  group,  if  present,  is  trichloroacetyl  or  trifluoroacetyl.  In  some  embodiments,  the  trihaloacetyl  group  is  trichloroacetyl.  In  some  embodiments, the trihaloacetyl group is trifluoroacetyl.  [0279] In the preceding embodiments, therefore, the base labile protecting group may be selected  from  trialkylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  The  base  labile  protecting  group may  be  selected from Si(C1‐C4 alkyl)3, acetyl, trihalo acetyl, and phthalimidyl. The base labile protecting group  may be  selected  from  Si(C1‐C4 alkyl)3,  acetyl,  trichloroacetyl,  trifluoroacetyl,  and phthalimidyl. The  base  labile  protecting  group  may  be  selected  from  Si(C1‐C4 alkyl)3,  acetyl,  trifluoroacetyl,  and  phthalimidyl. The base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl  (TES), tert‐butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trihalo acetyl, and phthalimidyl. The  base  labile  protecting  group  may  be  selected  from  trimethylsilyl  (TMS),  triethylsilyl  (TES),  tert‐ butyldimethylsilyl  (TBS),  triisopropylsilyl  (TIPS),  acetyl,  trichloroacetyl,  trifluoroacetyl,  and  phthalimidyl. The base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl  (TES), tert‐butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trifluoroacetyl, and phthalimidyl.  [0280] In some embodiments, R9 is H. In some embodiments, R10 is H. In some embodiments R11 is H.  In some embodiments, R9 and R10 are both H. In some embodiments, R9 and R11 are both H. In some  embodiments, R10 and R11 are both H. In some embodiments, R9, R10, and R11 are each H.  [0281] In some embodiments, R10 is F. In some embodiments, R11 is F. In some embodiments, R10 and  R11 are both F.  [0282] In some embodiments, R15  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R15 is selected from H, =O, and NR21R22. In some embodiments, R15 is H. In some  embodiments, R15 is =O. In some embodiments, R15 is NR21R22.  [0283] In some embodiments, R16  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R16 is selected from H, =O, and NR21R22. In some embodiments, R16 is H. In some  embodiments, R16 is =O. In some embodiments, R16 is NR21R22.  [0284] In some embodiments, R17  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R17 is selected from H, =O, and NR21R22. In some embodiments, R17 is H. In some  embodiments, R17 is =O. In some embodiments, R17 is NR21R22.    [0285] In some embodiments Z is absent and X and Y are each independently selected from C1‐30 alkyl,  C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m, C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl,  C6‐10 aryl, or C5‐10 heteroaryl; wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐ 10 aryl or C5‐10 heteroaryl is optionally substituted with at least one group selected from halo, C1‐10 alkyl,  OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3.  [0286] In  some embodiments, X  is  selected  from NR5, C3‐10  cycloalkyl, C3‐10 heterocycloalkyl, C5‐10  cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl; wherein each is optionally substituted with at least one  group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN,  NO2, and N3.  In some embodiments, X  is NR5.  In some embodiments, X  is C3‐10 cycloalkyl.  In some  embodiments,  X  is  C3‐10  heterocycloalkyl.  In  some  embodiments,  X  is  C5‐10  cycloalkenyl.  In  some  embodiments, X is C6‐10 aryl. In some embodiments, X is C5‐10 heteroaryl.  [0287] In  some embodiments, X  is  selected  from NR5, C3‐10  cycloalkyl, C3‐10 heterocycloalkyl, C5‐10  cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl. In some embodiments, X is NR5. In some embodiments, X  is C3‐10 cycloalkyl. In some embodiments, X is C3‐10 heterocycloalkyl. In some embodiments, X is C5‐10  cycloalkenyl. In some embodiments, X is C6‐10 aryl. In some embodiments, X is C5‐10 heteroaryl.  [0288] In  some  embodiments,  X  is  selected  from  NR5  and  C3‐10  heterocycloalkyl.  In  some  embodiments, X is NR5. In some embodiments, X is C3‐10 heterocycloalkyl.  [0289] In  some  embodiments,  X  is  selected  from  NR5,  pyrrolidine,  and  piperidine.  In  some  embodiments, X is NR5. In some embodiments, X is pyrrolidine. In some embodiments, X is piperidine.  [0290] In some embodiments, Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O)n, C3‐10 cycloalkyl, and  C3‐10 heterocycloalkyl; wherein each C3‐10 cycloalkyl or C3‐10 heterocycloalkyl is optionally substituted  with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6,  P(O)(OR6)2,  CN, NO2,  and  N3.  In  some  embodiments,  Y  is  C1‐30  alkyl.  In  some  embodiments,  Y  is  (CH2CH2O)m.  In some embodiments, Y  is C(O)n.  In some embodiments, Y  is C3‐10 cycloalkyl.  In some  embodiments, Y is C3‐10 heterocycloalkyl.  [0291] In some embodiments, Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O), C3‐10 cycloalkyl, and C3‐ 10 heterocycloalkyl. In some embodiments, Y is C1‐30 alkyl. In some embodiments, Y is (CH2CH2O)m. In  some embodiments, Y is C(O). In some embodiments, Y is C3‐10 cycloalkyl. In some embodiments, Y is  C3‐10 heterocycloalkyl.  [0292] In  some  embodiments,  Y  is  selected  from  C1‐30  alkyl,  C(O),  and  C3‐10  cycloalkyl.  In  some  embodiments  Y  is  C1‐30  alkyl.  In  some  embodiments,  Y  is  C(O).  In  some  embodiments,  Y  is  C3‐10  cycloalkyl.  [0293] In some embodiments, X‐Y‐Z comprises at least one tertiary amine.    [0294] In  some embodiments, X‐Y‐Z  comprises  at  least one of NR5, C(O), C3‐10  cycloalkyl, C3‐10 N‐ heterocycloalkyl, C6‐10 aryl, and C5‐10 heteroaryl.  [0295] In  some embodiments, X‐Y‐Z  comprises  at  least one of NR5, C(O), C3‐10  cycloalkyl, C3‐10 N‐ heterocycloalkyl, and C6‐10 aryl.  [0296] In some embodiments, X‐Y‐Z comprises NR5.  [0297] In some embodiments, X‐Y‐Z comprises C(O).  [0298] In some embodiments, X‐Y‐Z comprises C3‐10 cycloalkyl.  [0299] In some embodiments, X‐Y‐Z comprises C3‐10 N‐heterocycloalkyl.  [0300] In some embodiments, X‐Y‐Z comprises C6‐10 aryl.  [0301] In some embodiments, X‐Y‐Z comprises C5‐10 heteroaryl.  [0302] In some embodiments, X‐Y‐Z is selected from:  
Figure imgf000056_0001
[0303] In some embodiments, X‐Y‐Z is selected from:      
Figure imgf000056_0002
  [0306] In some embodiments, 
Figure imgf000057_0001
.   [0307] In some embodiments, 
Figure imgf000057_0002
.   [0308] In some embodiments, 
Figure imgf000057_0003
.   [0309] In some embodiments, 
Figure imgf000057_0004
.   [0310] In some embodiments, 
Figure imgf000057_0005
.  [0311] In some embodiments, 
Figure imgf000057_0006
.  [0312] In some embodiments, 
Figure imgf000057_0007
.  [0313] In some embodiments, 
Figure imgf000057_0008
.  [0314] In some embodiments, 
Figure imgf000057_0009
.  [0315] In some embodiments, 
Figure imgf000057_0010
.  [0316] In some embodiments, 
Figure imgf000057_0011
.  [0317] In some embodiments, X‐Y‐Z is selected from:  
 
Figure imgf000058_0001
[0318] In some embodiments, X‐Y‐Z is selected from:        
Figure imgf000058_0002
       
Figure imgf000059_0001
[0326] In some embodiments, 
Figure imgf000059_0002
.  [0327] In some embodiments, 
Figure imgf000059_0003
.  [0328] In some embodiments, 
Figure imgf000059_0004
.  [0329] In some embodiments, 
Figure imgf000059_0005
.  [0330] In some embodiments, 
Figure imgf000059_0006
.  [0331] In some embodiments, 
Figure imgf000059_0007
.  [0332] In some embodiments, 
Figure imgf000059_0008
.  [0333] In some embodiments, 
Figure imgf000059_0009
.  [0334] In some embodiments, 
Figure imgf000059_0010
.  [0335] In some embodiments, 
Figure imgf000059_0011
.    [0336] In some embodiments, the modified oligonucleotide is selected from: 
Figure imgf000060_0001
,    
Figure imgf000061_0002
.  [0337] In some embodiments, the modified oligonucleotide is selected from: 
Figure imgf000061_0001
,  
Figure imgf000062_0002
.    [0338] In  some  embodiments,  the  modified  oligonucleotide  is  selected  from:
Figure imgf000062_0001
,   ,  and  .  [0339] In  some  embodiments,  the  modified  oligonucleotide  is  selected  from: , , ,
Figure imgf000063_0001
, ,
 
Figure imgf000064_0001
.  [0341] In  some  embodiments,  the  modified  oligonucleotide  is:
Figure imgf000064_0002
.    [0343] In  some  embodiments,  the  modified  oligonucleotide  is:    
Figure imgf000065_0001
.  [0346] In  some  embodiments,  the  modified  oligonucleotide  is:   
Figure imgf000065_0002
.    [0348] In  some  embodiments,  the  modified  oligonucleotide  is:   
Figure imgf000066_0002
.  [0350] In some embodiments, the modified oligonucleotide is selected from:  ,
Figure imgf000066_0001
   
Figure imgf000067_0002
.  [0351] In some embodiments, the modified oligonucleotide is selected from: 
Figure imgf000067_0001
 
Figure imgf000068_0001
    [0352] In  some  embodiments,  the  modified  oligonucleotide  is  selected  from:
Figure imgf000068_0002
 
Figure imgf000069_0001
 
Figure imgf000070_0001
  [0354] In  some  embodiments,  the  modified  oligonucleotide 
Figure imgf000070_0002
   
Figure imgf000070_0003
Figure imgf000070_0005
.  [0356] In  some  embodiments,  the  modified  oligonucleotide  is: 
Figure imgf000070_0004
.    [0357] In  some  embodiments,  the  modified  oligonucleotide  is:   
Figure imgf000071_0002
.  [0359] In  some  embodiments,  the  modified  oligonucleotide  is:   
Figure imgf000071_0003
.  [0361] In  some  embodiments,  the  modified  oligonucleotide  is:   
Figure imgf000071_0004
.  [0363] In  some  embodiments,  the  modified  oligonucleotide  is: 
Figure imgf000071_0001
.    [0364] In  some  embodiments,  the  modified  oligonucleotide  is:       
Figure imgf000072_0003
.    modified  oligonucleotide 
Figure imgf000072_0001
 
Figure imgf000072_0002
.  [0369] In some embodiments, the modified oligonucleotide is selected from:   
Figure imgf000073_0001
 
Figure imgf000074_0001
  [0370] In some embodiments, the modified oligonucleotide is selected from: 
Figure imgf000074_0002
   
Figure imgf000075_0002
.    [0371] In some embodiments, the modified oligonucleotide is selected from: 
Figure imgf000075_0001
   
Figure imgf000076_0002
.  [0372] In some embodiments, the modified oligonucleotide is selected from: 
Figure imgf000076_0001
 
Figure imgf000077_0001
    the  modified  oligonucleotide  is 
Figure imgf000077_0002
.  [0374] In  some  embodiments,  the  modified  oligonucleotide 
Figure imgf000077_0003
   
Figure imgf000077_0004
 
Figure imgf000077_0006
.  [0376] In  some  embodiments,  the  modified  oligonucleotide  is 
Figure imgf000077_0005
.    [0377] In  some  embodiments,  the  modified  oligonucleotide  is   
Figure imgf000078_0002
.  [0379] In  some  embodiments,  the  modified  oligonucleotide  is   
Figure imgf000078_0003
.  [0381] In  some  embodiments,  the  modified  oligonucleotide  is   
Figure imgf000078_0004
.  [0383] In  some  embodiments,  the  modified  oligonucleotide  is 
Figure imgf000078_0001
.    [0384] In  some  embodiments,  the  modified  oligonucleotide  is     
Figure imgf000079_0002
.  [0387] In  some  embodiments,  the  modified  oligonucleotide  is   
Figure imgf000079_0003
.  [0389] In  some  embodiments,  the  modified  oligonucleotide  is 
Figure imgf000079_0001
.    [0390] In  some  embodiments,  the  modified  oligonucleotide  is     
Figure imgf000080_0002
.    Oligonucleotide conjugates    [0393] The modified  oligonucleotides  described  above  can  be  reacted  with  other  structures  of  interest, such as small molecules or peptides, which contain at  least one thiol group. The resulting  structures are oligonucleotide conjugates.   [0394] Accordingly, in one embodiment of the invention there is provided a modified oligonucleotide  conjugate, wherein a modified oligonucleotide as described herein is conjugated to a thiol‐containing  moiety (M‐SH) via the vinyl group to provide a conjugate of formula (II): 
Figure imgf000080_0001
  wherein Q is S or O.  [0395] In  some  embodiments,  the  oligonucleotide  is  an  antisense  oligonucleotide.  In  some  embodiments,  the  oligonucleotide  is  an  RNA  interference  (RNAi)  oligonucleotide.  In  some  embodiments, the oligonucleotide is an aptamer. In some embodiments, the oligonucleotide is a short  interfering  RNA  (siRNA)  oligonucleotide.  In  some  embodiments,  the  oligonucleotide  is  a  splice‐ switching  antisense  oligonucleotide.  In  some  embodiments,  the  oligonucleotide  is  an microRNA    (miRNA) oligonucleotide. In some embodiments, the oligonucleotide is a DNA oligonucleotide. In some  embodiments, the oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is an XNA.  In some embodiments, the oligonucleotide is an xRNA. In some embodiments, the oligonucleotide is  an xDNA. In some embodiments, the oligonucleotide is an saRNA.  [0396] In some embodiments, M  is a small molecule. For example,  in some embodiments, M  is a  pharmacologically active small molecule. In some embodiments, M is a probe. A probe is a molecule  which is used to investigate biological mechanisms or validate targets for drug discovery. Probes may  comprise a moiety which allows for their detection by an external source. For example, a probe may  be  radiolabelled,  i.e. may comprise  radioisotopes.  In another example,  the probe may comprise a  moiety which displays fluorescence or phosphorescence.  [0397] In some embodiments, M is a fluorophore.   [0398] In some embodiments, M is fluorescein or a fluorescein derivative. Fluorescein is a common  diagnostic  contrast  agent  with  many  derivatives  which  are  well  known  in  the  art.  Exemplary  derivatives include fluorescein 5‐isothiocyanate, fluorescein 6‐isothiocyanate, mixtures of fluorescein  5‐isothiocyanate  and  fluorescein  6‐isothiocyanate,  fluorescein  succinimidyl  esters,  carboxyfluorescein,  carboxyfluorescein  succinimidyl  ester,  fluorescein  pentafluorophenyl  esters,  fluorescein tetrafluorophenyl esters, and fluorescein amidites.  [0399] In some embodiments, M is rhodamine or a rhodamine derivative. Rhodamine derivatives are  a well‐known  family of dyes which  feature  the  rhodamine core  structure. Examples of  rhodamine  derivatives  include  carboxytetramethylrhodamine,  tetramethylrhodamine,  tetramethylrhodamine  isothiocyanate, sulforhodamine 101, sulforhodamine 101 acid chloride (Texas Red), and rhodamine  red.  [0400] In some embodiments, M is 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY).   [0401] In some embodiments, M  is a radiolabelled molecule. Radiolabelled molecules are typically  small  molecules  which  are  non‐biologically  active  and  which  are  isotopically  enriched  with  a  radioactive isotope such as 32P, 35S, 14C, and 3H.  [0402] In some embodiments, M  is a polypeptide.  In some embodiments, M  is a protein.  In some  embodiments, M is a polypeptide or protein connected to the modified oligonucleotide via a cysteine  residue.   [0403] In embodiments of the invention, the polypeptide or protein of the invention may be a carrier  protein. A carrier protein  is a protein or polypeptide which  is capable of transporting an  ion, small  molecule, or macromolecule across a biological membrane, e.g.  into a cell.  In embodiments of the  invention,  the carrier protein  is selected to  transport the modified oligonucleotide  into  the cell.  In  embodiments  of  the  invention,  the  oligonucleotide  is  an  antisense  oligonucleotide,  RNAi    oligonucleotide, aptamer, siRNA oligonucleotide, splice‐switching antisense oligonucleotide, miRNA  oligonucleotide,  DNA  oligonucleotide,  gapmer,  XNA  oligonucleotide,  xRNA  oligonucleotide,  xDNA  oligonucleotide, or saRNA oligonucleotide which is intended to be transported into cells or a specific  cell type.   [0404] In embodiments of the invention, the polypeptide or protein is an antigen binding fragment,  a  nanobody,  or  an  antibody.  Antibodies  comprise  antigen  binding  fragments.  Antigen  binding  fragments are polypeptide sequences which bind to antigens. Antigen binding fragments are antigen‐ specific, i.e. can only react to and bind to one specific antigen, or can cross‐react, i.e. can react to and  bind more  than  one  antigen.  Nanobodies,  or  single‐domain  antibodies,  are  a  type  of  antibody  fragment consisting of a single monomeric variable antibody domain. Nanobodies generally have a  molecular weight around 12‐15 kDa and so are significantly  lighter than common antibodies which  generally have a molecular weight around 150‐160 kDa. Nanobodies. Like antibodies, are capable of  selectively binding to a specific antigen. By linking an oligonucleotide to an antigen binding fragment,  nanobody, or an antibody, it is possible to provide a modified oligonucleotide conjugate which targets,  for  example,  cells  displaying  a  specific  antigen.  Delivery  of  a  therapeutic  oligonucleotide  can,  therefore, be targeted to only the cells in which the oligonucleotide is desired to have an effect.  [0405] In embodiments of  the  invention,  the polypeptide or protein  is a  cell‐penetrating peptide  (CPP). CPPs are usually  short peptides,  for example, of  less  than 30‐40 amino acids. They may be  derived from proteins or chimeric sequences or may be of completely artificial, synthetic or designed  origin. They are occasionally amphipathic, but almost always possess a net positive (cationic) charge  at physiological pH. CPPs are able to penetrate biological membranes, to trigger the movement of  various biomolecules across cell membranes  into  the cytoplasm and  to  improve  their  intracellular  routing, thereby facilitating interactions with the target. CPPs have been shown to be able to deliver  oligonucleotide cargos into a wide variety of cell types.  [0406] The  features  of  the  linker‐oligonucleotide  portion  of  the  above modified  oligonucleotide  conjugate correspond to the features of the above‐described modified oligonucleotides. As such, in  some embodiments:  A is mono‐ or bicyclic heteroaryl group in which at least one carbon ring‐atom is replaced with N;  X, Y, and Z are each independently selected from C1‐30 alkyl, C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m,  C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, C5‐10 heteroaryl, or absent;  wherein at least one of X, Y and Z is present;   wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl or C5‐10 heteroaryl  is  optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8,  SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3;     n is 1 or 2;  m is an integer from 1‐30;   each   is either a single bond or double bond;  R5 is absent, is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl;  R6 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R7 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R8 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R9, is selected from H, C1‐10 alkyl, halide, C(O)C1‐10 alkyl, C1‐10 haloalkyl, C(O)OR12, C(O)SR12, C(O)NR13R14,  CN, or NO2;   R10, and R11 are each independently selected from H, C1‐10 alkyl, halide, or C1‐10 haloalkyl;  R12 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R13 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  and  R14 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  [0407] In embodiments of the invention, X‐Y‐Z is not a furanosyl group.  [0408] In embodiments of the invention, X‐Y‐Z does not comprise a furanosyl group.  [0409] In embodiments of the invention, A is able to undertake Watson‐Crick base pairing.  [0410] In embodiments of  the  invention, A  is a natural or artificial nucleobase. A nucleobase, or  nitrogenous base,  is a nitrogen‐containing biological compound which are bonded to a five‐carbon  sugar (ribose or 2’‐deoxyribose) to form a nucleoside. These nucleosides are bonded to phosphate  groups to form nucleotides, which are the monomeric units of nucleic acid polymers, for example RNA  and DNA. Nucleobases generally have ring structures which are derived from purine (purine bases) or  pyrimidine  (pyrimidine  bases).  There  are  five  “primary”  or  “canonical”  nucleobases,  adenine  (A),  cytosine  (C),  guanine  (G),  thymine  (T),  and uracil  (U). These  five primary nucleobases  are natural  nucleobases. Non‐primary nucleobases may also be natural nucleobases,  i.e. modified nucleobases  which occur in nature, such as aminoadenine (Z). Other natural non‐primary nucleobases include the  modified nucleobases 5‐methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and  7‐methylguanosine  (m7G). A  large  number  of  artificial  nucleobases  exist,  such  as  isoguanine  and  isocytosine. Artificial nucleobases are capable of forming hydrogen bonds and therefore are capable  of taking part in base pairing via these hydrogen bonds. Other artificial nucleobases include aminoallyl  nucleobases such as aminoallyl uracil or aminoallyl cytosine which are used in post‐labelling of nucleic  acids by fluorescence detection, i.e. providing a non‐isotopic tag. Artificial nucleobases are commonly  used in oligonucleotides for use as anticancer or antiviral agents.     [0411] In embodiments of the invention A is selected from  and  ;  wherein:   each   is either a single bond or double bond;  each of A1, A2, A3 is selected from NR18 and CR19, where at least one of A1, A2, and A3 is NR18;  each of A4, A5, A6, and A7 is selected from NR18 and CR19 where at least one of A4, A5, A6, and A7 is NR18;  at  least one R15, R16, or R17 group  is present and  independently selected from H, OR20, SR20, =O, =S,  NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo, wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl is optionally substituted with at least one group selected from halo, OR5, C(O)OR5, C(O)NR6R7,  SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;   R18, if present, is H, an optionally substituted C1‐10 alkyl group, or a base labile protecting group;  R19  is  selected  from H, OR20, SR20, =O, =S, NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo,  wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl is optionally substituted with at least one group  selected from halo, OR5, C(O)OR5, C(O)NR6R7, SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;  R20 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R21 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl; and  R22 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In  these  embodiments, both 
Figure imgf000084_0001
  and X may be  attached  to A  at  any  appropriate point of 
Figure imgf000084_0002
connection. In some embodiments where A is monocyclic,   and X are attached in a meta  or para configuration. In some embodiments where A is monocyclic, 
Figure imgf000084_0003
 and X are attached 
Figure imgf000084_0004
in  a meta  configuration.  In  some  embodiments where  A  is monocyclic,    and  X  are  attached in a para configuration.  [0412] In some embodiments A, is selected from:   
Figure imgf000085_0001
  be attached to A at any appropriate point of connection, e.g. via the replacement of any hydrogen  within  the  structure of A.  In  some  embodiments where A  is monocyclic, 
Figure imgf000085_0002
  and X  are  attached in a meta or para configuration. In some embodiments where A is monocyclic, 
Figure imgf000085_0003
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments  where  A  is  monocyclic, 
Figure imgf000085_0004
 and X are attached in a para configuration.    [0413] In some embodiments, 
Figure imgf000085_0005
.  [0414] In some embodiments, 
Figure imgf000085_0006
.  [0415] In some embodiments, 
Figure imgf000085_0007
.  [0416] In some embodiments, 
Figure imgf000085_0008
.  [0417] In some embodiments, 
Figure imgf000085_0009
.  [0418] In some embodiments, 
Figure imgf000085_0010
.     
Figure imgf000086_0001
[0421] In some embodiments, 
Figure imgf000086_0002
.  [0422] In some embodiments, A is selected from:  , 
Figure imgf000086_0003
Figure imgf000086_0004
.      s,  both    and  X may  be  attached  to  A  at  any  appropriate point of connection, e.g. via the replacement of any hydrogen within the structure of A. 
Figure imgf000086_0005
In some embodiments where A  is monocyclic,   and X are attached  in a meta or para 
Figure imgf000086_0006
configuration.  In some embodiments where A  is monocyclic,   and X are attached  in a  meta configuration. In some embodiments where A is monocyclic, 
Figure imgf000086_0007
 and X are attached  in a para configuration.   
Figure imgf000086_0008
    [0424] In some embodiments, 
Figure imgf000087_0001
.  [0425] In some embodiments, 
Figure imgf000087_0002
.  [0426] In some embodiments, 
Figure imgf000087_0003
.  [0427] In some embodiments, 
Figure imgf000087_0004
.  [0428] In some embodiments, 
Figure imgf000087_0005
.  [0429] In some embodiments, 
Figure imgf000087_0006
.   [0430] In some embodiments, A is selected from: 
Figure imgf000087_0007
  embodiments, both 
Figure imgf000087_0008
 and X may be attached to A at any appropriate point of connection,  e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is    monocyclic, 
Figure imgf000088_0001
 and X are attached in a meta or para configuration. In some embodiments  where  A  is  monocyclic, 
Figure imgf000088_0002
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments where A is monocyclic, 
Figure imgf000088_0003
 and X are attached in a para configuration.  [0431] In some embodiments, A is selected from: 
Figure imgf000088_0004
  embodiments, both 
Figure imgf000088_0005
 and X may be attached to A at any appropriate point of connection,  e.g. via the replacement of any hydrogen within the structure of A. In some embodiments where A is  monocyclic, 
Figure imgf000088_0006
 and X are attached in a meta or para configuration. In some embodiments  where  A  is  monocyclic, 
Figure imgf000088_0007
  and  X  are  attached  in  a  meta  configuration.  In  some  embodiments where A is monocyclic, 
Figure imgf000088_0008
 and X are attached in a para configuration.   
Figure imgf000088_0009
.    [0434] In some embodiments, 
Figure imgf000089_0001
  [0435] In some embodiments, 
Figure imgf000089_0002
  [0436] In some embodiments, 
Figure imgf000089_0003
  [0437] In some embodiments, 
Figure imgf000089_0004
  [0438] In some embodiments, 
Figure imgf000089_0005
  [0439] In some embodiments, 
Figure imgf000089_0006
  [0440] In some embodiments, 
Figure imgf000089_0007
  [0441] In some embodiments, 
Figure imgf000089_0008
  [0442] In some embodiments, 
Figure imgf000089_0009
  [0443] Insomeembodiments
Figure imgf000089_0010
  [0444] In  some embodiments, R5  is absent, or  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  wherein R5  is absent.  In some embodiments, R5  is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl. In some embodiments, R5 is selected from H, C1‐10 alkyl, C2‐10  alkenyl, and C2‐10 alkynyl.  In some embodiments, R5  is trialkylsilyl. In some embodiments, R5  is tert‐ butyldiphenylsilyl.  In some embodiments, R5  is acetyl.  In some embodiments, R5  is trihaloacetyl.  In  some embodiments, R5 is phthalimidyl. In some embodiments, R5 is H. In some embodiments, R5 is C1‐ 10 alkyl. In some embodiments, R5 is C2‐10 alkenyl. In some embodiments, R5 is C2‐10 alkynyl.  [0445] In  some  embodiments,  R6  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R6  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R6 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R6 is trialkylsilyl. In some embodiments, R6 is tert‐butyldiphenylsilyl. In some embodiments, R6 is acetyl.  In  some  embodiments,  R6  is  trihaloacetyl.  In  some  embodiments,  R6  is  phthalimidyl.  In  some  embodiments, R6 is H. In some embodiments, R6 is C1‐10 alkyl. In some embodiments, R6 is C2‐10 alkenyl.  In some embodiments, R6 is C2‐10 alkynyl.  [0446] In  some  embodiments,  R7  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R7  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R7 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R7 is trialkylsilyl. In some embodiments, R7 is tert‐butyldiphenylsilyl. In some embodiments, R7 is acetyl.  In  some  embodiments,  R7  is  trihaloacetyl.  In  some  embodiments,  R7  is  phthalimidyl.  In  some  embodiments, R7 is H. In some embodiments, R7 is C1‐10 alkyl. In some embodiments, R7 is C2‐10 alkenyl.  In some embodiments, R7 is C2‐10 alkynyl.  [0447] In  some  embodiments,  R8  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R8  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R8 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R8 is trialkylsilyl. In some embodiments, R8 is tert‐butyldiphenylsilyl. In some embodiments, R8 is acetyl.  In  some  embodiments,  R8  is  trihaloacetyl.  In  some  embodiments,  R8  is  phthalimidyl.  In  some  embodiments, R8 is H. In some embodiments, R8 is C1‐10 alkyl. In some embodiments, R8 is C2‐10 alkenyl.  In some embodiments, R8 is C2‐10 alkynyl.  [0448] In  some  embodiments,  R12  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R12 is    selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R12 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R12  is trialkylsilyl.  In some embodiments, R12  is tert‐butyldiphenylsilyl.  In some embodiments, R12  is  acetyl. In some embodiments, R12 is trihaloacetyl. In some embodiments, R12 is phthalimidyl. In some  embodiments, R12  is H.  In  some embodiments, R12  is C1‐10 alkyl.  In  some embodiments, R12  is C2‐10  alkenyl. In some embodiments, R12 is C2‐10 alkynyl.  [0449] In  some  embodiments,  R13  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R13 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R13 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R13  is trialkylsilyl.  In some embodiments, R13  is tert‐butyldiphenylsilyl.  In some embodiments, R13  is  acetyl. In some embodiments, R13 is trihaloacetyl. In some embodiments, R13 is phthalimidyl. In some  embodiments, R13  is H.  In  some embodiments, R13  is C1‐10 alkyl.  In  some embodiments, R13  is C2‐10  alkenyl. In some embodiments, R13 is C2‐10 alkynyl.  [0450] In  some  embodiments,  R14  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments, R14 is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments, R14 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl. In some embodiments,  R14  is trialkylsilyl.  In some embodiments, R14  is tert‐butyldiphenylsilyl.  In some embodiments, R14  is  acetyl. In some embodiments, R14 is trihaloacetyl. In some embodiments, R14 is phthalimidyl. In some  embodiments, R14  is H.  In  some embodiments, R14  is C1‐10 alkyl.  In  some embodiments, R14  is C2‐10  alkenyl. In some embodiments, R14 is C2‐10 alkynyl.  [0451] In some embodiments, R18, when present, is selected from H, an optionally substituted C1‐10  alkyl  group,  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  In  some  embodiments,  R18  is  selected  from  H  and  an  optionally  substituted  C1‐10  alkyl  group.  In  some  embodiments,  R18  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R18 is H. In some embodiments, R18 is an optionally substituted  C1‐10  alkyl  group.  In  some  embodiments,  R18  is  trialkylsilyl.  In  some  embodiments,  R18  is  tert‐ butyldiphenylsilyl. In some embodiments, R18 is acetyl. In some embodiments, R18 is trihaloacetyl. In  some embodiments, R18 is phthalimidyl.  [0452] In some embodiments, R20, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R20  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R20 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    In some embodiments, R20 is trialkylsilyl. In some embodiments, R20 is tert‐butyldiphenylsilyl. In some  embodiments, R20 is acetyl. In some embodiments, R20 is trihaloacetyl. In some embodiments, R20 is  phthalimidyl.  In  some  embodiments,  R20  is  H.  In  some  embodiments,  R20  is  C1‐10  alkyl.  In  some  embodiments, R20 is C2‐10 alkenyl. In some embodiments, R20 is C2‐10 alkynyl.  [0453] In some embodiments, R21, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R21  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R21 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R21 is trialkylsilyl. In some embodiments, R21 is tert‐butyldiphenylsilyl. In some  embodiments, R21 is acetyl. In some embodiments, R21 is trihaloacetyl. In some embodiments, R21 is  phthalimidyl.  In  some  embodiments,  R21  is  H.  In  some  embodiments,  R21  is  C1‐10  alkyl.  In  some  embodiments, R21 is C2‐10 alkenyl. In some embodiments, R21 is C2‐10 alkynyl.  [0454] In some embodiments, R22, when present, is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl,  H,  C1‐10  alkyl,  C2‐10  alkenyl,  and  C2‐10  alkynyl.  In  some  embodiments,  R22  is  selected  from  trialkylsilyl,  tert‐butyldiphenylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl. In some embodiments, R22 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.  In some embodiments, R22 is trialkylsilyl. In some embodiments, R22 is tert‐butyldiphenylsilyl. In some  embodiments, R22 is acetyl. In some embodiments, R22 is trihaloacetyl. In some embodiments, R22 is  phthalimidyl.  In  some  embodiments,  R22  is  H.  In  some  embodiments,  R22  is  C1‐10  alkyl.  In  some  embodiments, R22 is C2‐10 alkenyl. In some embodiments, R22 is C2‐10 alkynyl.  [0455] In  the  preceding  embodiments,  the  trialkylsilyl  group,  if  present, may  have  the  formula  Si(C1‐C4 alkyl)3.  In further embodiments, trialkylsilyl group,  if present,  is selected from trimethylsilyl  (TMS),  triethylsilyl  (TES),  tert‐butyldimethylsilyl  (TBS),  and  triisopropylsilyl  (TIPS).  In  further  embodiments, the trialkylsilyl group  is trimethylsilyl (TMS). In further embodiments, the trialkylsilyl  group  is  triethylsilyl  (TES).  In  further embodiments,  the  trialkylsilyl group  is  tert‐butyldimethylsilyl  (TBS). In further embodiments, the trialkylsilyl group is triisopropylsilyl (TIPS).  [0456] In  the  preceding  embodiments,  the  trihaloacetyl  group,  if  present,  is  trichloroacetyl  or  trifluoroacetyl.  In  some  embodiments,  the  trihaloacetyl  group  is  trichloroacetyl.  In  some  embodiments, the trihaloacetyl group is trifluoroacetyl.  [0457] In the preceding embodiments, therefore, the base labile protecting group may be selected  from  trialkylsilyl,  acetyl,  trihaloacetyl,  and  phthalimidyl.  The  base  labile  protecting  group may  be  selected from Si(C1‐C4 alkyl)3, acetyl, trihalo acetyl, and phthalimidyl. The base labile protecting group  may be  selected  from  Si(C1‐C4 alkyl)3,  acetyl,  trichloroacetyl,  trifluoroacetyl,  and phthalimidyl. The  base  labile  protecting  group  may  be  selected  from  Si(C1‐C4 alkyl)3,  acetyl,  trifluoroacetyl,  and    phthalimidyl. The base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl  (TES), tert‐butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trihalo acetyl, and phthalimidyl. The  base  labile  protecting  group  may  be  selected  from  trimethylsilyl  (TMS),  triethylsilyl  (TES),  tert‐ butyldimethylsilyl  (TBS),  triisopropylsilyl  (TIPS),  acetyl,  trichloroacetyl,  trifluoroacetyl,  and  phthalimidyl. The base labile protecting group may be selected from trimethylsilyl (TMS), triethylsilyl  (TES), tert‐butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), acetyl, trifluoroacetyl, and phthalimidyl.  [0458] In some embodiments, R9 is H. In some embodiments, R10 is H. In some embodiments R11 is H.  In some embodiments, R9 and R10 are both H. In some embodiments, R9 and R11 are both H. In some  embodiments, R10 and R11 are both H. In some embodiments, R9, R10, and R11 are each H.  [0459] In some embodiments, R10 is F. In some embodiments, R11 is F. In some embodiments, R10 and  R11 are both F.  [0460] In some embodiments, R15  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R15 is selected from H, =O, and NR21R22. In some embodiments, R15 is H. In some  embodiments, R15 is =O. In some embodiments, R15 is NR21R22.  [0461] In some embodiments, R16  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R16 is selected from H, =O, and NR21R22. In some embodiments, R16 is H. In some  embodiments, R16 is =O. In some embodiments, R16 is NR21R22.  [0462] In some embodiments, R17  is selected from H, OR20, SR20, =O, =S, NR21R22, and C1‐10 alkyl. In  some embodiments, R17 is selected from H, =O, and NR21R22. In some embodiments, R17 is H. In some  embodiments, R17 is =O. In some embodiments, R17 is NR21R22.  [0463] In some embodiments Z is absent and X and Y are each independently selected from C1‐30 alkyl,  C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m, C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl,  C6‐10 aryl, or C5‐10 heteroaryl; wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐ 10 aryl or C5‐10 heteroaryl is optionally substituted with at least one group selected from halo, C1‐10 alkyl,  OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3.  [0464] In  some embodiments, X  is  selected  from NR5, C3‐10  cycloalkyl, C3‐10 heterocycloalkyl, C5‐10  cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl; wherein each is optionally substituted with at least one  group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN,  NO2, and N3.  In some embodiments, X  is NR5.  In some embodiments, X  is C3‐10 cycloalkyl.  In some  embodiments,  X  is  C3‐10  heterocycloalkyl.  In  some  embodiments,  X  is  C5‐10  cycloalkenyl.  In  some  embodiments, X is C6‐10 aryl. In some embodiments, X is C5‐10 heteroaryl.  [0465] In  some embodiments, X  is  selected  from NR5, C3‐10  cycloalkyl, C3‐10 heterocycloalkyl, C5‐10  cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl. In some embodiments, X is NR5. In some embodiments, X    is C3‐10 cycloalkyl. In some embodiments, X is C3‐10 heterocycloalkyl. In some embodiments, X is C5‐10  cycloalkenyl. In some embodiments, X is C6‐10 aryl. In some embodiments, X is C5‐10 heteroaryl.  [0466] In  some  embodiments,  X  is  selected  from  NR5  and  C3‐10  heterocycloalkyl.  In  some  embodiments, X is NR5. In some embodiments, X is C3‐10 heterocycloalkyl.  [0467] In  some  embodiments,  X  is  selected  from  NR5,  pyrrolidine,  and  piperidine.  In  some  embodiments, X is NR5. In some embodiments, X is pyrrolidine. In some embodiments, X is piperidine.  [0468] In some embodiments, Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O)n, C3‐10 cycloalkyl, and  C3‐10 heterocycloalkyl; wherein each C3‐10 cycloalkyl or C3‐10 heterocycloalkyl is optionally substituted  with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6,  P(O)(OR6)2,  CN, NO2,  and  N3.  In  some  embodiments,  Y  is  C1‐30  alkyl.  In  some  embodiments,  Y  is  (CH2CH2O)m.  In some embodiments, Y  is C(O)n.  In some embodiments, Y  is C3‐10 cycloalkyl.  In some  embodiments, Y is C3‐10 heterocycloalkyl.  [0469] In some embodiments, Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O), C3‐10 cycloalkyl, and C3‐ 10 heterocycloalkyl. In some embodiments, Y is C1‐30 alkyl. In some embodiments, Y is (CH2CH2O)m. In  some embodiments, Y is C(O). In some embodiments, Y is C3‐10 cycloalkyl. In some embodiments, Y is  C3‐10 heterocycloalkyl.  [0470] In  some  embodiments,  Y  is  selected  from  C1‐30  alkyl,  C(O),  and  C3‐10  cycloalkyl.  In  some  embodiments  Y  is  C1‐30  alkyl.  In  some  embodiments,  Y  is  C(O).  In  some  embodiments,  Y  is  C3‐10  cycloalkyl.  [0471] In some embodiments, X‐Y‐Z comprises at least one tertiary amine.  [0472] In  some embodiments, X‐Y‐Z  comprises  at  least one of NR5, C(O), C3‐10  cycloalkyl, C3‐10 N‐ heterocycloalkyl, C6‐10 aryl, and C5‐10 heteroaryl.  [0473] In  some embodiments, X‐Y‐Z  comprises  at  least one of NR5, C(O), C3‐10  cycloalkyl, C3‐10 N‐ heterocycloalkyl, and C6‐10 aryl.  [0474] In some embodiments, X‐Y‐Z comprises NR5.  [0475] In some embodiments, X‐Y‐Z comprises C(O).  [0476] In some embodiments, X‐Y‐Z comprises C3‐10 cycloalkyl.  [0477] In some embodiments, X‐Y‐Z comprises C3‐10 N‐heterocycloalkyl.  [0478] In some embodiments, X‐Y‐Z comprises C6‐10 aryl.  [0479] In some embodiments, X‐Y‐Z comprises C5‐10 heteroaryl.  [0480] In some embodiments, X‐Y‐Z is selected from:    
Figure imgf000095_0001
[0481] In some embodiments, X‐Y‐Z is selected from:  
Figure imgf000095_0002
[0482] In some embodiments, X‐Y‐Z is selected from:    
Figure imgf000095_0003
[0483] In some embodiments, X‐Y‐Z is selected from:    
Figure imgf000095_0004
[0485] In some embodiments, 
Figure imgf000095_0005
.   [0486] In some embodiments, 
Figure imgf000095_0006
.   [0487] In some embodiments, 
Figure imgf000095_0007
.    
Figure imgf000096_0001
.  [0490] In some embodiments, 
Figure imgf000096_0002
.  [0491] In some embodiments, 
Figure imgf000096_0003
.  [0492] In some embodiments, 
Figure imgf000096_0004
. 
Figure imgf000096_0005
  [0495] In some embodiments, X‐Y‐Z is selected from:  
Figure imgf000096_0006
  [0497] In some embodiments, X‐Y‐Z is selected from:   ,
Figure imgf000097_0001
[0498] In some embodiments, X‐Y‐Z is selected from:   , [
Figure imgf000097_0002
[ [   [
Figure imgf000097_0008
  [0503] In some embodiments, 
Figure imgf000097_0003
  [0504] In some embodiments, 
Figure imgf000097_0004
[0505] In some embodiments, 
Figure imgf000097_0005
[0506] In some embodiments, X
Figure imgf000097_0006
[0507] Insomeembodiments,X
Figure imgf000097_0007
  [0508] In some embodiments, 
Figure imgf000098_0001
.  [0509] In some embodiments, 
Figure imgf000098_0002
.  [0510] In some embodiments, 
Figure imgf000098_0003
.   
Figure imgf000098_0004
[0513] In some embodiments, 
Figure imgf000098_0005
.  [0514] In some embodiments, the modified oligonucleotide conjugate is selected from:  ,
Figure imgf000098_0006
   
Figure imgf000099_0001
.  [0515] In some embodiments, the modified oligonucleotide conjugate is selected from: 
Figure imgf000099_0002
   
Figure imgf000100_0002
.    [0516] In  some  embodiments,  the  modified  oligonucleotide  conjugate  is  selected  from: 
Figure imgf000100_0001
, ,   , ,
Figure imgf000101_0001
Figure imgf000101_0002
.  [0517] In  some  embodiments,  the  modified  oligonucleotide  conjugate  is  selected  from: 
Figure imgf000101_0003
Figure imgf000102_0001
[0519] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000102_0002
[0521] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000103_0001
Oligonucleotide
[0522] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000103_0002
Oligonucleotide [0523] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000103_0004
[0525] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000103_0003
Oligonucleotide) [0526] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000104_0002
[0528] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000104_0001
Figure imgf000105_0001
[0529] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000105_0002
Figure imgf000106_0001
[0530] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000106_0002
Figure imgf000107_0001
[0531] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000108_0001
[0532] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000109_0001
[0534] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000109_0002
[0536] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000109_0003
[0538] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000110_0001
[0540] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000110_0002
[0542] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000110_0003
[0544] In some embodiments, the modified oligonucleotide conjugate is:
Figure imgf000111_0001
Figure imgf000111_0002
Figure imgf000111_0004
[0547] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000111_0003
Figure imgf000112_0001
ʼnll [0548] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000113_0001
Figure imgf000114_0001
5 [0549] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000114_0002
Figure imgf000115_0001
[0550] In some embodiments, the modified oligonucleotide conjugate is selected from:
Figure imgf000116_0001
Figure imgf000117_0002
[0552] In some embodiments, the modified oligonucleotide conjugate is
Figure imgf000117_0001
[0553] In some embodiments, the modified oligonucleotide conjugate is
Figure imgf000117_0003
[0555] In some embodiments, the modified oligonucleotide conjugate
Figure imgf000118_0003
[0557] In some embodiments, the modified oligonucleotide conjugate
Figure imgf000118_0001
Figure imgf000118_0004
[0559] In some embodiments, the modified oligonucleotide conjugate is
Figure imgf000118_0005
[0561] In some embodiments, the modified oligonucleotide conjugate is
Figure imgf000118_0002
modified oligonucleotide conjugate
Figure imgf000119_0001
Figure imgf000119_0007
[0564] In some embodiments, the modified oligonucleotide conjugate
Figure imgf000119_0002
Figure imgf000119_0003
Figure imgf000119_0008
oligonucleotide conjugate is
Figure imgf000119_0004
[0567] In some embodiments, the modified oligonucleotide conjugate
Figure imgf000119_0005
Figure imgf000119_0006
modified oligonucleotide conjugate
Figure imgf000120_0001
Methods of conjugation
[0571] Provided herein is a method of conjugating the modified oligonucleotide described above to a thiol-containing molecule (M-SH) to form the modified oligonucleotide conjugate also described above, wherein the method comprises a thiol-ene reaction between the thiol and the vinyl group of the previously described modified oligonucleotide.
[0572] In some embodiments, the oligonucleotide is an antisense oligonucleotide. In some embodiments, the oligonucleotide is an RNA interference (RNAi) oligonucleotide. In some embodiments, the oligonucleotide is an aptamer. In some embodiments, the oligonucleotide is a short interfering RNA (siRNA) oligonucleotide. In some embodiments, the oligonucleotide is a spliceswitching antisense oligonucleotide. In some embodiments, the oligonucleotide is an microRNA (miRNA) oligonucleotide. In some embodiments, the oligonucleotide is a DNA oligonucleotide. In some embodiments, the oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is an XNA. In some embodiments, the oligonucleotide is an xRNA. In some embodiments, the oligonucleotide is an xDNA. In some embodiments, the oligonucleotide is an saRNA.
[0573] In some embodiments, M is a small molecule. For example, in some embodiments, M is a pharmacologically active small molecule. In some embodiments, M is a probe. A probe is a molecule which is used to investigate biological mechanisms or validate targets for drug discovery. Probes may comprise a moiety which allows for their detection by an external source. For example, a probe may be radiolabelled, i.e. may comprise radioisotopes. In another example, the probe may comprise a moiety which displays fluorescence or phosphorescence.
[0574] In some embodiments, M is a fluorophore.
[0575] In some embodiments, M is fluorescein or a fluorescein derivative. Fluorescein is a common diagnostic contrast agent with many derivatives which are well known in the art. Exemplary derivatives include fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, mixtures of fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate, fluorescein succinimidyl esters, carboxyfluorescein, carboxyfluorescein succinimidyl ester, fluorescein pentafluorophenyl esters, fluorescein tetrafluorophenyl esters, and fluorescein amidites.
[0576] In some embodiments, M is rhodamine or a rhodamine derivative. Rhodamine derivatives are a well-known family of dyes which feature the rhodamine core structure. Examples of rhodamine derivatives include carboxytetramethylrhodamine, tetramethylrhodamine, tetramethylrhodamine isothiocyanate, sulforhodamine 101, sulforhodamine 101 acid chloride (Texas Red), and rhodamine red.
[0577] In some embodiments, M is 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY).
[0578] In some embodiments, M is a radiolabelled molecule. Radiolabelled molecules are typically small molecules which are non-biologically active and which are isotopically enriched with a radioactive isotope such as 32P, 35S, 14C, and 3H.
[0579] In some embodiments, M is a polypeptide. In some embodiments, M is a protein. In some embodiments, M is a polypeptide or protein connected to the modified oligonucleotide via a cysteine residue.
[0580] In embodiments of the invention, the polypeptide or protein of the invention may be a carrier protein. A carrier protein is a protein or polypeptide which is capable of transporting an ion, small molecule, or macromolecule across a biological membrane, e.g. into a cell. In embodiments of the invention, the carrier protein is selected to transport the modified oligonucleotide into the cell. In embodiments of the invention, the oligonucleotide is an antisense oligonucleotide, RNAi oligonucleotide, aptamer, siRNA oligonucleotide, splice-switching antisense oligonucleotide, miRNA oligonucleotide, DNA oligonucleotide, gapmer, XNA oligonucleotide, xRNA oligonucleotide, xDNA oligonucleotide, or saRNA oligonucleotide which is intended to be transported into cells or a specific cell type.
[0581] In embodiments of the invention, the polypeptide or protein is an antigen binding fragment, a nanobody or an antibody. Antibodies comprise antigen binding fragments. Antigen binding fragments are polypeptide sequences which bind to antigens. Antigen binding fragments are antigen- specific, i.e. can only react to and bind to one specific antigen, or can cross-react, i.e. can react to and bind more than one antigen. Nanobodies, or single-domain antibodies, are a type of antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies generally have a molecular weight around 12-15 kDa and so are significantly lighter than common antibodies which generally have a molecular weight around 150-160 kDa. Nanobodies. Like antibodies, are capable of selectively binding to a specific antigen. By linking an oligonucleotide to an antigen binding fragment, a nanobody or an antibody, it is possible to provide a modified oligonucleotide conjugate which targets, for example, cells displaying a specific antigen. Delivery of a therapeutic oligonucleotide can, therefore, be targeted to only the cells in which the oligonucleotide is desired to have an effect.
[0582] In embodiments of the invention, the polypeptide or protein is a cell-penetrating peptide (CPP). CPPs are usually short peptides, for example, of less than 30-40 amino acids. They may be derived from proteins or chimeric sequences or may be of completely artificial, synthetic or designed origin. They are occasionally amphipathic, but almost always possess a net positive (cationic) charge at physiological pH. CPPs are able to penetrate biological membranes, to trigger the movement of various biomolecules across cell membranes into the cytoplasm and to improve their intracellular routing, thereby facilitating interactions with the target. CPPs have been shown to be able to deliver oligonucleotide cargos into a wide variety of cell types.
[0583] In some embodiments the method comprises the step of M-SH being dissolved in a buffered solution to provide a M-SH buffered solution. A buffered solution is a solution which contains a buffer which keeps the pH of the solution at a specific pH or within a specific pH range. In some embodiments the buffered solution is a commercially available buffer. In some embodiments, the buffered solution comprises a buffer and water. In some embodiments, the buffered solution has a pH around physiological pH. In some embodiments, the buffered solution has a pH between 7.3 and 7.5. In some embodiments, the buffered solution has a pH of about 7.4.
[0584] In some embodiments, the buffered solution comprises an aqueous buffer. In other embodiments, the buffered solution comprises a non-aqueous buffer. In some embodiments, the aqueous buffer is phosphate buffered saline (PBS).
[0585] In some embodiments, the method comprises the step of the modified oligonucleotide being dissolved in an aqueous solution to provide a modified oligonucleotide solution.
[0586] In some embodiments, the method comprises the step of the M-SH buffered solution being mixed with the modified oligonucleotide solution.
[0587] In some embodiments the reaction between the modified oligonucleotide and M-SH is performed between 10-50 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed between 20-40 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed between 25-40 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed at 25 °C. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed at physiological temperatures. In some embodiments, the reaction between the modified oligonucleotide and M-SH is performed at 37 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has its temperature altered following the mixing. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has a temperature between 10-50 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has a temperature between 20-40 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution is heated to a temperature between 20-40 °C or 25-40 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution has a temperature of 25 °C. In some embodiments, the solution resulting from the mixing of the M-SH buffered solution and the modified oligonucleotide solution is heated to a physiological temperature. In some embodiments, the solution resulting from the mixing of the M- SH buffered solution and the modified oligonucleotide solution is heated to 37 °C.
[0588] In some embodiments, the thiol-ene reaction step is performed in the presence of a radical initiator. A radical initiator is a substance or molecule which produces radical species under mild reaction conditions and therefor promotes radical reactions. These radical initiators will generally comprise a bond with a small bond dissociation energy, for example azo- or peroxide bonds. Common radical initiators include azobisisobutyronitrile (AIBN), l,l'-azobis(cyclohexanecarbonitrile) (ABCN), di=tert-butyl peroxide, benzoyl peroxide, methyl ethyl ketone peroxide, and acetone peroxide. In some embodiments, the thiol-ene reaction step is performed in the presence of dithiothreitol.
EXAMPLES
Linkers
[0589] The following linkers were synthesised.
Table 1
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
General Experimental Methods
[0590] All reactions were carried out under an atmosphere of argon. Reactions were monitored by TLC using aluminum backed silica gel 60 (F254) plates, visualized using UV 254 nm and vanillin dips. Flash column chromatography was carried out with silica gel (60-120 mesh). Reagents were used as received from commercial sources unless otherwise stated. 1H NMR spectra were recorded on a Bruker Avance 400 Spectrometer (400 MHz). Chemical shifts are reported in 6 units (parts per million) and coupling constants (J) are measured in Hertz. The residual solvent signals were used as references.   Liquid  Chromatography  High  Resolution  Mass  spectra  were  recorded  on  QTOF  Premier  mass  spectrometer (Waters Ltd, UK) and Synapt XS mass spectrometer (Waters Ltd, UK). Dry solvents were  purchased and used as received.  Example 1: Synthesis of Linkers  General method for synthesis of linkers:  Method A  Step 1   4‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclohexan‐1‐ol 
Figure imgf000127_0001
  [0591] To a solution of 2,4‐dichloro‐pyrimidine (1.00 g, 6.7 mmol) in THF (10 mL) was added Et3N (2.5  eq, 0.43 mL) and commercially available  trans‐4‐(Methylamino)cyclohexanol  (1.5 eq, 10.05 mmol).  The mixture was stirred at ambient temperature for 7 hr and concentrated under reduced pressure.  The residue was dissolved in DCM and the solution was poured into saturated aqueous NaHCO3. The  two layers were separated, and the aqueous layer was extracted with DCM (2x25 mL). The combined  organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by  flash  chromatography  (silica  gel,  0‐100%  Hexane/EtOAc)  to  afford  4‐((2‐chloropyrimidin‐4‐ yl)(methyl)amino)cyclohexan‐1‐ol  (major  product)  (925 mg,  57%)  as  pale  yellow  gum  and  4‐((4‐ chloropyrimidin‐2‐yl)(methyl)amino)cyclohexan‐1‐ol (minor product) (140 mg, 8.6%) as yellow viscous  oil.  [0592] 1H NMR for 4‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclohexan‐1‐ol (major product)  [0593]  NMR (400 MHz, CD3OD) δ 8.20 (d, J = 5.2 Hz, 1H), 6.58 (d, J = 5.1 Hz, 1H), 4.59 (p, J = 8.6 Hz,  1H), 3.58 (ddd, J = 15.3, 11.0, 4.3 Hz, 1H), 3.01 (s, 3H), 2.05 (d, J = 12.4 Hz, 2H), 1.72 (h, J = 3.3 Hz, 4H),  1.46 (dt, J = 21.5, 11.0 Hz, 2H). (One exchangeable proton of OH was not observed in CD3OD).  [0594] HRMS (ESI) m/z found [M+H]+ 242.104, C11H17ClN3O+ required 242.105.  [0595] 1H NMR for 4‐((4‐chloropyrimidin‐2‐yl)(methyl)amino)cyclohexan‐1‐ol (minor product)  [0596]   [0597] 1H NMR (400 MHz, CD3OD) δ 8.09 (d, J = 6.4 Hz, 1H), 6.65 (dd, J = 17.3, 10.4 Hz, 1H), 6.58 –  6.41 (m, 1H), 5.63 (dd, J = 10.4, 2.1 Hz, 1H), 3.62 (dt, J = 11.1, 4.3 Hz, 1H), 2.97 (s, 3H), 2.09 – 2.01 (m,    2H), 1.74  (tt,  J = 6.1, 3.0 Hz, 3H), 1.57 – 1.44  (m, 2H).  (One exchangeable proton of OH was not  observed in CD3OD).  [0598] HRMS (ESI) m/z found [M+H]+ 242.109, C11H17ClN3O+ required 242.105.  Step 2: Procedure 1  4‐(methyl(2‐vinylpyrimidin‐5‐yl)amino)cyclohexan‐1‐ol 
Figure imgf000128_0001
  [0599] To a solution of 4‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)cyclohexan‐1‐ol (140 mg, 0.58 mmol)  and potassium trifluorovinyl borate (1.67 eq, 130 mg) in ethanol (8 mL) under argon atmosphere was  added Et3N (2.1 eq, 0.17 ml). The mixture was degassed for 5 mins. Then the catalyst Pd(dppf)Cl2 (0.1  eq, 43 mg) was added  to  the mixture and  further degassed  for 5 mins. The  reaction mixture was  warmed upto 80 oC and stirred at this temperature for 6 hours. The reaction mixture was cooled to  room temperature, diluted with DCM  (10 mL) and  filtered through a bed of celite washing several  times with DCM (25 mL). Solvent was removed under vacuum. The residue was subjected to column  chromatography  (silica  gel,  0‐100%  Heptane/Ethyl  acetate)  to  afford  the  product  4‐(methyl(2‐ vinylpyrimidin‐4‐yl)amino)cyclohexan‐1‐ol (128 mg, 95%) as a colourless gum.  [0600] 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 5.0 Hz, 1H), 6.67 – 6.50 (m, 2H), 6.37 (dd, J = 17.4, 1.7  Hz, 1H), 5.57 (dd, J = 10.5, 1.7 Hz, 1H), 4.63 (tt, J = 10.1, 4.9 Hz, 1H), 3.56 (tt, J = 11.0, 4.3 Hz, 1H), 2.99  (s, 3H), 2.13 – 1.95 (m, 2H), 1.85 – 1.55 (m, 4H), 1.55 – 1.37 (m, 2H). (One exchangeable proton of OH  was not observed in CD3OD).  [0601] HRMS (ESI) m/z found [M+H]+ 234.161, C13H20N3O+ required 234.160.  Step 2: Procedure 2  4‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclohexan‐1‐ol 
Figure imgf000128_0002
  [0602] To  a  solution  of  4‐((4‐chloropyrimidin‐2‐yl)(methyl)amino)cyclohexan‐1‐ol  (140  mg,  0.58  mmol) and vinyl boronate pinacol ester (3 eq, 0.3 mL) in Dioxane (4 mL) and water (1 mL) under argon    catalyst Pd(dppf)Cl2.DCM (0.1 eq, 49 mg) was added to the mixture and further degassed for 5 mins.  The reaction mixture was warmed upto 85 oC and stirred at this temperature for 18 hours. The reaction  mixture was  cooled  to  room  temperature, diluted with DCM and  filtered  through a bed of  celite  washing several times with DCM. Solvent was removed under vacuum. The residue was subjected to  column chromatography (silica gel, 0‐100% Heptane/Ethyl acetate) to afford the product 4‐(methyl(4‐ vinylpyrimidin‐2‐yl)amino)cyclohexan‐1‐ol (130 mg, 96%) as a pale yellow gum.  [0603] 1H NMR (400 MHz, CD3OD) δ 8.08 (d, J = 6.3 Hz, 1H), 7.67 – 7.46 (m, 1H), 6.65 (dd, J = 17.4,  10.4 Hz, 1H), 6.60 – 6.37 (m, 2H), 5.63 (dd, J = 10.4, 2.1 Hz, 1H), 3.60 (tt, J = 11.1, 4.4 Hz, 1H), 2.95 (s,  3H), 2.18 – 1.97 (m, 2H), 1.72 (h, J = 3.4 Hz, 4H), 1.48 (dtt, J = 17.4, 11.5, 6.7 Hz, 2H). (One exchangeable  proton of OH was not observed in CD3OD).  [0604] HRMS (ESI) m/z found [M+H]+ 234.168, C13H20N3O+ required 234.160.  Step 3  2‐cyanoethyl  (4‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)cyclohexyl)  diisopropylphosphoramidite  (Compound 5)   
Figure imgf000129_0001
  [0605] A  stirred  solution  of  4‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclohexan‐1‐ol  (128 mg,  0.55  mmol) (dried in high vac) in DCM (8 mL) was degassed by bubbling with argon for 5 mins. The solution  was cooled to 0 oC in ice bath and DIPEA (3 eq, 0.29 mL) was added followed by dropwise addition of  2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite  (1.5  eq,  0.18 mL).  The  reaction mixture was  allowed to gradually warm to room temperature and stirred for 6 hours. Solvent was removed under  vacuum.  The  residue  was  subjected  to  column  chromatography  (silica  gel,  0‐100%  Heptane  (1%Et3N)/EtOAc)  to afford product 2‐cyanoethyl  (4‐(methyl(2‐vinylpyrimidin‐5‐yl)amino)cyclohexyl)  diisopropylphosphoramidite (136 mg, 57.2%) as a colourless gum.   [0606] 1H NMR (400 MHz, CD3CN) δ 8.29 (d, J = 4.9 Hz, 1H), 6.62 (dd, J = 17.4, 10.5 Hz, 1H), 6.54 (d, J  = 4.9 Hz, 1H), 6.42 (dd, J = 17.4, 1.8 Hz, 1H), 5.57 (dd, J = 10.5, 1.8 Hz, 1H), 4.76 – 4.63 (m, 1H), 3.78  (dtdd, J = 14.2, 10.5, 7.1, 5.1 Hz, 3H), 3.63 (dh, J = 10.0, 6.8 Hz, 2H), 3.01 (s, 3H), 2.70 – 2.65 (m, 2H),  2.14 (dt, J = 12.9, 3.3 Hz, 1H), 2.06 (dq, J = 10.6, 3.6 Hz, 1H), 1.75 – 1.66 (m, 4H), 1.54 (tdd, J = 11.5,  8.6, 4.4 Hz, 2H), 1.20 (dd, J = 6.8, 2.2 Hz, 12H).  [0607] 31P NMR (162 MHz, CD3CN) δ 145.45.    [0608] HRMS (ESI) m/z found [M+H]+ 434.260, C22H37N5O2P+ required 434.268.  Method B  Step 1  2‐chloro‐4‐vinylpyrimidine 
Figure imgf000130_0001
  [0609] To  a  solution of 2,4‐dichlotopyrimidine  (519 mg, 3.48 mmol)  and potassium  trifluorovinyl  borate (1.1 eq, 500 mg)  in anhydrous 2‐propanol (10 mL) under argon atmosphere  in a microwave  tube  was  added  Et3N  (1  eq,  0.5 ml).  The mixture  was  degassed  for  5 mins.  Then  the  catalyst  Pd(dppf)Cl2.DCM (0.05 eq, 142 mg) was added to the mixture and further degassed for 5 mins. The  tube was sealed and the reaction mixture was warmed upto 90 oC and stirred at this temperature for  5 hours. The reaction mixture was cooled to room temperature, diluted with Ethyl acetate (25 mL)  and filtered through a bed of celite washing several times with Ethyl acetate (3x10 mL). Solvent was  removed  under  vacuum.  Column  chromatography  (silica  gel,  0‐100%  Heptane/EtOAc)  afforded  product 2‐chloro‐4‐vinylpyrimidine (296 mg, 60%) as pale yellow oil. 1H NMR data was consistent with  the literature data (Ref: Molecules 2010, 15(3), 1973‐1984).   [0610] 1H NMR (400 MHz, CDCl3) δ 8.57 (dd, J = 5.1, 1.3 Hz, 1H), 7.23 (dd, J = 5.1, 0.9 Hz, 1H), 6.71  (ddd, J = 17.4, 10.6, 1.4 Hz, 1H), 6.54 (ddd, J = 17.4, 1.7, 1.0 Hz, 1H), 5.80 (dt, J = 10.5, 1.3 Hz, 1H).  Step 2  1‐(4‐vinylpyrimidin‐2‐yl)piperidin‐4‐ol   
Figure imgf000130_0002
  [0611] A solution of 2‐chloro‐4‐vinylpyrimidine (100 mg, 0.711 mmol), piperidin‐4‐ol (1.2 eq, 0.854  mmol) and anhydrous DIPEA (2.5 eq, 0.35 mL) in anhydrous DMF (1.5 mL) was stirred at 100 oC in a  sealed tube for 18 hours. The reaction mixture was cooled to room temperature and solvent removed  under vacuum. Column chromatography (silica gel, 0‐100% Heptane/Ethyl acetate) afforded product  1‐(4‐vinylpyrimidin‐2‐yl)piperidin‐4‐ol (49 mg, 34%) as pale brown gum.  [0612] 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 5.0 Hz, 1H), 6.59 (dd, J = 17.4, 10.5 Hz, 1H), 6.48 (d, J =  5.0 Hz, 1H), 6.37 (dd, J = 17.4, 1.6 Hz, 1H), 5.57 (dd, J = 10.5, 1.6 Hz, 1H), 4.62 – 4.40 (m, 2H), 3.94 (tt,    J = 8.7, 4.0 Hz, 1H), 3.31 (ddd, J = 13.4, 10.0, 3.3 Hz, 2H), 1.96 (dtdd, J = 8.5, 7.0, 4.0, 2.3 Hz, 3H), 1.63  – 1.42 (m, 2H).  [0613] HRMS (ESI) m/z found [M+H]+ 206.122, C11H16N3O+ required 206.129.  Step 3 – same as Method A    Synthesis of specific linkers:  2‐cyanoethyl (1‐(4‐vinylpyrimidin‐2‐yl)piperidin‐4‐yl) diisopropylphosphoramidite (Compound 8)  [0614] Starting  from  1‐(4‐vinylpyrimidin‐2‐yl)piperidin‐4‐ol,  compound  2‐cyanoethyl  (1‐(4‐ vinylpyrimidin‐2‐yl)piperidin‐4‐yl)  diisopropylphosphoramidite  was  synthesized  in  67%  yield  as  a  colourless gum, following same synthetic procedure as mentioned in Method A Step 3. 
Figure imgf000131_0001
  [0615] 1H NMR (400 MHz, CD2Cl2) δ 8.29 (d, J = 5.0 Hz, 1H), 6.61 (dd, J = 17.3, 10.5 Hz, 1H), 6.50 (d, J  = 5.0 Hz, 1H), 6.40 (dd, J = 17.3, 1.7 Hz, 1H), 5.58 (dd, J = 10.5, 1.7 Hz, 1H), 4.24 – 4.10 (m, , 3H), 3.91  – 3.76 (m, 2H), 3.75 – 3.60 (m, 4H), 2.67 (t, J = 6.3 Hz, 2H), 2.02 – 1.87 (m, 2H), 1.71 (dqd, J = 12.0, 7.9,  3.8 Hz, 2H), 1.23 (dd, J = 6.8, 1.2 Hz, 12H).  [0616] 31P NMR (162 MHz, CD2Cl2) δ 146.14.  [0617] HRMS (ESI) m/z found [M+H]+ 406.235, C20H33N5O2P+ required 406.237.  2‐cyanoethyl  (4‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclohexyl)  diisopropylphosphoramidite  (Compound 6)  [0618] Method  A  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (4‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclohexyl)  diisopropylphosphoramidite  from  4‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclohexan‐1‐ol in 68% yield as a colourless gum. 
Figure imgf000131_0002
    [0619] 1H NMR (400 MHz, CD3CN) δ 8.15 (d, J = 6.2 Hz, 1H), 6.65 (dd, J = 17.3, 10.4 Hz, 1H), 6.48 –  6.33 (m, 1H), 5.57 (dd, J = 10.4, 2.4 Hz, 1H), 3.88 – 3.72 (m, 3H), 3.64 (dp, J = 10.1, 6.8 Hz, 2H), 2.90 (s,  3H), 2.67 (t, J = 6.0 Hz, 2H), 2.14 (dq, J = 10.7, 3.5 Hz, 1H), 2.06 (dt, J = 12.4, 3.5 Hz, 1H), 1.78 – 1.63 (m,  4H), 1.62 – 1.44 (m, 2H), 1.35 – 1.24 (m, 2H), 1.21 (dd, J = 6.8, 2.3 Hz, 12H).  [0620] 31P NMR (162 MHz, CD3CN) δ 145.49.  [0621] HRMS (ESI) m/z found [M+H]+ 434.263, C22H37N5O2P+ required 434.268.  1‐(2‐chloropyrimidin‐4‐yl)azetidin‐3‐ol  [0622] Method A Step 1 was followed to synthesise 1‐(2‐chloropyrimidin‐4‐yl)azetidin‐3‐ol, starting  from  commercially  available  compounds  2,4‐dichloro‐pyrimidine  and  3‐Hydroxyazetidine  hydrochloride. Flash column chromatography (silica gel, 0‐50% DCM/MeOH) afforded compounds 1‐ (2‐chloropyrimidin‐4‐yl)azetidin‐3‐ol  (major  product)  in  78%  yield  as  yellow  gum  and  1‐(4‐ chloropyrimidin‐2‐yl)azetidin‐3‐ol (minor product) in 4% yield as yellow oil. 
Figure imgf000132_0001
  [0623] 1H NMR for 1‐(2‐chloropyrimidin‐4‐yl)azetidin‐3‐ol (major product)  [0624] 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 5.8 Hz, 1H), 6.10 (d, J = 5.9 Hz, 1H), 4.84 (brs, 1H), 4.49  – 4.27 (m, 2H), 4.11 – 3.97 (m, 2H), 2.54 (brs, 1H).  [0625] HRMS (ESI) m/z found [M+H]+ 186.049, C7H9ClN3O+ required 186.043.  [0626] 1H NMR for 1‐(4‐chloropyrimidin‐2‐yl)azetidin‐3‐ol (minor product)  [0627] 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 5.2 Hz, 1H), 6.58 (d, J = 5.1 Hz, 1H), 4.81 (dtd, J = 10.7,  6.4, 4.3 Hz, 1H), 4.44 (ddd, J = 10.0, 6.6, 1.4 Hz, 2H), 4.05 (ddd, J = 10.0, 4.3, 1.3 Hz, 2H), 2.40 (brs, 1H).  1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐3‐ol  [0628] Method A Step 2 Procedure 2 was followed to synthesise 1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐3‐ ol  from  1‐(2‐chloropyrimidin‐4‐yl)azetidin‐3‐ol  in  70%  yield  as  pale  brown  gum.  Flash  column  chromatography (silica gel) was performed for purification using (0‐50%) DCM‐MeOH solvent system. 
Figure imgf000132_0002
    [0629] 1H NMR (400 MHz, CD3OD) δ 8.08 (d, J = 6.0 Hz, 1H), 7.71 – 7.39 (m, 1H), 6.64 (dd, J = 17.3,  10.5 Hz, 1H), 6.46 (dd, J = 17.3, 2.1 Hz, 1H), 6.25 (d, J = 6.0 Hz, 1H), 5.64 (dd, J = 10.5, 2.0 Hz, 1H), 4.73  (td, J = 6.8, 3.4 Hz, 1H), 4.35 (dd, J = 9.7, 6.6 Hz, 2H), 3.91 (dd, J = 9.8, 4.3 Hz, 2H).  [0630] HRMS (ESI) m/z found [M+H]+ 178.099, C9H12N3O+ required 178.097.  2‐cyanoethyl (1‐(2‐vinylpyrimidin‐4‐yl)azetidin‐3‐yl) diisopropylphosphoramidite (Compound 4)  [0631] Method A Step 3 was followed to synthesise 2‐cyanoethyl (1‐(2‐vinylpyrimidin‐4‐yl)azetidin‐ 3‐yl) diisopropylphosphoramidite from 1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐3‐ol in 51% yield as colourless  gum. 
Figure imgf000133_0001
  [0632] 1H NMR (400 MHz, CD3CN) δ 8.17 (d, J = 5.8 Hz, 1H), 6.64 (dd, J = 17.3, 10.4 Hz, 1H), 6.46 (dd,  J = 17.3, 2.4 Hz, 1H), 6.20 (d, J = 5.9 Hz, 1H), 5.59 (dd, J = 10.4, 2.4 Hz, 1H), 4.85 (dtt, J = 9.0, 6.6, 4.4  Hz, 1H), 4.35 (ddt, J = 9.6, 6.6, 1.5 Hz, 2H), 4.00 (td, J = 10.4, 4.3 Hz, 2H), 3.82 (dddt, J = 22.4, 10.4, 7.8,  6.1 Hz, 2H), 3.66 (dq, J = 10.3, 6.8 Hz, 2H), 2.69 (t, J = 6.0 Hz, 2H), 1.21 (dd, J = 6.8, 2.1 Hz, 12H).  [0633] 31P NMR (162 MHz, CD3CN) δ 147.25.  [0634] HRMS (ESI) m/z found [M+H]+ 378.208, C18H29N5O2P+ required 378.205.  1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐3‐ol 
Figure imgf000133_0002
  [0635] Method B Step 2 was followed to synthesise 1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐3‐ol in 15% yield  as  pale  brown  viscous  oil  starting  from  2‐chloro‐4‐vinylpyrimidine  and  commercially  available  3‐ Hydroxyazetidine  hydrochloride.  Flash  column  chromatography  (silica  gel)  was  performed  for  purification using (0‐100%) DCM‐MeOH solvent system.  [0636] 1H NMR (400 MHz, CD3OD) δ 8.25 (d, J = 5.2 Hz, 1H), 6.76 (d, J = 5.3 Hz, 1H), 6.70 – 6.55 (m,  1H), 6.40 (dd, J = 17.4, 1.4 Hz, 1H), 5.66 (dd, J = 10.7, 1.4 Hz, 1H), 4.69 (tt, J = 6.6, 4.4 Hz, 1H), 4.36  (ddd, J = 9.5, 6.6, 1.3 Hz, 2H), 3.93 (ddd, J = 7.8, 3.9, 2.6 Hz, 2H). (One exchangeable proton of OH was  not observed in CD3OD).  [0637] HRMS(ESI)m/zfound[M+H]+178.099,C9H12N3O+required178.097.   2‐cyanoethyl (1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐3‐yl) diisopropylphosphoramidite (Compound 3)  [0638] Method B Step 3 was followed to synthesise 2‐cyanoethyl (1‐(4‐vinylpyrimidin‐2‐yl)azetidin‐ 3‐yl) diisopropylphosphoramidite from 1‐(2‐vinylpyrimidin‐4‐yl)azetidin‐3‐ol in 50% yield as colourless  gum.  
Figure imgf000134_0001
    1H NMR (400 MHz, CD3CN) δ 8.31 (d, J = 5.1 Hz, 0.5H), 8.21 (d, J = 5.0 Hz, 0.5H), 6.74 – 6.44 (m, 1H),  6.40 (dd, J = 17.4, 1.7 Hz, 0.5H), 5.61 (dd, J = 10.6, 1.7 Hz, 0.5H), 4.81 (dddt, J = 11.1, 9.2, 6.6, 4.6 Hz,  1H), 4.41 – 4.23 (m, 2H), 4.00 (qd, J = 9.6, 4.4 Hz, 2H), 3.91 – 3.75 (m, 2H), 3.75 – 3.55 (m, 3H), 2.69 (t,  J = 5.9 Hz, 2H), 2.62 ‐ 2.56 (m, 1H), 1.23 – 1.19 (m, 12H).  [0639] 31P NMR (162 MHz, CD3CN) δ 147.02.  [0640] HRMS (ESI) m/z found [M+H]+ 378.202, C18H29N5O2P+ required 378.205.  3‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclopentan‐1‐ol  [0641] Method  B  Step  2  was  followed  to  synthesise  3‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclopentan‐1‐ol  from  2‐chloro‐4‐vinylpyrimidine  and  commercially  available  3‐ Methylamino‐cyclopentanol hydrochloride in 61% yield as pale brown gum. 
Figure imgf000134_0002
  [0642] 1H NMR (400 MHz, CDCl3) δ 8.26 (dd, J = 16.3, 5.0 Hz, 1H), 6.74 – 6.26 (m, 3H), 5.57 (td, J =  10.6, 1.6 Hz, 1H), 4.76 – 4.25 (m, 2H), 3.23 (s, 1.5H), 3.03 (s, 1.5H), 2.37 – 1.99 (m, 3H), 1.98 – 1.62 (m,  4H).  [0643] HRMS (ESI) m/z found [M+H]+ 220.146, C12H18N3O+ required 220.144.  2‐cyanoethyl  (3‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclopentyl)  diisopropylphosphoramidite  (Compound 9)  [0644] Method  B  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (3‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclopentyl)  diisopropylphosphoramidite  from  3‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclopentan‐1‐ol in 70% yield as colourless gum.   
Figure imgf000135_0001
  [0645] 1H NMR (400 MHz, CD2Cl2) δ 8.28 – 8.10 (m, 1H), 6.49 (ddd, J = 17.4, 10.5, 1.7 Hz, 1H), 6.41 –  6.22 (m, 2H), 5.51 – 5.40 (m, 1H), 4.59 – 4.20 (m, 1H), 3.80 – 3.65 (m, 2H), 3.54 (dtdt, J = 13.8, 6.9, 3.6,  1.5 Hz, 2H), 2.99 (d, J = 3.2 Hz, 1.5H), 2.92 (d, J = 1.9 Hz, 1.5H), 2.55 (dtd, J = 11.8, 6.8, 3.2 Hz, 2H), 2.23  – 1.51 (m, 7H), 1.13 – 1.08 (m, 12H).  [0646] 31P NMR (162 MHz, CD2Cl2) δ 146.62, 146.54, 146.43.  [0647] HRMS (ESI) m/z found [M+H]+ 420.258, C21H35N5O2P+ required 420.252.  3‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclobutan‐1‐ol  [0648] Method  B  Step  2  was  followed  to  synthesise  3‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclobutan‐1‐ol  from  2‐chloro‐4‐vinylpyrimidine  and  commercially  available  3‐ (methylamino)cyclobutan‐1‐ol in 44% yield as yellow gum. 
Figure imgf000135_0002
  [0649] 1H NMR (400 MHz, CDCl3) δ 8.30 (dd, J = 5.0, 2.0 Hz, 1H), 6.67 – 6.58 (m, 1H), 6.50 (d, J = 5.0  Hz, 1H), 6.39 (ddd, J = 17.4, 4.7, 1.6 Hz, 1H), 5.65 – 5.51 (m, 1H), 4.70 (tt, J = 9.7, 7.4 Hz, 1H), 4.21 –  4.06 (m, 1H), 3.17 (d, J = 5.6 Hz, 3H), 2.79 – 2.52 (m, 2H), 2.40 – 2.11 (m, 3H).  [0650] HRMS (ESI) m/z found [M+H]+ 206.121, C11H16N3O+ required 206.129.    2‐cyanoethyl  (3‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)cyclobutyl)  diisopropylphosphoramidite  (Compound 10)  [0651] Method  B  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (3‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclobutyl)  diisopropylphosphoramidite  from  3‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)cyclobutan‐1‐ol in 72% yield as a colourless gum. 
Figure imgf000135_0003
  [0652] 1H NMR (400 MHz, CD2Cl2) δ 8.30 (t, J = 4.7 Hz, 1H), 6.62 (ddd, J = 17.3, 10.5, 1.3 Hz, 1H), 6.52  (d, J = 5.0 Hz, 1H), 6.47 – 6.36 (m, 1H), 5.58 (dd, J = 10.5, 1.7 Hz, 1H), 4.87 – 4.76 (m, 1H), 4.17 (dddd,  J = 14.3, 9.2, 7.6, 6.6 Hz, 1H), 3.94 – 3.76 (m, 2H), 3.76 – 3.54 (m, 2H), 3.17 (s, 3H), 2.82 – 2.54 (m, 4H),  2.51 – 2.23 (m, 2H), 1.23 (ddd, J = 6.8, 3.3, 1.5 Hz, 12H).  [0653] 31P NMR (162 MHz, CD2Cl2) δ 146.26, 145.38.  [0654] HRMS (ESI) m/z found [M+H]+ 406.239, C20H33N5O2P+ required 406.237.  2‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)ethan‐1‐ol  [0655] Method  A  Step  2  Procedure  2  was  followed  to  synthesise  2‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)ethan‐1‐ol  from commercially available 2‐((4‐chloropyrimidin‐2‐yl)(methyl)amino)ethan‐1‐ ol in 79% yield as pale brown viscous oil. 
Figure imgf000136_0001
  [0656] 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.0, 1H), 6.57 (dd, J = 17.4, 10.6, 1H), 6.51 (d, J = 5.0,  1H), 6.36 (dd, J = 17.4, 1.4, 1H), 5.59 (dd, J = 10.6, 1.4, 1H), 4.53 (s, 1H), 3.91 (dd, J = 5.4, 3.9, 2H), 3.80  (dd, J = 5.5, 4.0, 2H), 3.25 (s, 3H).  [0657] HRMS (ESI) m/z found [M+H]+ 180.117, C9H14N3O+ required 180.113.  2‐cyanoethyl  (2‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)ethyl)  diisopropylphosphoramidite  (Compound 7)  [0658] Method  A  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (2‐(methyl(4‐vinylpyrimidin‐2‐ yl)amino)ethyl)  diisopropylphosphoramidite  from  2‐(methyl(4‐vinylpyrimidin‐2‐yl)amino)ethan‐1‐ol  in 69% yield as colourless gum. 
Figure imgf000136_0002
  [0659] 1H NMR (400 MHz, CD2Cl2) δ 8.17 ‐ 8.05 (m, 1H), 6.54 – 6.22 (m, 1.65 H), 5.45 (dd, J = 10.5, 1.7  Hz, 0.37 H), 3.88 – 3.60 (m, 6H), 3.57 – 3.36 (m, 2H), 3.12 (d, J = 10.1 Hz, 3H), 2.67 – 2.42 (m, 2H), 1.20  – 0.97 (m, 14H).  [0660] 31P NMR (162 MHz, CD2Cl2) δ 147.79, 147.63.  [0661] HRMS(ESI)m/zfound[M+H]+380225 C18H31N5O2P+required380221   2‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)ethan‐1‐ol  [0662] Method  A  Step  2  Procedure  1  was  followed  to  synthesise  2‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)ethan‐1‐ol  from commercially available 2‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)ethan‐1‐ ol in 70% yield as pale brown oil. 
Figure imgf000137_0001
  [0663] 1H NMR (400 MHz, CDCl3) δ 6.63 – 6.51 (m, 1H), 6.36 (dd, J = 17.4, 1.4, 2H), 5.59 (dd, J = 10.5,  1.4,  1H),  4.53  (s,  1H),  3.91  (dd,  J  =  5.6,  3.8,  2H),  3.80  (dd,  J  =  5.6,  3.8,  2H),  3.25  (s,  3H).  (One  exchangeable proton of OH was not observed).  [0664] HRMS (ESI) m/z found [M+H]+ 180.112, C9H14N3O+ required 180.113.  2‐cyanoethyl  (2‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)ethyl)  diisopropylphosphoramidite  (Compound 1)  [0665] Method  A  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (2‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)ethyl)  diisopropylphosphoramidite  from  2‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)ethan‐1‐ol  in 53% yield as a colourless gum. 
Figure imgf000137_0002
  [0666] 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 6.2, 1H), 6.69 (dd, J = 17.3, 10.4, 1H), 6.48 (dd, J = 17.3,  2.1, 1H), 6.32 (d, J = 6.2, 1H), 5.58 (dd, J = 10.4, 2.1, 1H), 3.87 – 3.71 (m, 6H), 3.57 (dt, J = 10.2, 6.8, 2H),  3.13 (s, 3H), 2.59 (dd, J = 6.3, 0.7, 2H), 1.16 (d, J = 6.8, 6H), 1.12 (d, J = 6.8, 6H).  [0667] 31P NMR (162 MHz, CDCl3) δ 148.0.  [0668] HRMS (ESI) m/z found [M+H]+ 380.222, C18H31N5O2P+ required 380.221.  (S)‐1‐(2‐chloropyrimidin‐4‐yl)pyrrolidin‐3‐ol  [0669] Method  A  Step  1  was  followed  to  synthesise  (S)‐1‐(2‐chloropyrimidin‐4‐yl)pyrrolidin‐3‐ol  starting from commercially available compounds 2,4‐dichloropyrimidine and (S)‐3‐hydroxypyrrolidine.  Flash  column  chromatography  (silica  gel,  0‐100%  Heptane/EtOAc)  afforded  compounds  (S)‐1‐(2‐ chloropyrimidin‐4‐yl)pyrrolidin‐3‐ol  (major  product)  in  55%  yield  as  a  white  solid  and  (S)‐1‐(4‐ chloropyrimidin‐2‐yl)pyrrolidin‐3‐ol (minor product) in 4% yield as a pale yellow solid.     
Figure imgf000138_0001
.06 (m,  1H), 4.61 – 4.22 (m, 1H), 3.62 – 3.02 (m, 4H), 2.16 – 1.63 (m, 2H).  [0672] HRMS (ESI) m/z found [M+H]+ 200.059, C8H11ClN3O+ required 200.058.  [0673] 1H NMR for (S)‐1‐(4‐chloropyrimidin‐2‐yl)pyrrolidin‐3‐ol (minor product)  [0674] 1H NMR (400 MHz, DMSO‐d6) δ 8.27 (s, J = 5.1, 1H), 6.67 (d, J = 5.1, 1H), 4.99 (s, 1H), 4.36 (tt,  J = 4.6, 2.4, 1H), 4.06 – 3.04 (m, 4H), 1.99 (dtd, J = 13.1, 8.9, 4.5, 1H), 1.88 (dddd, J = 12.7, 6.8, 3.7, 1.5,  1H).  (S)‐1‐(2‐vinylpyrimidin‐4‐yl)pyrrolidin‐3‐ol  [0675] Method  A  Step  2  Procedure  1  was  followed  to  synthesise  (S)‐1‐(2‐vinylpyrimidin‐4‐ yl)pyrrolidin‐3‐ol from (S)‐1‐(2‐chloropyrimidin‐4‐yl)pyrrolidin‐3‐ol in 60% yield as sticky yellow oil. 
Figure imgf000138_0002
  [0676] 1H NMR (400 MHz, DMSO‐d6) δ 8.13 (d, J = 6.0, 1H), 6.58 (dd, J = 17.3, 10.3, 1H), 6.40 (dd, J =  17.3, 2.5, 1H), 6.34 (d, J = 6.0, 1H), 5.56 (dd, J = 10.3, 2.5, 1H), 5.09 – 4.84 (m, 1H), 4.38 (s, 1H), 3.66 –  3.16 (m, 4H), 2.12 – 1.78 (m, 2H)  [0677] HRMS (ESI) m/z found [M+H]+ 192.114, C10H14N3O+ required 192.113    2‐cyanoethyl (1‐(2‐vinylpyrimidin‐4‐yl)pyrrolidin‐3‐yl) diisopropylphosphoramidite (Compound 3)  [0678] Method A Step 3 was followed to synthesise 2‐cyanoethyl (1‐(2‐vinylpyrimidin‐4‐yl)pyrrolidin‐ 3‐yl) diisopropylphosphoramidite  from  (S)‐1‐(2‐vinylpyrimidin‐4‐yl)pyrrolidin‐3‐ol  in 47%  yield  as  a  colourless sticky gum.   
Figure imgf000139_0001
  [0679] 1H NMR (400 MHz, CD3CN) δ 8.13 (dd, J = 6.0, 1.6, 1H), 6.62 (ddd, J = 17.3, 10.4, 3.0, 1H), 6.45  (ddd, J = 17.4, 5.2, 2.4, 1H), 6.28 (d, J = 5.8, 1H), 5.55 (dt, J = 10.4, 2.3, 1H), 4.66 – 4.61 (m, 1H), 3.85 –  3.74 (m, 2H), 3.67 – 3.52 (m, 4H), 2.63 (dt, J = 9.0, 6.0, 2H), 2.19 – 2.15 (m, 2H), 1.94 (t, J = 2.48, 2H),  1.17 (dd, J = 6.8, 2.9, 12H).  [0680] 31P NMR (162 MHz, CD3CN) δ 147.1.  [0681] HRMS (ESI) m/z found [M+H]+ 392.226, C19H31N5O2P+ required 392.221.  (R)‐1‐(4‐vinylpyrimidin‐2‐yl)pyrrolidin‐3‐ol 
Figure imgf000139_0002
  [0682] Method  A  Step  2  Procedure  2  was  followed  to  synthesise  (R)‐1‐(4‐vinylpyrimidin‐2‐ yl)pyrrolidin‐3‐ol  from  commercially  available  (R)‐1‐(4‐chloropyrimidin‐2‐yl)pyrrolidin‐3‐ol  in  45%  yield as yellow gum. Flash column chromatography (silica gel) was performed for purification using (0‐ 100%) Hexane‐EtOAc solvent system.  [0683] 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 5.1 Hz, 1H), 6.50 (dd, J = 17.4, 10.5 Hz, 1H), 6.40 (d, J =  5.1 Hz, 1H), 6.28 (dd, J = 17.4, 1.6 Hz, 1H), 5.48 (dd, J = 10.5, 1.6 Hz, 1H), 4.62 – 4.37 (m, 1H), 3.77 –  3.48 (m, 4H), 2.79 (brs, 1H), 2.12 – 1.84 (m, 2H).  [0684] HRMS (ESI) m/z found [M+H]+ 192.119, C10H14N3O+ required 192.113.            2‐cyanoethyl ((R)‐1‐(4‐vinylpyrimidin‐2‐yl)pyrrolidin‐3‐yl) diisopropylphosphoramidite   
Figure imgf000140_0001
  [0685] Method  A  Step  3  was  followed  to  synthesise  2‐cyanoethyl  ((R)‐1‐(4‐vinylpyrimidin‐2‐ yl)pyrrolidin‐3‐yl)  diisopropylphosphoramidite  from  (R)‐1‐(4‐vinylpyrimidin‐2‐yl)pyrrolidin‐3‐ol    in  74% yield as colourless gum.  [0686] 1H NMR (400 MHz, CD3CN) δ 8.29 (dd, J = 5.0, 2.7 Hz, 1H), 6.76 – 6.54 (m, 2H), 6.41 (ddd, J =  17.4, 4.5, 1.8 Hz, 1H), 5.58 (ddd, J = 10.5, 2.4, 1.7 Hz, 1H), 3.98 – 3.43 (m, 9H), 2.73 – 2.59 (m, 2H), 2.18  – 2.10 (m, 2H), 1.21 – 1.14 (m, 12H).  [0687] 31P NMR (162 MHz, CD3CN) δ 147.05, 146.98.  1‐(2‐chloropyrimidin‐4‐yl)piperidin‐4‐ol 
Figure imgf000140_0002
  [0688] Method A Step 1 was followed to synthesise 1‐(2‐chloropyrimidin‐4‐yl)piperidin‐4‐ol starting  from    commercially  available  compounds  2,4‐dichloropyrimidine  and  piperidin‐4‐ol.  Flash  column  chromatography  (silica  gel,  0‐100%  Heptane/EtOAc)  afforded  compounds  1‐(2‐chloropyrimidin‐4‐ yl)piperidin‐4‐ol (major product) in 65% yield as a white solid and 1‐(4‐chloropyrimidin‐2‐yl)piperidin‐ 4‐ol (minor product) in 8% yield as a pale yellow gum.  [0689] 1H NMR for 1‐(2‐chloropyrimidin‐4‐yl)piperidin‐4‐ol (major product)  [0690] 1H NMR (400 MHz, MeOD) δ 7.96 (d, J = 6.3 Hz, 1H), 6.71 (d, J = 6.4 Hz, 1H), 4.28 – 3.98 (m,  2H), 3.93 (tt, J = 8.3, 3.9 Hz, 1H), 3.43 – 3.33 (m, 2H), 2.00 – 1.87 (m, 2H), 1.62 – 1.44 (m, 2H). [One  exchangeable proton of OH was not observed in methanol‐d4].  [0691] HRMS (ESI) m/z found [M+H]+ 214.077, C9H13ClN3O+ required 214.074.  [0692] 1H NMR of 1‐(4‐chloropyrimidin‐2‐yl)piperidin‐4‐ol (minor product)  [0693] 1H NMR (400 MHz, MeOD) δ 8.18 (d, J = 5.1 Hz, 1H), 6.56 (d, J = 5.1 Hz, 1H), 4.49 – 4.25 (m,  2H), 3.88 (tt, J = 8.7, 4.0 Hz, 1H), 3.33 (dt, J = 10.2, 3.3 Hz, 2H), 2.04 – 1.82 (m, 2H), 1.47 (dtd, J = 13.2,  9.4, 4.0 Hz, 2H). [One exchangeable proton of OH was not observed in methanol‐d4].  [0694] HRMS (ESI) m/z found [M+H]+ 214.071, C9H13ClN3O+ required 214.074.  1‐(2‐vinylpyrimidin‐4‐yl)piperidin‐4‐ol   
Figure imgf000141_0001
  [0695] Method A Step 2 Procedure 2 was followed to synthesise 1‐(2‐vinylpyrimidin‐4‐yl)piperidin‐ 4‐ol from 1‐(2‐chloropyrimidin‐4‐yl)piperidin‐4‐ol in 52% yield as pale brown gum.  [0696] 1H NMR (400 MHz, MeOD) δ 8.08 (t, J = 6.2 Hz, 1H), 7.74 – 7.26 (m, 1H), 6.60 – 6.51 (m, 2H),  6.47 (dd, J = 17.3, 2.1 Hz, 1H), 5.76 – 5.55 (m, 1H), 4.23 (d, J = 13.6 Hz, 2H), 3.55 – 3.07 (m, 3H), 1.98 –  1.91 (m, 2H), 1.61 – 1.41 (m, 2H).  [0697] HRMS (ESI) m/z found [M+H]+ 206.125, C11H16N3O+ required 206.129.  2‐cyanoethyl (1‐(2‐vinylpyrimidin‐4‐yl)piperidin‐4‐yl) diisopropylphosphoramidite 
Figure imgf000141_0002
  [0698] Method A Step 3 was followed to synthesise 2‐cyanoethyl (1‐(2‐vinylpyrimidin‐4‐yl)piperidin‐ 4‐yl)  diisopropylphosphoramidite  from  1‐(2‐vinylpyrimidin‐4‐yl)piperidin‐4‐ol  in  78%  yield  as  colourless gum.  [0699] 1H NMR (400 MHz, CD2Cl2) δ 8.12 – 8.01 (m, 1H), 6.63 – 6.48 (m, 1H), 6.37 (dd, J = 17.3, 2.3 Hz,  1H), 6.32 (d, J = 6.2 Hz, 1H), 5.53 – 5.41 (m, 1H), 4.15 – 3.95 (m, 1H), 3.87 – 3.62 (m, 4H), 3.63 – 3.34  (m, 4H), 2.54 (t, J = 6.2 Hz, 2H), 1.86 – 1.75 (m, 2H), 1.69 – 1.55 (m, 2H), 1.11 (d, J = 6.8 Hz, 12H).  [0700] 31P NMR (162 MHz, CD2Cl2) δ 146.39.  3‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclobutan‐1‐ol 
Figure imgf000141_0003
  [0701] Method  A  Step  1  was  followed  to  synthesise  3‐((2‐chloropyrimidin‐4‐ yl)(methyl)amino)cyclobutan‐1‐ol  starting  from  commercially  available  compounds  2,4‐ dichloropyrimidine  and  3‐(methylamino)cyclobutanol.  Flash  column  chromatography  (silica  gel,  0‐ 100% Heptane/EtOAc) afforded compounds 3‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclobutan‐1‐   ol  (major  product)  in  62%  yield  as  a  sticky  white  solid  and  3‐((4‐chloropyrimidin‐2‐ yl)(methyl)amino)cyclobutan‐1‐ol (minor product) in 5% yield as yellow gum.  [0702] 1H NMR for 3‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclobutan‐1‐ol (major product)   [0703] 1H NMR (400 MHz, CDCl3) δ 7.89 (dd, J = 7.4, 5.9 Hz, 1H), 6.34 – 6.19 (m, 1H), 4.68 – 4.32 (m,  1H), 4.11 – 4.03 (m, 1H), 2.97 (d, J = 5.2 Hz, 3H), 2.69 – 2.61 (m, 2H), 2.46 – 2.24 (m, 1H), 2.22 – 1.97  (m, 2H).  [0704] HRMS (ESI) m/z found [M+H]+ 214.079, C9H13ClN3O+ required 214.074.  [0705] 1H NMR for 3‐((4‐chloropyrimidin‐2‐yl)(methyl)amino)cyclobutan‐1‐ol (minor product)  [0706] 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 5.1 Hz, 1H), 6.43 (dd, J = 5.0, 1.0 Hz, 1H), 4.55 (tt, J =  9.7, 7.5 Hz, 1H), 4.11 – 3.97 (m, 1H), 3.05 (d, J = 6.7 Hz, 3H), 2.66 ‐ 2.58 (m, 2H), 2.21 – 1.97 (m, 3H).  [0707] HRMS (ESI) m/z found [M+H]+ 214.078, C9H13ClN3O+ required 214.074.  3‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)cyclobutan‐1‐ol 
Figure imgf000142_0001
  [0708] Method  A  Step  2  Procedure  2  was  followed  to  synthesise  3‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)cyclobutan‐1‐ol from 3‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclobutan‐1‐ol in 50% yield  as pale brown gum.  [0709] 1H NMR (400 MHz, CDCl3) δ 8.09 (t, J = 6.0 Hz, 1H), 6.67 (dd, J = 17.3, 10.4 Hz, 1H), 6.56 – 6.40  (m, 1H), 6.32 – 6.15 (m, 1H), 5.59 (dd, J = 10.4, 2.0 Hz, 1H), 4.55 – 4.30 (m, 1H), 4.10 (tt, J = 7.6, 6.5 Hz,  1H), 3.17 – 2.83 (m, 4H), 2.73 – 2.62 (m, 2H), 2.15 – 2.06 (m, 2H).  [0710] HRMS (ESI) m/z found [M+H]+ 206.128, C11H16N3O+ required 206.129.  2‐cyanoethyl (3‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)cyclobutyl) diisopropylphosphoramidite 
Figure imgf000142_0002
  [0711] Method  A  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (3‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)cyclobutyl)  diisopropylphosphoramidite  from  3‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)cyclobutan‐1‐ol in 79% yield as colourless gum.    [0712] 1H NMR (400 MHz, CD2Cl2) δ 8.18 (d, J = 6.1 Hz, 1H), 6.80 – 6.63 (m, 1H), 6.60 – 6.43 (m, 1H),  6.43 – 6.19 (m, 1H), 5.58 (dd, J = 10.4, 2.3 Hz, 1H), 4.67 – 4.41 (m, 1H), 4.27 – 4.09 (m, 1H), 3.92 – 3.73  (m, 2H), 3.70 – 3.60 (m, 2H), 3.05 (d, J = 5.6 Hz, 3H), 2.91 – 2.53 (m, 4H), 2.39 – 2.16 (m, 2H), 1.27 –  1.17 (m, 12H).  [0713] 31P NMR (162 MHz, CD2Cl2) δ 145.70, 145.59.  (3‐(6‐vinyl‐9H‐purin‐9‐yl)phenyl)methanol 
Figure imgf000143_0001
  [0714] To a solution of (3‐(6‐chloro‐9H‐purin‐9‐yl)phenyl)methanol (120 mg, 0.46 mmol) and vinyl  boronate pinacol ester (3 eq, 0.25 mL) in Dioxane (5 mL) and water (0.5 mL) in microwave tube under  argon atmosphere was added K2CO3 (4 eq, 262 mg). The mixture was degassed for 5 mins. Then the  catalyst Pd(dppf)Cl2.DCM (0.1 eq, 38 mg) was added to the mixture and further degassed for 5 mins.  The tube was sealed and the reaction mixture was warmed up to 120 oC and stirred at this temperature  for 18 hours. The reaction mixture was cooled to room temperature, diluted with DCM (5 mL) and  filtered through a bed of celite washing several times with DCM (3 x 10 mL). Solvent was removed  under  vacuum.  The  residue  was  subjected  to  column  chromatography  (silica  gel,  0‐100%  Heptane/Ethyl acetate) to afford the product (3‐(6‐vinyl‐9H‐purin‐9‐yl)phenyl)methanol (60 mg, 50%)  as a pale yellow gum.  [0715] 1H NMR (400 MHz, MeOD) δ 8.87 (d, J = 7.5 Hz, 1H), 8.79 (s, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.75  (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.35 (dd, J = 17.5, 11.0 Hz, 1H), 7.05  (dd, J = 17.5, 1.7 Hz, 1H), 6.02 (dd, J = 11.0, 1.7 Hz, 1H), 4.76 (s, 2H). (One exchangeable proton of OH  was not observed in methanol‐d4).  [0716] HRMS (ESI) m/z found [M+H]+ 253.109, C14H13N4O+ required 253.108.  2‐cyanoethyl (3‐(6‐vinyl‐9H‐purin‐9‐yl)benzyl) diisopropylphosphoramidite 
Figure imgf000143_0002
  [0717] Method A Step 3 was  followed to synthesise 2‐cyanoethyl  (3‐(6‐vinyl‐9H‐purin‐9‐yl)benzyl)  diisopropylphosphoramidite  from  (3‐(6‐vinyl‐9H‐purin‐9‐yl)phenyl)methanol  in  76%  yield  as  colourless gum.  [0718] 1H NMR (400 MHz, CD2Cl2) δ 8.81 (d, J = 13.3 Hz, 1H), 8.30 (s, 1H), 7.77 – 7.69 (m, 1H), 7.64 –  7.53 (m, 1H), 7.53 – 7.43 (m, 1H), 7.37 (dt, J = 7.9, 1.2 Hz, 1H), 7.26 (dd, J = 17.5, 10.9 Hz, 1H), 6.98 (dd,  J = 17.5, 1.8 Hz, 1H), 5.98 – 5.79 (m, 1H), 4.86 – 4.62 (m, 2H), 3.85 – 3.71 (m, 2H), 3.64 ‐ 3.53 (m, 2H),  2.64 – 2.47 (m, 2H), 1.17 – 1.07 (m, 12H).  [0719] 31P NMR (162 MHz, CD2Cl2) δ 148.79. 
Figure imgf000144_0001
  2‐((4‐hydroxycyclohexyl)(methyl)amino)pyrimidine‐5‐carbaldehyde  [0720] Method  B  Step  2  was  followed  to  synthesise  2‐((4‐ hydroxycyclohexyl)(methyl)amino)pyrimidine‐5‐carbaldehyde  from  commercially  available  2‐ chloropyrimidine‐5‐carbaldehyde and 4‐(methylamino)cyclohexan‐1‐ol  in 65% yield as yellow gum.  Flash column chromatography (silica gel) was performed for purification using (0‐100%) Hexane‐EtOAc  solvent system.  [0721] 1H NMR (400 MHz, CDCl3) δ 9.70 (s, 1H), 8.67 (s, 2H), 4.75 (tt, J = 11.7, 4.0 Hz, 1H), 3.60 (tt, J =  10.8, 4.3 Hz, 1H), 3.07 (s, 3H), 2.65 (s, 1H), 2.12 – 2.01 (m, 2H), 1.81 – 1.40 (m, 6H).  [0722] HRMS (ESI) m/z found [M+H]+ 236.149, C12H18N3O2 + required 236.140.  4‐((5‐(2,2‐difluorovinyl)pyrimidin‐2‐yl)(methyl)amino)cyclohexan‐1‐ol 
Figure imgf000144_0002
  [0723] 2‐((4‐hydroxycyclohexyl)(methyl)amino)pyrimidine‐5‐carbaldehyde (165 mg, 0.70 mmol) and  triphenylphosphine  (2 eq, 368 mg) were dissolved  in DMF  (2.5 mL) under argon atmosphere and  heated to 100 oC. Sodium chlorodifluoroacetate (3 eq, 314 mg) was added portionwise at 100 oC and  thereactionmixturewasstirredatthistemperaturefor24hours.Thereactionmixturewascooledto   room temperature and solvent was removed under vacuum. The residue was dissolved in DCM (10  mL) and washed with water (2.5 mL), 3% H2O2 in water (2.5 mL) and brine (2.5 mL). The organic layer  was dried over sodium sulphate and solvent was removed under vacuum. Column chromatography  with  (0‐100%)  Heptane/Ethyl  acetate afforded  4‐((5‐(2,2‐difluorovinyl)pyrimidin‐2‐ yl)(methyl)amino)cyclohexan‐1‐ol (76 mg, 45%) as a pale brown oil.   [0724] 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 2H), 4.99 (dd, J = 27.3, 2.8 Hz, 1H), 4.56 (tt, J = 11.7, 4.0  Hz, 1H), 3.55 (tt, J = 10.6, 4.3 Hz, 1H), 2.93 (s, 3H), 2.07 – 1.80 (m, 3H), 1.78 – 1.60 (m, 2H), 1.59 – 1.33  (m, 4H).  [0725] 19F NMR (376 MHz, CDCl3) δ ‐83.19 (dd, J = 36.1, 27.2 Hz, 1F), ‐85.40 (dd, J = 36.1, 2.7 Hz, 1F).  [0726] HRMS (ESI) m/z found [M+H]+ 270.148, C13H18F2N3O+ required 270.141.  2‐cyanoethyl  (4‐((5‐(2,2‐difluorovinyl)pyrimidin‐2‐yl)(methyl)amino)cyclohexyl)  diisopropylphosphoramidite 
Figure imgf000145_0001
  [0727] Method B Step 3 was followed to synthesise 2‐cyanoethyl (4‐((5‐(2,2‐difluorovinyl)pyrimidin‐ 2‐yl)(methyl)amino)cyclohexyl)  diisopropylphosphoramidite  from  4‐((5‐(2,2‐difluorovinyl)pyrimidin‐ 2‐yl)(methyl)amino)cyclohexan‐1‐ol in 75% yield as colourless gum.  [0728] 1H NMR (400 MHz, CD2Cl2) δ 8.18 (s, 2H), 5.01 (dd, J = 27.6, 3.0 Hz, 1H), 4.54 (tt, J = 11.3, 4.1  Hz, 1H), 3.80 – 3.61 (m, 3H), 3.57 – 3.48 (m, 2H), 2.90 (s, 3H), 2.55 (t, J = 6.3 Hz, 2H), 2.15 – 1.87 (m,  2H), 1.67 – 1.40 (m, 6H), 1.10 (dd, J = 6.9, 1.2 Hz, 12H).  [0729] 31P NMR (162 MHz, CD2Cl2) δ 145.90.  [0730] 19F NMR (376 MHz, CD2Cl2) δ ‐84.16 (dd, J = 37.8, 27.6 Hz, 1F), ‐86.60 (dd, J = 38.1, 3.4 Hz, 1F).  3‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclopentan‐1‐ol 
Figure imgf000145_0002
  [0731] Method  A  Step  1  was  followed  to  synthesise  3‐((2‐chloropyrimidin‐4‐ l)( th l) i ) l t 1 l t ti f i ll il bl d 24   dichloropyrimidine  and  3‐(methylamino)cyclopentan‐1‐ol  hydrochloride.  Flash  column  chromatography  (silica  gel,  0‐100% Heptane/EtOAc)  afforded  compounds  3‐((2‐chloropyrimidin‐4‐ yl)(methyl)amino)cyclopentan‐1‐ol  (major  product)  in  68%  yield  as  a  sticky white  solid  and  3‐((4‐ chloropyrimidin‐2‐yl)(methyl)amino)cyclopentan‐1‐ol (minor product) in 5% yield as yellow solid.  [0732] 1H NMR for 3‐((2‐chloropyrimidin‐4‐yl)(methyl)amino)cyclopentan‐1‐ol (major product)  [0733] 1H NMR (400 MHz, MeOD) δ 7.96 (dd, J = 6.3, 3.2 Hz, 1H), 6.70 – 6.50 (m, 1H), 4.48 – 4.25 (m,  1H), 3.04 (s, 1.5H) 2.95 (s, 1.5H), 2.25 (ddd, J = 14.0, 9.5, 5.9 Hz, 1H), 2.16 – 2.05 (m, 1H), 1.97 – 1.84  (m, 2H), 1.79 (dt, J = 10.4, 5.3 Hz, 1H), 1.77 – 1.46 (m, 2H). [One exchangeable proton of OH was not  observed in methanol‐d4].  [0734] HRMS (ESI) m/z found [M+H]+ 228.709, C10H15ClN3O+ required 228.700.  [0735] 1H NMR for 3‐((4‐chloropyrimidin‐2‐yl)(methyl)amino)cyclopentan‐1‐ol (minor product)  [0736] 1H NMR (400 MHz, CDCl3) δ 8.07 (t, J = 5.2 Hz, 1H), 6.41 (dd, J = 7.5, 5.1 Hz, 1H), 4.68 (p, J = 9.2  Hz, 1H), 4.26 (tt, J = 5.1, 2.5 Hz, 1H), 3.09 (s, 1.5H), 2.92 (s, 1.5H),  2.19 (ddd, J = 14.3, 10.3, 5.9 Hz,  0.5H), 2.01 (dddd, J = 12.6, 10.0, 7.9, 5.4 Hz, 1.5H), 1.92 – 1.49 (m, 5H).  [0737] HRMS (ESI) m/z found [M+H]+ 228.698, C10H15ClN3O+ required 228.700.  3‐(methyl(2‐vinylpyrimidin‐4‐yl)amino)cyclopentan‐1‐ol       
Figure imgf000146_0001
  [0741] Method  A  Step  3 was  followed  to  synthesise  2‐cyanoethyl  (3‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)cyclopentyl)  diisopropylphosphoramidite  from  3‐(methyl(2‐vinylpyrimidin‐4‐ yl)amino)cyclopentan‐1‐ol in 50% yield as colourless gum.  [0742] 1H NMR (400 MHz, CD2Cl2) δ 8.16 – 7.96 (m, 1H), 6.69 – 6.50 (m, 1H), 6.50 – 6.28 (m, 1H), 6.30  – 6.11 (m, 1H), 5.47 – 5.37 (m, 1H), 5.31 – 5.11 (m, 1H), 4.44 – 4.27 (m, 1H), 3.82 – 3.62 (m, 2H), 3.59  – 3.49 (m, 2H), 2.89 – 2.79 (m, 3H), 2.64 – 2.45 (m, 2H), 2.18 (dddd, J = 14.2, 9.7, 5.9, 2.0 Hz, 1H), 2.03  – 1.89 (m, 1H), 1.85 – 1.57 (m, 4H), 1.13 – 1.09 (m, 12H).  [0743] 31P NMR (162 MHz, CD2Cl2) δ 146.78, 146.53.    Example 2: Modified Oligonucleotides  General method for oligonucleotide synthesis and linker‐oligonucleotide conjugation  [0744] DNA phosphoramidites were dissolved in acetonitrile (0.08M). Linker phosphoramidites were  dissolved in a mixture 50:50 THF:DCM (0.08M). Solvents were moisture controlled with less than 30  ppm water content.   [0745] The oligonucleotide synthesis was performed using conventional solid phase synthesis on a  base‐functionalised solid support. This procedure consisted of deblocking (detritylation) using an acid  solution (normally TCA 3% in DCM); activation/coupling of the phosphoramidite using a tetrazole‐like  reagent  such  as  BTT  or  ETT;  capping  using  a  solution  containing  acetic  anhydride  and  N‐ methylimidazole; and oxidation using iodine in a THF/pyridine/water solution or sulphurisation.   [0746] For the addition of the linker to the 5’ end of oligonucleotide, the synthetic cycle was modified  removing the capping steps and the final addition of TCA since there was no DMT group and to avoid  degradation of the linker compound. Also, the coupling time for the phosphoramidites were increased  to 8 mins using 3 consecutive coupling steps.  [0747] To exemplify, the following oligonucleotides (DNA sequences) were used during the synthesis.  Oligo 1 – SEQ. ID NO: 1 – CGACGCTTGCAGCT  Oligo 2 – SEQ. ID NO: 2 – CTACACTTCCATCT  [0748] The linker addition to the oligonucleotide was performed using the following compounds.        
Figure imgf000147_0001
 
Figure imgf000148_0001
 
Figure imgf000149_0001
 
Figure imgf000150_0001
  [0749] The conjugation and reactivity of Oligo 1 and Oligo 2 with compounds of the invention were  tested as set out in Example 3.  General method for the deprotection (cleavage from the solid support and removal of the protecting  groups) of linker‐conjugated oligonucleotides.  Method A:  [0750] Representative example: Deprotection of Compound 5‐Oligo1 conjugate  [0751] 5 mg of Compound 5‐Oligo1 conjugate on CPG solid support was placed in a vial, treated with  100 μL of 0.4 M NaOH in MeOH/water (4:1) solution and shaken (Eppendorf ThermoMixerC) for 20  min at 80 °C. The sample was cooled down to room temperature, spun down and filtered. The filtrate  was  frozen  and  freeze‐dried.  The  sample was  desalted  on  Glen  Gel‐Pak™  0.2  Desalting  Column  (catalogue no 61‐5002‐50) following the manufacturer’s protocol. The collected sample was frozen,    LCMS (Waters LCMS system with Acquity QDa detector, ACQUITY PREMIER Oligonucleotide BEH C18  column (130 Å; 2.1 x 50 mm, 1.7 µm; Waters) at 65 °C with a flow rate of 0.3 mL/min. Eluent A: 7 mM  TEA, 80 mM HFIP in water; eluent B: 3.5 mM TEA, 40 mM HFIP in 50% ACN; gradient 5 ‐ 30% B in 8  min).  [0752] Compound 5_oligo 1: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4534.07, mass found: 4534.09.  Method B:  [0753] Representative example: Deprotection of Compound 7‐Oligo2 conjugate.  [0754] 5 mg of Compound 7 ‐ Oligo 2 conjugate on CPG solid support was placed in vial, treated with  100  μL  of  32%  aq.  ammonia  solution  and  shaken  (Eppendorf  ThermoMixerC)  overnight  at  room  temperature. Sample was spun down and filtered. The filtrate was frozen and freeze‐dried. Sample  was  desalted  on  Glen  Gel‐Pak™  0.2  Desalting  Column  (catalogue  no  61‐5002‐50)  following  manufacturer’s protocol. Collected sample was frozen, freeze‐dried and analysed on LCMS. Sample  was dissolved  in 150 µL of water and analysed on  LCMS  (Waters  LCMS  system with Acquity QDa  detector, ACQUITY PREMIER Oligonucleotide BEH C18 column (130 Å; 2.1 x 50 mm, 1.7 µm; Waters)  at 65 °C with a flow rate of 0.3 mL/min. Eluent A: 7 mM TEA, 80 mM HFIP in water; eluent B: 3.5 mM  TEA, 40 mM HFIP in 50% ACN; gradient 5 ‐ 30% B in 8 min).  [0755] Compound 7_oligo 2: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4371.77, mass found: {[M‐H]‐ +H2O} 4389.77.  Specific deprotection examples:  [0756] Following Method B, Compound 1‐Oligo2 was deprotected and analysed.  [0757] Compound 1_oligo 2: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4371.77, mass found: {[M‐H]‐ +H2O} 4389.75.  [0758] Following Method A, Compound 5 – oligo 2 conjugate was deprotected and analysed.  [0759] Compound 5_oligo 2: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4428.02, mass found: 4428.09.  [0760] Following Method A, Compound 6 – oligo 2 conjugate was deprotected and analysed.  [0761] Compound 6_oligo 2: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4428.02, mass found: {[M‐H]‐ +MeOH} 4460.03.  [0762] Following Method A, Compound 6 – oligo 1 conjugate was deprotected and analysed.  [0763] Compound 6_oligo 1: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4534.07, mass found: {[M‐H]‐ +MeOH} 4566.12.  [0764] Following Method B, Compound 9‐oligo 1 conjugate was deprotected and analysed.  [0765] Compound 9_oligo 1: LCMS (ESI‐) m/z [M‐H]‐ mass calculated: 4520.05, mass found: 4520.09.  [0766] Following Method B, Compound 9‐oligo 2 conjugate was deprotected and analysed.  [0767] Compound9 oligo2:LCMS(ESI‐)m/z[M‐H]‐masscalculated:441401 massfound:441409 [0768] Following Method A, Compound 8-oligo 1 conjugate was deprotected and analysed.
[0769] Compound 8_oligo 1: LCMS (ESI-) m/z [M-H]' mass calculated: 4506.02, mass found: 4506.09.
[0770] Following Method A, Compound 8-oligo 2 conjugate was deprotected and analysed.
[0771] Compound 8_oligo 2: LCMS (ESI-) m/z [M-H]' mass calculated: 4400.00, mass found: 4400.08.
[0772] Following Method B, Compound 3-oligo 1 conjugate was deprotected and analysed.
[0773] Compound 3_oligo 1: LCMS (ESI-) m/z [M-H]' mass calculated: 4479.01, mass found: 4479.03.
[0774] Following Method A, Compound 4 - oligo 2 conjugate was deprotected and analysed.
[0775] Compound 4_oligo 2: LCMS (ESI-) m/z [M-H]' mass calculated: 4371.69, mass found: 4371.68.
[0776] Following Method A, Compound 2 - oligo 2 conjugate was deprotected and analysed.
[0777] Compound 2_oligo 2: LCMS (ESI-) m/z [M-H]' mass calculated: 4387.01, mass found: 4387.08.
[0778] Following Method B, Compound 3_Oligo 2 conjugate was deprotected and analysed.
[0779] Compound 3_Oligo 2: LCMS (ESI ) m/z [M]' mass found: 4371.03, mass calculated: 4371.76.
[0780] Following Method A, Compound 7_Oligo 1 conjugate was deprotected and analysed.
[0781] Compound 7_Oligo 1: LCMS (ESI ) m/z {[M]'+MeOH]} mass found: 4511.73, mass calculated: 4511.90.
[0782] Following Method A, Compound 10_Oligo 1 conjugate was deprotected and analysed.
[0783] Compound 10_Oligo 1: LCMS (ESI-) m/z [M-H]- mass found: 4504.50, mass calculated:
4504.96.
[0784] Following Method A, Compound 12_Oligo 1 conjugate was deprotected and analysed.
[0785] Compound 12_oligo 1: LCMS (ESI ) m/z [M]' mass found: 4505.03, mass calculated: 4505.95.
[0786] Following Method A, Compound 12_Oligo 2 conjugate was deprotected and analysed.
[0787] Compound 12_oligo 2: LCMS (ESI ) m/z [M-H]' mass found: 4398.50, mass calculated: 4398.94.
[0788] Following Method A, Compound ll_Oligo 1 conjugate was deprotected and analysed.
[0789] Compound ll_oligo 1: LCMS (ESI ) m/z [M]' mass found: 4491.80, mass calculated: 4491.96.
[0790] Following Method A, Compound ll_Oligo 2 conjugate was deprotected and analysed.
[0791] Compound ll_oligo 2: LCMS (ESI ) m/z [M]' mass found: 4385.50, mass calculated: 4385.91.
[0792] Following Method A, Compound 14_Oligo 1 conjugate was deprotected and analysed.
[0793] Compound 14_oligo 1: LCMS (ESI ) m/z {[M]'+MeOH} mass found: 4585.80, mass calculated:
4585.95.
[0794] Following Method A, Compound 14_Oligo 2 conjugate was deprotected and analysed.
[0795] Compound 14_oligo 2: LCMS (ESI ) m/z {[M]'+MeOH} mass found: 4478.50, mass calculated:
4478.96.
[0796] Following Method B, Compound 15_Oligo 1 conjugate was deprotected and analysed.
[0797] Compound 15_oligo 1: LCMS (ESI ) m/z [M]' mass found: 4570.00, mass calculated: 4569.99. [0798] Following Method A, Compound 15_Oligo 2 conjugate was deprotected and analysed.
[0799] Compound 15_oligo 2: LCMS (ESI ) m/z {[M] +MeOH } mass found: 4495.60, mass calculated: 4495.98.
[0800] Following Method A, Compound 13_Oligo 1 conjugate was deprotected and analysed.
[0801] Compound 13_oligo 1: LCMS (ESI ) m/z [M-H]' mass found: 4504.50, mass calculated 4504.96. [0802] Following Method A, Compound 13_Oligo 2 conjugate was deprotected and analysed.
[0803] Compound 13_oligo 2: LCMS (ESI ) m/z [M]’ mass found: 4399.50, mass calculated 4399.94.
Example 3: Thiol-Ene Click Reactions
Method A
Representative example: Click reaction with Compound 5_oligo 1.
[0804] A solution of 100 mM P-mercaptoethanol (P-ME) in the conjugation buffer was freshly prepared: 1.4 pL of P-mercaptoethanol was added to 200 pL of 5x PBS (phosphate-buffered saline) buffer. Then, the oligo sample (dissolved in around 50 pL of water) was mixed with 50 pL of water and 25 pL of conjugation buffer (with P-mercaptoethanol) and incubated for 2 h at 37 °C. LCMS (conditions as described previously) analysis was performed after 1 h (by mixing 10 pL of sample with 10 pL of water).
[0805] Compound 5_oligo 1 + P-ME: LCMS (ESI) m/z {[M-H]+P-ME}_ mass calculated: 4611.20, mass found: 4611.27.
Method B
General Method for Thiol-ene click reaction with Glutathione
[0806] The glutathione (GSH) reaction was performed by mixing 50 pM in IX PBS with 250 pM of the freshly prepared glutathione solution in water. The reaction was prepared in a vial by mixing 24 pL of the linker containing oligo (83.4 pM in water), 8 pL of 5X PBS (prepared from tablets), and 8 pL of the freshly prepared glutathione solution (1.25 mM in water) at 37 - 55 °C for 6 - 24 hours.
Representative example: Click reaction with Compound 2-oligo2
[0807] The glutathione (GSH) reaction was performed, according to Method B, by mixing 50 pM in IX PBS with 250 pM of the freshly prepared glutathione solution in water. The reaction was prepared in a vial by mixing 24 pL of Compound 2_oligo 2 (83.4 pM in water), 8 pL of 5X PBS (prepared from tablets), and 8 pL of the freshly prepared glutathione solution (1.25 mM in water).
[0808] The reaction was monitored by HPLC every 30 minutes. For this, HPLC was performed using a Waters Acquity system equipped with an ACQUITY PREMIER Oligonucleotide BEH C18 (Pore size: 130 A, particle size: 1.70 pm, Inner diameter:2.1 mm, length: 50 mm). Buffer A: 7 mM Et3N 80 mM hexafluoro-2-propanol (HFIP) in water, Buffer B: Acetonitrile. 0%B to 15%B in 8 min at 0.5 mL/min. Injection volume = 2 uL. The HPLC time course monitoring is shown in Figure 1.
[0809] The area under the oligo peak and the product (oligo-protein conjugate) peak was automeasured using the integrate function on MassLynx. The area under curve for the oligo peak and oligo- protein conjugate versus time is shown in Figure 2. The mass spectra of the two peaks were determined using the LCMS (conditions as described previously) for the unconjugated oligo [M-H]': 4387.01 and for the conjugated oligonucleotide [M-H+GSH]': 4693.39 confirming that the conjugate formed. A control of oligo 50 pM in 1XPBS was also analyzed in between each reaction sample. No reduction in the oligo peak was observed for the control experiments.
[0810] Compound 2_oligo2_GSH: LCMS (ESI-) m/z [M-H+GSH]' mass calculated: 4693.33, mass found: 4693.39.
[0811] Following Method A, Compound 5_oligo 2 was subjected to the thiol-ene click reaction and analysed.
[0812] Compound 5_oligo 2 + P-ME: LCMS (ESI ) m/z {[M]+P-ME}‘ mass found: 4506.98, mass calculated: 4506.15.
[0813] Following Method A, Compound 6_oligo 1 was subjected to the thiol-ene click reaction and analysed.
[0814] Compound 6_oligo 1 + P-ME: LCMS (ESI ) m/z {[M]+P-ME}‘ mass found: 4612.90, mass calculated: 4612.25.
[0815] Following Method A, Compound 6_oligo 2 was subjected to the thiol-ene click reaction and analysed.
[0816] Compound 6_oligo 2 + P-ME: LCMS (ESI ) m/z {[M]+P-ME}‘ mass found: 4506.00, mass calculated: 4506.15.
[0817] Following Method A, Compound 7_oligo 1 was subjected to the thiol-ene click reaction and analysed.
[0818] Compound 7_oligo 1 + P-ME: LCMS (ESI ) m/z {[M]+P-ME}- mass found: 4506.00, mass calculated: 4506.15.
[0819] Following Method B, Compound 5_oligo 2 was subjected to the thiol-ene click reaction and analysed.
[0820] Compound 5_oligo 2 + GSH: LCMS (ESI ) m/z [M+GSH]' mass found: 4735.98, mass calculated: 4735.30.
[0821] Following Method B, Compound 12_Oligo 1 was subjected to the thiol-ene click reaction and analysed. [0822] Compound 12_Oligo 1 + GSH: LCMS (ESI ) m/z [M+GSH]' mass found: 4813.30, mass calculated: 4813.00.
[0823] Following Method B, Compound ll_Oligo 1 was subjected to the thiol-ene click reaction and analysed.
[0824] Compound ll_Oligo 1 + GSH: LCMS (ESI-) m/z [M+GSH]' mass found: 4799.60, mass calculated: 4799.28.
[0825] Following Method B, Compound 14_Oligo 1 was subjected to thiol-ene click reaction (in this case the reaction was carried out at an elevated temperature of 75 °C) and analysed.
[0826] Compound 14_Oligo 1 + GSH: LCMS (ESI ) m/z [M+GSH]' mass found: 4860.80, mass calculated: 4860.25.
Conjugation of linker-oligonucleotide with Human Serum Albumen (HSA) protein (Compound 2- oligo2 and HSA)
[0827] The reaction with HSA was performed using Dithiothreitol (DTT, 3.62 pL, 5 mM in IX PBS) in a solution of HSA (35.8 pL, 253 pM) in PBS (200 pL, xl). The resulting solution was vortexed and incubated at 37 °C for 2 h. Removal of excess reagents and buffer exchange to the required solvent was achieved by repeated ultracentrifugation into IX PBS using an Amicon Ultra centrifugal filter (10k MWCO, Merck Millipore)
[0828] Electrophoresis was performed using a NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well with IX NuPAGE MES SDS running buffer.
[0829] The 0.2 ug/well samples were prepared as follows:
[0830] 1 pL of the reaction was diluted with 100 pL of water and 33.3 pL of 4X NuPage LDS sample buffer was added. The sample was heated to 70 °C for 10 min, cooled to room temperature, and 10 pL was loaded per well on to the gel.
[0831] For 1 ug/well, samples were prepared as follows:
[0832] 1 pL was diluted with 20 pL of water and 7 pL of the 4X NuPage LDS sample buffer was added. The sample was heated to 70 °C for 10 min, cooled to room temperature, and 10 pL was loaded per well on to the gel.
[0833] After electrophoresis, the gel was stained using 20 mL of SimpleBlueTM SafeStain, with gentle rocking for 2 h and washed three times with 15 mL water for 15 min.
[0834] The SDS-PAGE for the formation of the Compound 2-Oligo2+HAS is shown in Figure 3.
[0835] The lanes of the SDS-page display (from left to right):
1. 3 uL of Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards #1610375 from
BioRad 2. 0.2 ug of HSA incubated over the weekend in IX PBS.
3. 0.2 ug of HSA incubated with 1 eq of Compound 2-Oligo2 in IX PBS over the weekend.
4. 0.2 ug of HSA incubated with 10 eq of Compound 2-Oligo2 in IX PBS over the weekend.
5. 0.2 ug of HSA incubated with 50 eq of Compound 2-Oligo2 in IX PBS over the weekend. 6. 50 eq of Compound 2-Oligo2 in IX PBS over the weekend.
7. 3 uL of Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards #1610375 from BioRad
8. 1 ug of HSA incubated over the weekend in IX PBS.
9. 1 ug of HSA incubated with 1 eq of Compound 2-Oligo2 in IX PBS over the weekend. 10. 1 ug of HSA incubated with 10 eq of Compound 2-Oligo2 in IX PBS over the weekend.
11. 1 ug of HSA incubated with 50 eq of Compound 2-Oligo2 in IX PBS over the weekend.
12. 50 eq of Compound 2-Oligo2 in IX PBS over the weekend.
[0836] The gel shows the formation of a second band in lanes 4, 5, 10, and 11 (the oligo-protein conjugate) appearing with increasing intensity just above the HSA. The results are consistent with similar experiments in the literature (References: Bioconjugate Chem. 2022, 33, 1254-1260, Chem. Sci. 2021, 12, 9060-9068).

Claims

  CLAIMS  1.  A compound of formula (I): 
Figure imgf000157_0001
  A is mono‐ or bicyclic heteroaryl group in which at least one carbon ring‐atom is replaced with N;  X, Y, and Z are each independently selected from C1‐30 alkyl, C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m,  C(O), C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, C5‐10 heteroaryl, or absent;  wherein at least one of X, Y and Z is present;  wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl or C5‐10 heteroaryl is  optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6,  C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3;   n is 1 or 2;  m is an integer from 1‐30;   each   is either a single bond or double bond;  W is OR2 or NR3R4;  R1 and R2 are each independently selected from H and a base labile protecting group;  R3 and R4 are each independently selected from H and an optionally substituted C1‐10 alkyl;  R5 is absent, is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl;  R6 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R7 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R8 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R9, is selected from H, C1‐10 alkyl, halide, C(O)C1‐10 alkyl, C1‐10 haloalkyl, C(O)OR12, C(O)SR12,  C(O)NR13R14, CN, or NO2;   R10, and R11 are each independently selected from H, C1‐10 alkyl, halide, or C1‐10 haloalkyl;  R12 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  R13 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;  and  R14 is a base labile protecting group, or is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    2.  The compound of claim 1 wherein A is a natural or artificial nucleobase.    3.  The compound of claim 1 or 2, wherein A is selected from 
Figure imgf000158_0001
;  wherein:   each   is either a single bond or double bond;  each of A1, A2, A3 is selected from NR18 and CR19, where at least one of A1, A2, and A3 is NR18;  each of A4, A5, A6, and A7 is selected from NR18 and CR19 where at least one of A4, A5, A6, and A7 is  NR18;  at least one R15, R16, or R17 group is present and independently selected from H, OR20, SR20, =O, =S,  NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo, wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl is optionally substituted with at least one group selected from halo, OR5, C(O)OR5, C(O)NR6R7,  SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;   R18, if present, is H, an optionally substituted C1‐10 alkyl group, or a base labile protecting group;  R19 is selected from H, OR20, SR20, =O, =S, NR21R22, C1‐10 alkyl, C2‐10 alkenyl, C2‐10 alkynyl, and halo,  wherein the C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl is optionally substituted with at least one group  selected from halo, OR5, C(O)OR5, C(O)NR6R7, SR5, S(O)R5, S(O)2R5, P(O)(OR5)2, CN, NO2, and N3;  R20 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl;   R21 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl; and  R22 is a base labile protecting group, or selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    4.  The compound of claim 3, wherein A is selected from:   
Figure imgf000158_0002
  5.  The compound of claim 4, wherein A is selected from:   
Figure imgf000159_0001
.    6.  The compound of claim 5, wherein A is selected from: 
Figure imgf000159_0002
  7.  The compound of claim 1, wherein A is selected from: 
Figure imgf000159_0003
    8.  The compound of any preceding claim, wherein R1 is selected from H, cyanoethyl,  trialkylsilyl, tert‐butyldiphenylsilyl, acetyl, trihaloacetyl, or phthalimidyl.    9.  The compound of any preceding claim, wherein R2 is selected from H, cyanoethyl,  trialkylsilyl, tert‐butyldiphenylsilyl, acetyl, trihaloacetyl, or phthalimidyl.    10.  The compound of any preceding claim, wherein R3 and R4 are each an optionally substituted  C1‐10 alkyl.    11.  The compound of claim 10, wherein R3 and R4 are each an unsubstituted branched chain C1‐ 10 alkyl.    12.  The compound of claim 11, wherein R3 and R4 are each isopropyl.    13.  The compound of any preceding claim, wherein R5 is absent, or is selected from trialkylsilyl,  tert‐butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    14.  The compound of claim 13, wherein R5 is absent.    15.  The compound of claim 13, wherein R5 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    16.  The compound of claim 13, wherein R5 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    17.  The compound of any preceding claim, wherein R6 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    18.  The compound of claim 17, wherein R6 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    19.  The compound of claim 17, wherein R6 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.     20.  The compound of any preceding claim, wherein R7 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    21.  The compound of claim 20, wherein R7 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    22.  The compound of claim 20, wherein R7 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    23.  The compound of any preceding claim, wherein R8 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    24.  The compound of claim 23, wherein R8 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    25.  The compound of claim 23, wherein R8 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    26.  The compound of any preceding claim, wherein R12 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    27.  The compound of claim 26, wherein R12 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    28.  The compound of claim 26, wherein R12 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    29.  The compound of any preceding claim, wherein R13 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    30.  The compound of claim 29, wherein R13 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    31.  The compound of claim 29, wherein R13 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    32.  The compound of any preceding claim, wherein R14 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    33.  The compound of claim 32, wherein R14 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    34.  The compound of claim 32, wherein R14 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    35.  The compound of any one of claims 3‐34, wherein R18 is selected from H, an optionally  substituted C1‐10 alkyl group, trialkylsilyl, tert‐butyldiphenylsilyl, acetyl, trihaloacetyl, and  phthalimidyl.    36.  The compound of claim 35, wherein R18 is selected from H and an optionally substituted C1‐10  alkyl group.    37  The compound of claim 35, wherein R18 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    38.  The compound of any one of claims 3‐37, wherein R20 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    39.  The compound of claim 38, wherein R20 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    40.  The compound of claim 38, wherein R20 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    41.  The compound of any one of claims 3‐40, wherein R21 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    42.  The compound of claim 41, wherein R21 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    43.  The compound of claim 41, wherein R21 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    44.  The compound of any one of claims 3‐43, wherein R22 is selected from trialkylsilyl, tert‐ butyldiphenylsilyl, acetyl, trihaloacetyl, and phthalimidyl, H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10 alkynyl.    45.  The compound of claim 44, wherein R22 is selected from trialkylsilyl, tert‐butyldiphenylsilyl,  acetyl, trihaloacetyl, and phthalimidyl.    46.  The compound of claim 44, wherein R22 is selected from H, C1‐10 alkyl, C2‐10 alkenyl, and C2‐10  alkynyl.    47.  The compound of any of claims 3‐46, wherein the trialkylsilyl group, if present, has the  formula Si(C1‐C4 alkyl)3.    48.  The compound of claim 47, wherein the trialkylsilyl group, if present, is selected from  trimethylsilyl (TMS), triethylsilyl (TES), tert‐butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS).    49.  The compound of any of claims 4‐48, wherein the trihaloacetyl group, if present, is  trichloroacetyl or trifluoroacetyl.    50.  The compound of claim 49, wherein the trihaloacetyl group, if present, is trifluoroacetyl.     51.  The compound of any preceding claim, wherein R9, R10, and R11 are each H.    52.  The compound of any one of claims 1‐50, wherein R10 and R11 are each F.    53.  The compound of any one of claims 3‐52, wherein R15 is selected from H, OR20, SR20, =O, =S,  NR21R22, and C1‐10 alkyl.    54.  The compound of claim 53, wherein R15 is selected from H, =O, and NR21R22.    55.  The compound of any one of claims 3‐54, wherein R16 is selected from H, OR20, SR20, =O, =S,  NR21R22, and C1‐10 alkyl.    56.  The compound of claim 55, wherein R16 is selected from H, =O, and NR21R22.    57.  The compound of any one of claims 3‐56, wherein R17 is selected from H, OR20, SR20, =O, =S,  NR21R22, and C1‐10 alkyl.    58.  The compound of claim 57, wherein R17 is selected from H, =O, and NR21R22.    59.  The compound of any preceding claim, wherein Z is absent and X and Y are each  independently selected from C1‐30 alkyl, C1‐30 alkenyl, (S)n, (O)n, NR5, (CH2CH2O)m, C(O), C3‐10 cycloalkyl,  C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, or C5‐10 heteroaryl;   wherein each C3‐10 cycloalkyl, C3‐10 heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl or C5‐10 heteroaryl is  optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6, C(O)OR6,  C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3.    60.  The compound of any preceding claim, wherein X is selected from NR5, C3‐10 cycloalkyl, C3‐10  heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl;   wherein each is optionally substituted with at least one group selected from halo, C1‐10 alkyl, OR6,  C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2, and N3.    61.  The compound of claim 60, wherein X is selected from NR5, C3‐10 cycloalkyl, C3‐10  heterocycloalkyl, C5‐10 cycloalkenyl, C6‐10 aryl, and C5‐10 heteroaryl.    62.  The compound of claim 60 or 61, wherein X is selected from NR5 and C3‐10 heterocycloalkyl.    63.  The compound of claim 62, wherein X is selected from NR5, azetidine, pyrrolidine, and  piperidine.    64.  The compound of any preceding claim, wherein Y is selected from C1‐30 alkyl, (CH2CH2O)m,  C(O)n, C3‐10 cycloalkyl, and C3‐10 heterocycloalkyl;   wherein each C3‐10 cycloalkyl or C3‐10 heterocycloalkyl is optionally substituted with at least one group  selected from halo, C1‐10 alkyl, OR6, C(O)OR6, C(O)NR7R8, SR6, S(O)R6, S(O)2R6, P(O)(OR6)2, CN, NO2,  and N3.    65.  The compound of claim 64, wherein Y is selected from C1‐30 alkyl, (CH2CH2O)m, C(O), C3‐10  cycloalkyl, and C3‐10 heterocycloalkyl.    66.  The compound of claim 64 or 65, wherein Y is selected from C1‐30 alkyl, C(O), and C3‐10  cycloalkyl.  group.      68.  The compound of any preceding claim, wherein X‐Y‐Z comprises at least one tertiary amine.    69.  The compound of any preceding claim, wherein X‐Y‐Z comprises at least one of NR5, C(O), C3‐ 10 cycloalkyl, C3‐10 N‐heterocycloalkyl, C6‐10 aryl, and C5‐10 heteroaryl.    70.  The compound of any preceding claim, wherein X‐Y‐Z is selected from: 
Figure imgf000165_0001
  71.  The compound of claim 70, wherein X‐Y‐Z is selected from: 
Figure imgf000165_0002
  72.  The compound of any preceding claim, wherein the compound is selected from: 
Figure imgf000165_0003
 
Figure imgf000166_0001
    73.  The compound of claim 72, wherein the compound is selected from: 
Figure imgf000166_0002
 
Figure imgf000167_0001
  75.  The compound of claim 74, wherein the compound is selected from: 
Figure imgf000167_0002
   
Figure imgf000168_0001
  76.  The compound of any preceding claim, wherein the compound is selected from Table 1.    77.  A modified oligonucleotide, wherein the 5’ end of the oligonucleotide is attached to a  compound of any preceding claim via a covalent bond which replaces the W moiety to give a  modified oligonucleotide of formula (II): 
Figure imgf000168_0002
  wherein Q is S or O.    78.  A modified oligonucleotide conjugate, wherein the modified oligonucleotide of claim 77 is  conjugated to a thiol‐containing moiety (M‐SH) via the vinyl group to provide a conjugate of formula  (II): 
Figure imgf000168_0003
  wherein Q is S or O.    79.  The modified oligonucleotide conjugate of claim 78, wherein M is a small molecule.    80.  The modified oligonucleotide conjugate of claim 78, wherein M is a polypeptide.    ein.      82.  The modified oligonucleotide conjugate of claim 80 or 81, wherein the modified  oligonucleotide is connected to M via a cysteine residue.    83.  A method of conjugating the modified oligonucleotide of claim 77 to a thiol‐containing  molecule (M‐SH) to form the modified oligonucleotide conjugate of any one of claims 78‐82,  wherein the method comprises a thiol‐ene reaction between the M‐SH thiol and the vinyl group of  the modified oligonucleotide of claim 77.    84.  The method of claim 83, wherein M is a small molecule, polypeptide, or protein.    85.  The method of claim 83 or 84, wherein the method comprises the step of M‐SH being  dissolved in a buffered solution to provide a M‐SH buffered solution.    86.  The method of claim 85, wherein the buffered solution comprises an aqueous buffer, or a  non‐aqueous buffer.    87.  The method of any one of claims 83‐86, wherein the method comprises the step of the  modified oligonucleotide being dissolved in an aqueous solution to provide a modified  oligonucleotide solution.    88.  The method of claim 87, wherein the method comprises the step of the M‐SH buffered  solution being mixed with the modified oligonucleotide solution.    89.  The method of any one of claims 83‐88, wherein the reaction between the modified  oligonucleotide and M‐SH is performed between 10‐50 °C.    90.  The method of claim 89, wherein the reaction between the modified oligonucleotide and  M‐SH is performed between 20‐40 °C.    91.  The method of any one of claims 83‐90, wherein the thiol‐ene reaction step is performed in  the presence of a radical initiator.      92.  The method of claim 91, wherein the thiol‐ene reaction step is performed in the presence of  dithiothreitol.     
PCT/EP2024/070454 2023-07-21 2024-07-18 Phosphoramidite linkers Pending WO2025021654A1 (en)

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