New Alkyne and Amine Linkers for Versatile Multiple Conjugation of Oligonucleotides

Abstract

Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly. This report describes the simplified methodology allowing for multiple labeling of ONs on a solid support and is compatible with phosphodiester as well as phosphorothioate (PS) ONs. The current approach utilizes two novel alkyne- and amino-functionalized linker phosphoramidites that can be readily synthesized from a common aminodiol intermediate in three steps. The combination of new linkers provides orthogonal functionalities, which allow for multiple attachments of similar or varied moieties. The linkers are incorporated into ONs during automated solid-phase ON synthesis, and the conjugation with functional entities is achieved by either amide bond formation or by copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The versatility of the approach is demonstrated by the synthesis of 5′-site ON multiconjugates with small molecules, peptides, and fatty acids as well as in the preparation of an internal peptide–ON conjugate.

Lipid and Peptide-Oligonucleotide Conjugates for Therapeutic Purposes: From Simple Hybrids to Complex Multifunctional Assemblies

“… Chemical synthesis of multifunctional peptide and/or lipid oligonucleotide conjugates. ( A ) Modified phosphoramidite for the incorporation of two ligands, ( B ) combination of amino-protection and click chemistry allowing the addition of two different ligands [ 200 ], ( C ) base-labile and photolabile protecting groups allow the successive combination of two different ligands [ 201 ]. …”

“…A tridentate derivative carrying three cyclic RGD peptides have been developed and incorporated into siRNAs showing an increase in inhibitory properties [ 124 ]. In recent research, novel reagents that allow the incorporation of multiple peptides by click chemistry have been developed [ 200 ]. In addition, they allow the preparation of heterofunctional conjugates through a clever use of click chemistry and amino-protection ( Figure 7 B) [ 200 ].…”

Evolution of complexity in non-viral oligonucleotide delivery systems: from gymnotic delivery through bioconjugates to biomimetic nanoparticles

“…Solid-phase reactions have the advantage of user-friendly procedures which often provide good purity. Due to full biocompatibility and orthogonality, Cu-assisted azide-alkyne cycloaddition (CuAAC) or Copper-free ring-strain promoted azide-alkyne cycloaddition (SPAAC) are among the most popular reactions for ON modifications allowing for fast and efficient conjugation of small molecules, carbohydrates, peptides, hydrophobic compounds (including lipids), aptamers, and antibodies [ 43 ]. Examples of various conjugation strategies of different compounds are described in the following subsections, showing the vast potential of bioconjugation in developing efficient RNA therapeutics.…”

Transition Metal‐Catalyzed Reactions and Solid‐Phase Synthesis: A Convenient Blend

“…The attachment of small molecules [79,80] fluorophores [81] like biotin, α-tocopherol and also N-acetylgalactosamin [82] and polyethyleneglycol [83] has demonstrated the importance and usefulness of this strategy to introduce functionalities in oligonucleotides in a simple manner. Recently, a work published by Honcharenko et al [84,85] reported a versatile approach for multiple labeling of oligonucleotides on a solid support. The methodology is compatible with automated solid-phase synthesis and with phosphodiester, as well as phosphorothioate chemistry.…”