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.

Figure 1

Schematic representation of synthesized ON conjugates, structures of alkyne and amino linker phosphoramidites, and azido-modified moieties of benzylguanine (BG-N₃), P4 peptide (P4-N₃), MIF peptide (Ac-MIF-N₃), and palmitic acid (PA).

Scheme 1. Synthesis of 4-Monomethoxytrityl- and 4,4′-Dimethoxytrityl-Protected Alkyne Linker Phosphoramidites 7 and 11

Scheme 2. Synthesis of Fmoc-Protected Amino Linker Phosphoramidite 16

Figure 2

Synthesis of tetra-benzylguanine-labeled ON1-(BG)₄.

Figure 3

Synthesis of mono-, bis-, and tris-P4 peptide PS-ON conjugates ON2-P4, ON3-(P4)₂, and ON4-(P4)₃, via 1–3 cycles of linker 11 incorporation.

Figure 4

Synthesis of ON internal conjugate with P4 peptide (ON5-P4) utilizing ON5 with internally incorporated linker 11 and P4-aza peptide.

Figure 5

Synthesis of ON tris-conjugate ON6-(MIF)-(PA)-(MIF) with attached palmitoyl residue and two units of the MIF peptide. Chromatograms A–C demonstrate the RP-HPLC profile of (A) crude ON6 after Fmoc deprotection and cleavage from the solid support, (B) crude ON6-(PA) conjugate, and (C) final crude tris-conjugate ON6-(MIF)-(PA)-(MIF) (see enlarged Figures S36–S38 in the Supporting Information). RP-HPLC is performed on a C18 column (Supelco Discovery BIO Wide Pore C18-5) with a linear gradient from 0 to 50% (for A), and from 50 to 100% (for B and C) of buffer B in buffer A over 45 min at 25 °C.