Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters

摘要

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI.P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-Ga1NAc alkyne gives oligonucleotide-GaINAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GaINAc derivatives of 2'-modified siRNA.