The Solution Synthesis of Antisense Oligonucleotide‐Peptide Conjugates Directly Linked via Phosphoramide Bond by Using a Fragment Coupling Approach

Abstract

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell‐penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1‐hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2′‐dithiopyridine and 4‐dimethylaminopyridine in organic media. Several oligonucleotides, including a 18‐mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.

Keywords:

• Oligonucleotide‐peptide conjugates
• Antisense oligonucleotides
• Conjugate synthesis

Acknowledgments

This work was supported by the Russian Foundation for Basic Research (grants RFBR 02‐04‐48797, 03‐04‐48927 and RFBR‐CNRS 00‐04‐22003) and Hungarian Academy of Sciences Grant (AKP 98‐89 2,4). We also acknowledge the Centre National de la Recherche Scientifique for the support (grant PICS No. 1131) and ARC (grant No. 4310). We thank Prof. T. Oretskaya and coworkers for the oligonucleotide synthesis.