Synthesis and cellular uptake of fluorescently labeled multivalent hyaluronan disaccharide conjugates of...

Clustered hyaluronan disaccharides were studied as mediators of cellular delivery of antisense oligonucleotides through receptor-mediated endocytosis. For this purpose, a synthetic route for preparation of an appropriately protected hyaluronic acid dimer bearing an aldehyde tether (1) was devised. Up to three non-nucleosidic phosphoramidite building blocks (2), each bearing two phthaloyl protected aminooxy groups, were then inserted into the 3'-terminus of the desired phosphorothioate oligodeoxyribonucleotide, and 6-FAM phosphoramidite was introduced into the 5'-terminus. After completion of the chain assembly, the aldehyde-tethered sugar ligands were attached to the deprotected aminooxy functions by on-support oximation. Three fluorescein-labeled phosphorothioate oligonucleotide glycoconjugates (28-30) containing two, four, or six hyaluronan disaccharides were prepared. The influence of the hyaluronan moieties on the cellular uptake of the thioated oligonucleotides was tested in a cell line expressing the hyaluronan receptor CD44. Specific uptake was not detected with this combination of multiple hyaluronan disaccharides.