Synthesis and Cellular Uptake of Fluorescently Labeled Multivalent Hyaluronan Disaccharide Conjugates of Oligonucleotide Phosphorothioates

Abstract

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.

DNA glycoclusters and DNA-based carbohydrate microarrays: From design to applications

“…The reaction between an aldehyde and an aminooxy to give rise to an oxime, known as click-oxime (click-O), has been used several times in the field. [54][55][56][57][58][59] It is generally argued that one advantage of the CuAAC reaction is the tolerance of the azide and alkyne functions to a broad diversity of organic functions and reaction conditions. Thus, the synthesis of DNA-glycoconjugates through CuAAC can be theoretically achieved either from alkynyl-functionalized saccharides with a DNA-like scaffold bearing azido functions or from azido-functionnalized saccharides with a DNA-like scaffold bearing alkynyl functions.…”

Immobilized Carbohydrates for Preparation of 3′‐Glycoconjugated Oligonucleotides

“…The oligonucleotide is usually modified by an amino oxy group and used for on‐support oximation with aldehyde‐modified carbohydrates (Katajisto, Virta, & Lönnberg, 2004). By using this strategy, multivalent hyaluronic acid and galactose‐oligonucleotide conjugates have been successively prepared (Karskela, Virta, Malinen, Urtti, & Lönnberg, 2008, 2010; Mäkilä et al., 2014). Both alkyne‐ and azide‐modified carbohydrates have been used for Cu(I)‐catalyzed Huisgen 1,3‐dipolar azide‐alkyne cycloaddition (click reaction) with oligonucleotides (Kiviniemi, Virta, & Lönnberg, 2008, 2010, 2011).…”

Synthesis of Biotinylated Multipodal Glycoclusters on a Solid Support

“…[33][34][35]39,40] Accordingly, d-biotin and N-(4-methoxytrityl)-1,4-phenylenedimethanamine [41] (17) were first coupled by 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) activation to obtain 18. Besides biotin, the ligand contained a 1,4-phenylene chromophore and a Boc-protected aminooxy function.…”

“…It should be noted that the high efficiency of solid-supported oximation has been demonstrated by the syntheses of numerous structurally complicated bioconjugates. [33][34][35]39,40] Accordingly, d-biotin and N-(4-methoxytrityl)-1,4-phenylenedimethanamine [41] (17) were first coupled by 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) activation to obtain 18. Ligand 19 was then obtained by removal of the 4methoxytrityl protection with TFA and coupling of N-Bocaminooxyacetic acid to the exposed amino group by DCC activation with use of 1-hydroxybenzotriazole (HOBt) as an auxiliary nucleophile.…”

“…The clusters were further conjugated with a biotin ligand to allow convenient immobilization of the conjugates (Figure 1) on avidin-or streptavidin-coated surfaces. Besides biotin, the approach should be extendable to conjugation of the glycoclusters with any aminooxy-functionalized compound, such as DOTA derivatives, [32] oligonucleotides, [33][34][35] and peptides. [36] The advantage of solid-phase synthesis -the easy removal of excess of reagents by simple washing -is a remarkable benefit in multistep conjugate synthesis of this kind, consisting of several removals of protecting groups and couplings of building blocks.…”