title: Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.
abstract: Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application.
authors.affiliation: University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090, Vienna, Austria.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Dirin
authors.affiliation: University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090, Vienna, Austria.
authors.forename: Ernst
authors.initials: E
authors.lastname: Urban
authors.affiliation: University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090, Vienna, Austria.
authors.forename: Christian R
authors.initials: CR
authors.lastname: Noe
authors.affiliation: University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090, Vienna, Austria. Electronic address: johannes.winkler@univie.ac.at.
authors.forename: Johannes
authors.initials: J
authors.lastname: Winkler
mesh_terms: D004220:Disulfides / Q000737:chemistry; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry*; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000627:therapeutic use; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D004220:Disulfides; D008024:Ligands; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D010455:Peptides; D011107:Polylysine; D011092:Polyethylene Glycols
keywords: Antisense oligonucleotides; Disulfide bioconjugates; Gene silencing; PEGylation; Solid phase synthesis