In vivo bioavailability and delivery of nucleic acids to the site of action is a severe limitation in oligonucleotide (ON) therapeutics. Equipping the ONs with cell penetrating, homing or endosomal escape peptides can enhance specificity and/or uptake efficiencies. We describe here a general procedure for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support utilizing a novel activated alkyne containing linker which enhances the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition. Conjugation reaction is efficient in millimolar concentration and submicromolar amounts at ambient temperature. The route for POC preparation involves two subsequent conjugation steps: to solid-supported ONs containing a 5'-amino modifier (1) the triple bond donor (p-(N-propynoylamino)toluic acid (PATA), p-([2-(propynyloxy)acetamido]methyl)benzoic acid (PAMBA) or 2-(propynyloxy)acetic acid (PAA)) is first coupled and then (2) an azido-functionalized peptide is attached via a triazole linkage by copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition. The fragment-conjugated POC is released from the solid support by concentrated ammonia. The method gives high conversion of ON to the POC and only involves a single purification step after complete assembly and release from the solid support. The synthesis is flexible and designed to utilize commercially available oligonucleotide and peptide derivatives without the need for specific automated synthesizers.
Keywords: Activated triple bond donor; Azido-containing peptides; Click chemistry; Conjugation; Huisgen 1,3-dipolar cycloaddition; Peptide–oligonucleotide conjugates; Solid-phase synthesis.