Direct Dual-Functionalisation of Amine-Oligonucleotides for Conjugation Purposes

Abstract The optimisation and further expansion of methods for the synthesis of oligonucleotide conjugates is receiving increased attention due to their importance for further advancement of therapeutic and diagnostic nucleic acid-based applications. Current methodologies, particularly those relying on maleimide-type linkers, are often hampered by multi-step synthetic procedures and linker instability. Herein, we present a versatile method for the direct functionalisation of readily available amino-modified oligonucleotides (AONs), where a 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) Michael acceptor is directly formed in a rapid and efficient manner on a free primary amine. The methodology demonstrates broad applicability, tolerating various amino-modifiers and their positions within different oligonucleotide types, including DNA, LNA, PNA, and phosphorothioate-modified oligonucleotide strands. Most importantly, the possibility to introduce an additional second orthogonal reactive handle uniquely enables a direct dual-functionalisation of AONs for the assembly of complex constructs, as exemplified by the synthesis of a fluorescent peptide-oligonucleotide construct.