Synthesis of Hydroxamate-Type Siderophores and Their Conjugates with PNA for Evaluation of Iron Binding and Bacterial Uptake.

Applications of peptide nucleic acids (PNAs) against bacteria are limited by their poor cellular uptake. We used a Trojan horse strategy for PNA delivery into gram-negative bacteria via TonB-dependent transport systems. To apply this strategy, iron-chelators (siderophores) covalently linked to PNA oligomers are used as PNA carriers. These siderophores bind ferric iron and are recognized and taken up by cells together with the attached PNA. Here, we describe the synthesis of PNA conjugates with hydroxamate-type siderophores that can be synthesized based on peptide chemistry. The conjugates are prepared through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), enabled by the incorporation of azide and alkyne functionalities into the siderophore and PNA scaffolds, respectively. Synthetic procedures, including solid-phase synthesis of PNA oligomers and siderophores, are provided. We also outline protocols for evaluating the iron-binding capacity of the siderophores, as well as methods for confirming their ability to form octahedral iron complexes with defined Δ/Λ chirality. This is to ensure that upon conjugation with PNA these ferric siderophore-PNA complexes are recognized by the TonB-dependent receptors. This strategy provides a foundation for developing targeted PNA-based antimicrobial agents using native bacterial uptake pathways.