Phosphate triester-based multifunctional handles for post-synthetic oligonucleotide functionalization

The continued advancement of oligonucleotide-based strategies in research and therapeutics relies on expanding the repertoire of chemical modifications to overcome persistent challenges, such as improving cellular uptake and delivery. Addressing these obstacles requires innovative bioconjugation approaches that integrate seamlessly with oligonucleotide modalities. Here, we report the development of a novel phosphotriester trifunctional probe based on the H-phosphonate derivative ammonium (9H-fluoren-9-yl)methyl, introducing significant advancements in synthetic phosphate chemistry. This platform supports robust and versatile chemical transformations, enabling the incorporation of diverse functionalities, such as biotin, fluorescent markers, G4-stabilizing ligands, and azido groups, into oligonucleotide backbones. The resulting multifunctional probes are compatible with different conjugation strategies and phosphorothioate modifications, allowing late-stage functionalization in solution without requiring solid-phase synthesis. We demonstrate the utility of this approach through the synthesis of G4-ligand-conjugated oligonucleotides (GL-Os) designed to target individual G4 structures. However, the strategy's adaptability ensures compatibility with a wide range of oligonucleotide-based applications that benefit from the addition of functional probes. This flexibility broadens accessibility and applicability, facilitating the development of oligonucleotide tools for advanced chemical biology studies, including fluorescence-based imaging and pull-down experiments.