Expanding the scope of a new bio-orthogonal templated reaction

The desire to explore biomolecules and biological processes within their natural contexts has driven the development of reactions that are compatible with such environments. This pursuit presents a substantial challenge owing to the complexity of cellular systems. Reactions capable of proceeding within a biological system without disrupting native biological processes are termed “bioorthogonal reactions”. Components involved must exhibit rapid and selective reactivity in water under phisiological conditions, in contexts of high molecular crowding. Despite the remarkable advancements in bioorthogonal chemistries, so far very few ligation reactions have proven to be efficient at cellular level. Within current methodologies, challenges related to interference amidst the numerous functionalities present in vivo, and the requirement to remain non-toxic to the biological system, persists. Our aim is to improve a recently discovered bio-orthogonal templated reaction which requires “proximiy” as sole trigger and expand his reactivity, ultimately extending its applicability to cellular studies.1,2 The chosen reaction involves the ligation between 2,5-dioxopentanyl (DOP) moiety and an alpha nucleophile, resulting in the formation of piridazinium or pyrrole linkage (Fig.1). This reaction, which has been successfully tested in cell lysate, has demonstrated its ability to proceed exclusively under the guidance of a template, such as (peptide) nucleic acids or coiled-coil system formation, with high specificity and without off-target reactivity. Here, we will present preliminar results regarding the synthesis of various alpha nucleophiles building blocks and preliminary ligation experiments performed on peptide nucleic acids (PNAs) models.