SALICYLALDEHYDE-TAGGED PEPTIDES FOR THE REVERSIBLE-COVALENT ENGAGEMENT OF PROTEIN LYSINE RESIDUES

Covalent drug discovery has recently re-emerged as a powerful approach for targeting clinically-relevant proteins, offering enhanced potency and selectivity through controlled covalent interactions. In particular, reversible covalent (RC) chemistry combines the strength of covalent binding with the tunability of non-covalent interactions, mitigating the risks of permanent off-target reactivity. Within this context, this PhD thesis explores the use of salicylaldehyde (SA) as a reversible electrophilic warhead for peptide-based ligand design. The first part of this thesis focuses on the synthesis of SA-modified amino acids and their incorporation into model peptides. Later on, a more straightforward and versatile approach was devised, consisting in the late-stage peptide functionalization with copper-catalyzed azide- alkyne cycloaddition (CuAAC). This second strategy enabled efficient installation of SA moieties onto pre-assembled peptides specific for two protein targets, namely SHP2 and NEMO. Moreover, using human serum albumin as model target, the CuAAC protocol was instrumental for the design of a new combinatorial approach to SA-tagged small molecules. The final part of the work, carried out at the University of Tokyo, integrates SA-based reversible covalent chemistry into the RaPID (Random nonstandard Peptides Integrated Discovery) platform, paving the way to a novel strategy towards macrocyclic peptide libraries displaying a Lys-engaging electrophile. Overall, this work expands the chemical space of peptide-based RC ligands, providing new synthetic and methodological tools to access selective, tunable covalent binders to target specific proteins or to inhibit clinically-relevant protein-protein interactions.