Development of ArgTag for Scalable Solid-Phase Synthesis of Aggregating Peptides

Aggregation during solid-phase peptide synthesis (SPPS) remains a key limitation, often leading to low coupling efficiencies and poor crude purities. Our previously introduced synthesis tag ("SynTag") for chemical protein synthesis combines six C-terminal Arg(Pbf) residues with a MeDbz linker to suppress aggregation via helical structure induction and serves as a handle for native chemical ligation (NCL). To apply the concept to short, yet aggregation-prone sequences, some practical limitations need to be addressed: Tag removal needs to be simplified, its utility demonstrated on more commonly used resin types and loadings, and the method must be effective on larger scale. To this end, we developed a simplified C-terminal hexaarginine tag ("ArgTag") and refined an enzymatic method for efficient removal with Carboxypeptidase B, enabling selective cleavage under mild, linker-free conditions. We evaluated the ArgTag across six solid supports (resins) of varying polarity and loading. Using automated fast-flow SPPS (AFPS), we observed consistent aggregation suppression and improved crude purities across all resin types. We finally demonstrated the efficiency of our ArgTag on larger scale using more economical synthesis parameters. This work broadens the applicability of the SynTag strategy to short, yet difficult peptide sequences and offers a more scalable solution to improve SPPS efficiency for challenging targets.