A preassembled backbone-anchored solubilizing tag to simplify native chemical ligation-based protein synthesis

Chemical protein synthesis (CPS) has become an essential tool in chemical biology, offering access to homogeneous proteins bearing modifications beyond the reach of biological expression systems. At the heart of CPS lies native chemical ligation (NCL), which enables the chemoselective coupling of unprotected peptide segments under mild aqueous conditions. However, the scope and efficiency of NCL are often severely constrained by the poor solubility or aggregation propensity of certain peptide segments leading to inneficient ligations and challenging purifications. To address these limitations, a wide range of temporary hydrophilizing tags has been developed, yet existing systems frequently require trade-offs between ease of installation, stability, and cleavage efficiency. Backbone substitution strategies such as Liu's removable backbone modification (RBM) platforms, based on N-2-hydroxy-4-methoxybenzyl (Hmb) derivatives, offer additional advantages of disrupting secondary structures thus preventing aggreagtion. We report here a new class of backbone-anchored solubilizing tags, termed Hmb , designed for automated introduction by straightforward on-resin reductive amination during standard Fmoc-SPPS. This tag integrates a hydrophilic tri-lysine module, N-Boc-protected for compatibility with SPPS, preassembled with a Hmb-derived backbone modification, thereby eliminating the need for additional non-standard solid-phase assembly steps. O-GABAylation ensures its stability towards TFA cleavage to deprotect and release the peptide from the solid support. Neutral pH incubation triggers selfimmolative de-GABAylation, restoring the acid sensitivity required for tag removal after NCL. The utility of this strategy is demonstrated through the synthesis of the challenging protein target human SUMO-2, and its modularity is further demonstrated by the successful incorporation of multiple Hmb units within a single peptide segment.