Total Chemical Synthesis of Interleukin‐15 and Interleukin‐2: Taming Protein Hydrophobicity and Aggregation by a Versatile Solubilizing Strategy

Hydrophobic and aggregation‐prone proteins still present obstacles for protein chemical synthesis and engineering. The chemical synthesis of Interleukin‐15 (IL‐15) is a formidable challenge due to inherent sequence hydrophobicity and severe peptide aggregation, which impedes downstream protein engineering and chemical biology studies. Here, we report the first total synthesis of IL‐15 using a versatile solubilizing strategy (RST‐2.0), which enables the preparation and ligation of aggregation‐prone segments while being readily removable during folding. Remarkably, this strategy is fully compatible with glycopeptide synthesis, allowing for the synthesis of homogeneously N79‐glycosylated IL‐15. The effectiveness of RST‐2.0 was further demonstrated through the efficient synthesis of wild‐type and azide‐labeled Interleukin‐2 (IL‐2) analogs. Moreover, the bioactivity of IL‐15 and IL‐2 analogs was validated by CTLL‐2 proliferation assays and microscale thermophoresis (MST). This work provides a de novo synthesis approach to elucidate the role of N‐glycosylation on IL‐15‐mediated immune regulation and lays the foundation for developing next‐generation cancer immunotherapies based on synthetic IL‐15 variants.