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.