Supporting data for PhD thesis: Advancing Protein Chemical Synthesis: Knorr Pyrazole Synthesis Mediated Salicylaldehyde Esters Regeneration In TIM-1 Synthesis and Prior Disulfide Bonds Formation Assisted Protein Refolding In IL-25 Synthesis

<p dir="ltr">Protein chemical synthesis is a powerful tool for studying protein structure, function, and interactions. However, it faces several significant challenges including low solubility of peptides, inefficient ligation and poor refolding efficiency. This dissertation focuses on providing some new strategies to overcome these issues. We chemically synthesized two biologically significant proteins: T-cell immunoglobulin and mucin domain-1 (TIM-1) and Interleukin-25 (IL-25), each presenting distinct synthetic challenges. Through our novel approaches, we achieved the first successful chemical synthesis of these proteins, enabling their futural detailed structure-function relationship and opening new avenues for their therapeutic applications. Our work also provides additional solutions to longstanding problems in the chemical synthesis of difficult proteins.</p><p dir="ltr">TIM-1 IgV domain, characterized by its strong β-sheet secondary structure, presented considerable challenges in chemical synthesis. To overcome issues of aggregation during ligation, we employed aggregation-disrupting building blocks. Additionally, we identified a previously unnoticed side reaction between tryptophan (Trp) residues and pyruvic acid in salicylaldehyde ester regeneration step in Ser/Thr Ligation, which further destroyed the desired product. To address this, we introduced acetylacetone (AcAc) as a novel reagent to regenerate the salicylaldehyde ester by catching semi-carbazide through Knorr Pyrazole Synthesis, and it also prevented the side reaction with tryptophan. This strategy not only resolved the side-reaction issue but also facilitated the production of high-quality peptide fragments. Through total chemical synthesis, we successfully produced TIM-1 with native conformation and exhibited biological activity.</p><p dir="ltr">IL-25, a pivotal mediator in type 2 immune responses, adopts a dimeric configuration and relies on two conserved disulfide bonds among its ten cysteine residues to maintain structural and functional integrity. Traditional refolding methods frequently resulted in disulfide bond mismatching due to the high number of cysteines, often leading to inactive protein. To overcome this challenge, we developed a new strategy involving the precise formation of disulfide bonds prior to refolding, thereby eliminating the usage of redox systems and ensuring the acquisition of high-fidelity native disulfide arrangement. We successfully achieved the total chemical synthesis of IL-25, obtaining the protein with native secondary structure and biological activity. Moreover, we observed that the disulfide bonds organization in IL-25 may differ from previously reported literature, and it necessitates further experimental validation.</p><p dir="ltr">The synthetic approaches developed for the TIM-1 IgV domain and IL-25 will provide some useful strategies for addressing certain challenges in the chemical synthesis of complex proteins. For TIM-1, the use of AcAc as a novel reagent addresses a critical synthetic challenge from Trp-pyruvic acid side reaction. For IL-25, the strategy of forming disulfide bonds prior to refolding ensures the production of a biologically active and structurally accurate protein. These methodologies provide a robust framework for the chemical synthesis of other complex proteins and advance our understanding of their functions and therapeutic potential.</p>