Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O‑Linked β‑N‑Acetylglucosamine Strategy

• Published in: Journal of the American Chemical Society Vol. 144; no. 1; pp. 349 - 357
• Main Authors: Shi, Wei-Wei, Shi, Chaowei, Wang, Tong-Yue, Li, Yu-Lei, Zhou, Yong-Kang, Zhang, Xu-Han, Bierer, Donald, Zheng, Ji-Shen, Liu, Lei
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.01.2022
• Subjects: Acetylglucosamine
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.1c10091

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked β-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.