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.
Long‐chain scorpion toxin AaH‐II isolated from Androctonus australis Hector can selectively inhibit mammalian voltage‐gated sodium ion channel Nav1.7 responsible for pain sensation. Efficient chemical synthesis of AaH‐II and its derivatives is beneficial to the study of the function and mechanism of Nav1.7 and the development of potential peptide inhibitors. Herein, we compared three different strategies, namely, direct solid‐phase peptide synthesis, hydrazide‐based two‐segment native chemical ligation, and hydrazide‐based three‐segment native chemical ligation for the synthesis of AaH‐II. The hydrazide‐based two‐segment native chemical ligation affords the target toxin with the optimal efficiency, which provides a practically robust procedure for the preparation of tool molecules derived from AaH‐II to study the biological functions and modulation of Nav1.7. Our work highlights the importance of selecting suitable segment condensation approach in the chemical synthesis of protein toxins.
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