Fujita Kei, Ichihara Rintaro, Kobayashi Masakazu, Matsuda Kenichi, Wakimoto Toshiyuki
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Methods Mol Biol. 2025;2931:105-121. doi: 10.1007/978-1-0716-4562-8_10.
Nonribosomal peptide cyclases possess enormous biocatalytic potential for cyclizing peptides, including those shorter than ten residues. However, N-acetylcysteamine (SNAC) or related low molecular weight thiol leaving groups, which have traditionally been used as surrogates for a pantetheine group on the substrate C-terminus, necessitate a labor-intensive process to prepare the substrate for cyclases. Our group recently established a faster, automation-compatible synthetic method for cyclase substrates, using ethylene glycol (EG) as a new pantetheine surrogate. This approach significantly streamlines substrate preparation while maintaining cyclization efficiency. In this chapter, we describe the synthetic pathway for the solid-phase peptide synthesis of EG-functionalized peptides, followed by enzymatic cyclization using nonribosomal peptide cyclases.
非核糖体肽环化酶在环化肽方面具有巨大的生物催化潜力，包括那些少于十个残基的肽。然而，传统上用作底物C端泛酰巯基乙胺基团替代物的N - 乙酰半胱胺（SNAC）或相关的低分子量硫醇离去基团，需要一个劳动密集型的过程来为环化酶制备底物。我们小组最近建立了一种更快、与自动化兼容的环化酶底物合成方法，使用乙二醇（EG）作为新的泛酰巯基乙胺替代物。这种方法在保持环化效率的同时，显著简化了底物制备过程。在本章中，我们描述了EG功能化肽的固相肽合成途径，随后使用非核糖体肽环化酶进行酶促环化。
