Division of Diabetes and Endocrinology, Department of Pediatrics, Stanford University, Palo Alto, CA, USA.
Department of Biochemistry, University of Utah, Salt Lake City, UT, USA.
Methods Mol Biol. 2022;2530:81-107. doi: 10.1007/978-1-0716-2489-0_7.
Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS) remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.
在化学蛋白质合成 (CPS) 中保持每个肽段的高溶解性，甚至是足够的溶解性仍然是一个关键挑战；只要有一个肽段的溶解性不佳，就可能会使整个合成尝试失败。为了解决这个挑战，已经采用了多种方法，最常用的方法是使用化学工具来临时提高肽的溶解性。在本章中，我们讨论了用于在 Lys 和 Glu 残基的侧链上引入半永久性增溶序列（称为帮助手）的化学工具。我们描述了这两种工具的合成、Fmoc-SPPS 中的掺入以及切割条件。对于 Lys 位点，我们讨论了基于 Fmoc-Ddap-OH 二亚甲基酮的 linker，它是手性的，一步合成，可直接引入到伯胺上，并使用羟胺（或肼）去除。对于 Glu 位点，我们详细介绍了新的 Fmoc-SPPS 构建块 Fmoc-Glu(AlHx)-OH，它可以通过两步纯化以高效的方式制备。增溶序列可直接在树脂上引入，然后在水相条件下使用钯催化转移进行切割，以恢复天然的 Glu 侧链。这两种化学工具易于制备和实施，我们预计在按照本文所述方案进行“困难”肽段时会继续使用。
