An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation
An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation
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DOI：
10.1002/anie.200705471
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Dawson, Philip E.
中科院分区：
文献类型：
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作者：
Blanco-Canosa, Juan B.;Dawson, Philip E.
在固体支持物上组装的肽的直接C-末端修饰仍然是肽和蛋白质化学中的重大挑战。特别地，C-末端硫酯肽是用于产生活性酯、酰胺和酰肼的重要中间体[1，2]，并且是用于蛋白质合成的许多合成策略的必要组分。[3]目前，合成肽基硫酯的最有效方法是使用硫酯接头的Boc固相肽合成（Boc-SPPS）的原位中和方案[4]。[2，5]然而，许多实验室仅使用Fmoc-SPPS，并且在合成糖肽和磷酸肽时，这些方案是有利的。用于Boc-SPPS的硫酯接头对于Fmoc-SPPS具有有限的效用，因为需要在碱性条件下重复去除Fmoc。已经付出了相当大的努力来解决这一挑战[6]，包括优化的Fmoc脱保护混合物，[7]硫醇不稳定的安全捕获接头，[8]在溶液中活化受保护的肽，[9]最近已经使用O至S [10]或N至S [11]酰基转移产生硫酯。尽管有这些显著的进展，通过Fmoc-SPPS合成硫酯肽仍然比合成相应的酸或酰胺肽更具挑战性。在这里，我们描述了一种用于天然化学连接（NCL）[12]的肽的Fmoc合成的替代方法，该方法基于C-末端N-酰基脲官能团的形成。N-酰基脲是温和的酰化剂，Pascal [13，14]在肽合成中以及Rapoport [15]和Zacharie在硫代酰胺合成中已经进行了探索。[16]然而，由于N-酰基脲产物对氨解的低反应性和先前N-酰基脲形成接头的碱不稳定性，这些基团作为酰化剂的效用受到限制。[13]受这项工作的启发，我们发现邻氨基苯胺1是稳定的合成中间体，在链延长后可以有效地转化为芳香族N-酰基脲部分2 [14，16]（方案1）。由于该基团先前已被描述为N-酰基-苯并咪唑啉酮，[13-16]我们建议使用缩写Nbz来表示该离去基团。树脂结合的酰基脲肽2可以脱保护，并使用标准酸不稳定接头（例如Rink或Wang）用TFA从树脂上裂解。所得的轻度活化的肽-Nbz 3对用于肽处理和纯化的酸性条件是稳定的。然而，在中性水性缓冲液中，完全未保护的酰脲肽经历快速硫解，使得硫酯肽4能够在纯化之前或在天然化学连接期间原位产生。重要的是，接头在链组装期间是稳定的酰胺，并且关键活化步骤利用固相肽合成中最稳健的反应：胺的酰化。因此，在本发明中，
The straightforward C-terminal modification of peptides assembled on a solid support remains a significant challenge in peptide and protein chemistry. In particular, C-terminal thioester peptides are important intermediates for the generation of active esters, amides and hydrazides [1, 2] and are an essential component of many synthetic strategies for protein synthesis.[3] Currently, the most effective approach for the synthesis of peptidyl thioesters is the in situ neutralization protocol for Boc solid phase peptide synthesis (Boc-SPPS)[4] using thioester linkers.[2, 5] However, many laboratories use Fmoc-SPPS exclusively and such protocols are favored when synthesizing glyco-and phosphopeptides. The thioester linkers used for Boc-SPPS have limited utility for Fmoc-SPPS due to the requirement for repeated Fmoc removal under basic conditions. Considerable effort has been applied to address this challenge [6] including optimized Fmoc deprotection cocktails,[7] thiol labile safety catch linkers,[8] activation of protected peptides in solution,[9] and recently thioesters have been generated using O to S [10] or N to S [11] acyl transfer. Despite these notable advances, the synthesis of thioester peptides by Fmoc-SPPS remains significantly more challenging than the synthesis of the corresponding acid or amide peptide.Here we describe an alternative approach for the Fmoc synthesis of peptides for use in native chemical ligation (NCL)[12] that is based on the formation of a C-terminal N-acylurea functionality. N-acylureas are mild acylating agents that have previously been explored in peptide synthesis by Pascal [13, 14] and for thioamide synthesis by Rapoport [15] and Zacharie.[16] However, the utility of these groups as acylating agents has been limited due to the low reactivity of N-acylurea products towards aminolysis and the base lability of previous N-acylurea forming linkers.[13] Inspired by this work, we have found that o-aminoanilides 1, are stable synthetic intermediates that can be efficiently transformed into an aromatic N-acylurea moiety 2 [14, 16] following chain elongation (Scheme 1). Since this group has been previously described as an N-acyl-benzimidazolinone,[13–16] we propose the use of the abbreviation Nbz to indicate this leaving group. The resin-bound acylurea peptide 2, can be deprotected and cleaved from the resin with TFA using standard acid labile linkers (eg Rink or Wang). The resulting mildly activated peptide-Nbz 3, is stable to the acidic conditions used for peptide handling and purification. However, in neutral aqueous buffers, the fully unprotected acylurea peptides undergo rapid thiolysis, enabling thioester peptide 4 to be generated before purification or in situ during a native chemical ligation. Importantly, the linker is a stable amide during chain assembly and the key activation step utilizes the most robust reaction in solid phase peptide synthesis: the acylation of an amine. As a result,