Extending the scope of native chemical peptide coupling
Extending the scope of native chemical peptide coupling
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DOI：
10.1002/anie.200704886
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Seitz, Oliver
中科院分区：
文献类型：
--
作者：
Haase, Christian;Seitz, Oliver
在20世纪90年代初，天然功能蛋白质的全合成被认为是一个几乎无法实现的目标。然而，由于肯特和同事们开发了天然化学连接，化学蛋白质合成已经进入了可实现的领域。[1]该成功方法的基础是Wieland等人描述的肽硫酯与半胱氨酰肽的化学选择性反应。[2]该反应发生在水性缓冲体系中，并产生"天然"肽键。肽区段可以以未保护的形式彼此偶联。也可以合成糖基化或磷酸化肽。通过与分子生物学方法相结合，可以通过表达的蛋白质连接来合成位点特异性修饰的蛋白质，其提供高达52 kDa的摩尔质量（F1-AT相的β亚基）。[3]天然化学连接的过程原理上在方案1中说明。最初，半胱氨酸残基的硫醇侧链参与可逆交换反应，其中肽硫酯的硫醇RSH也被半胱氨酰肽2取代。新形成的硫酯中间体3在S! N酰基通过五元过渡态转移到偶联产物4。硫醇交换控制了该反应序列中的速率。加入硫醇添加剂，如苄基硫醇、苯硫酚或2-（4-巯基苯酚）乙酸（MPAA）[4]以加速反应。这些添加剂导致在初始平衡中形成反应性硫酯。在一个实施例中，实现了具有令人印象深刻的203个氨基酸的共价偶联的HIV-1蛋白酶二聚体的全合成。[5]天然化学连接的适用性在两个方面受到限制。碱不稳定肽硫酯1的合成并不总是像肽酸和肽酰胺的常规合成那样简单。此外，半胱氨酸是相对罕见的氨基酸（含量为1.4%），因此在某些靶蛋白的情况下，必须经常插入人工半胱氨酸残基以提供合适的偶联位点。本亮点关注当前克服这两个障碍的进展。肽硫酯的获取受到限制是天然化学连接的主要障碍之一。考虑到硫酯结构的碱不稳定性，肽硫酯主要通过叔丁氧羰基（Boc）固相合成来制备。[6a]然而，必需使用强酸从聚合物载体上切割肽与酸敏感性侧链修饰（如糖基化或磷酸化）不相容。因此，允许使用更温和的9-芴甲氧羰基（Fmoc）固相合成的方法正在被深入研究。因此，已经寻找了保留硫酯官能团的Fmoc裂解的替代条件，[6b，c]并且已经开发了在合成的后期阶段构建硫酯的方法。[6d-r]最近，引入了具有自纯化效果的方法，其使得能够在没有制备纯化步骤的情况下以高纯度合成肽硫酯。[七]《中国日报》
In the early 1990s, a total synthesis of native, functional proteins was considered an almost unachievable goal. However, thanks to Kent and co-workers development of native chemical ligation, chemical protein synthesis has shifted into the realms of the achievable.[1] The basis of this successful method is the chemoselective reaction of a peptide thioester with a cysteinyl peptide described by Wieland et al.[2] This reaction takes place in aqueous buffer systems, and produces a “natural” peptide bond. The peptide segments can be coupled with one another in unprotected form. It is also possible to synthesize glycosylated or phosphorylated peptides. By combination with molecular biology methods, sitespecifically modified proteins can be synthesized by expressed protein ligation, which provides molar masses of up to 52 kDa (β-subunit of F1-ATPhase).[3] The course of native chemical ligation is illustrated in principle in Scheme 1. Initially the thiol side chains of cysteine residues participate in reversible exchange reactions in which the thiol RSH of the peptide thioester is also replaced by the cysteinyl peptide 2. The newly formed thioester intermediate 3 reacts in an S! N acyl transfer to the coupled product 4 via a five-membered transition state. The thiol exchange governs the rate in this reaction sequence. Thiol additives, such as benzylmercaptan, thiophenol, or 2-(4-mercaptophenol) acetic acid (MPAA),[4] are added to accelerate the reaction. These additives lead to the formation of reactive thioesters in an initial equilibrium. In one example, the total synthesis of a covalently coupled HIV-1 protease dimer with the impressive number of 203 amino acids was achieved.[5] The applicability of the native chemical ligation is restricted in two respects. The synthesis of base-labile peptide thioesters 1 is not always as simple as is customary for peptide acids and peptide amides. Furthermore, cysteine is a relatively rare amino acid (1.4% content), so that in the case of a certain target protein, an artificial cysteine residue must frequently be inserted to provide a suitable coupling site. The present Highlight is concerned with current advances in overcoming these two obstacles.The restricted access to peptide thioesters is one of the main obstacles of native chemical ligation. Taking into account the base lability of the thioester structure, peptide thioesters were mostly prepared by tert-butoxycarbonyl (Boc) solid-phase synthesis.[6a] However, the necessary use of strong acids for the cleavage of the peptide from the polymeric support is not compatible with acid-sensitive side chain modifications, such as glycosylation or phosphorylation. Therefore, methods that allow the use of the milder 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis are being intensively investigated. Thus, alternative conditions for Fmoc cleavage under which the thioester function is retained have been sought,[6b, c] and methods have been developed in which the thioester is constructed at a late stage of the synthesis.[6d–r] Recently, a method with a self-purification effect was introduced, which enabled the synthesis of peptide thioesters in high purity without a preparative purification step.[7]