Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis
Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis
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DOI：
10.1002/cbic.200700452
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发表时间：
2008-01-04
期刊：
影响因子：
3.2
通讯作者：
Kolmar, Harald
中科院分区：
文献类型：
--
作者：
Avrutina, Olga;Schmoldt, Hans-Ulrich;Kolmar, Harald
环肽是一类重要的天然存在或合成化合物，具有多种生物活性，如激素、离子载体、抗癌剂、抗生素、抗真菌剂或毒素生物学研究表明，与线性肽相比，环肽具有更高的代谢稳定性、更好的受体选择性和更好的活性谱在近年来从微生物、植物甚至人体中分离出的天然环状肽和蛋白质中，[3]环核苷酸提供了一种特别有趣的拓扑结构该家族的环状植物蛋白具有首尾环化肽骨架和基于由6个保守的胱氨酸残基形成的二硫键的胱氨酸结（CK）基序（图1）。两个二硫键及其连接的主键段形成一个环，被第三个二硫键穿透，形成伪结结构，这种结构与附近的b片结构不可避免地联系在一起胱氨酸结与环状主链结合似乎是一个高效的结构稳定基序，导致异常的构象刚性，以及对变性条件的稳定性，以及对蛋白质水解降解的稳定性。含有ck的肽存在于近20个不同的蛋白质家族中，具有离子通道阻断（conotoxin和spider toxins）、蛋白酶抑制（squash inhibitors）和抗虫活性（plant cyclotides）等活性。头部到尾部的大环胱氨酸结肽已从茜草科、堇菜科和葫芦科植物中分离出来。这些家族的一些成员已被引入作为药物设计和生物分子工程的多功能支架。由于ACHTUNGTRENNUNGcyclotides的大小，在30-40个氨基酸的范围内，既可以通过细菌表达重组生产，也可以通过化学合成在这两种途径中，合成后的两个步骤——六个半胱氨酸氧化形成三个二硫键和首尾环化——都需要得到最终的环化产物。虽然环肽主链环化的过程在很大程度上是未知的，但有两种主要的策略被应用于合成大环CK肽。第一种方法依赖于重组合成，并利用修饰的蛋白质剪接元件（称为interins）来形成c端硫酯，该硫酯与N端反应导致大环化。[10,11]第二种策略是基于目标肽的固相合成，然后是氧化和环化。完全去保护的肽已经成功地“压缩”到大环中，随后氧化和胱氨酸结形成在这里，我们提出了一种基于稳定腙形成的已经折叠的微小蛋白质的主链环化策略这种方法利用了廉价和高产的重组生产已经折叠和氧化的线性肽前体的优势。化学合成有效地提供了末端的人工连接，而不干扰由二硫键稳定的结基序的折叠。在线性和环化衍生物的比较中，据报道，具有代表性的亚胺环肽在胰蛋白酶抑制方面效率提高；这也表明
Cyclic peptides form an important class of naturally occurring or synthetic compounds with a large variety of biological activities as, for example, hormones, ion carriers, cancerostatics, antibiotics, antimycotics, or toxins.[1] Biological studies with cyclopeptides have often indicate increased metabolic stability, improved receptor selectivity, and improved activity profiles in comparison with their linear counterparts.[2] Among the group of natural circular peptides and proteins isolated in the last few years from microorganisms, plants, and even from humans,[3] cyclotides provide an especially interesting topology.[4] This family of circular plant proteins displays a head-totail cyclized peptide backbone together with a cystine knot (CK) motif based on disulfide bonds formed by six conserved Cys residues (Figure 1). Two disulfide bonds and their connecting backbone segments form a ring that is penetrated by the third disulfide bond to give a pseudo-knot structure that is ACHTUNGTRENNUNGinvariably associated with the nearby b sheet structure.[5] The cystine knot in combination with the cyclic backbone appears to be a highly efficient motif for structure stabilization, resulting in exceptional conformational rigidity, together with stability against denaturing conditions, as well as against proteolytic degradation. CK-containing peptides are found in almost 20 different protein families with activities such as ion channel blocking (conotoxins and spider toxins), protease inhibition (squash inhibitors), and antiinsecticidal activity (plant cyclotides). Head-to-tail macrocyclic cystine knot peptides have been isolated from plants in the Rubiaceae, Violaceae, and Cucurbitaceae families. Several members of these family have been introduced as versatile scaffolds in drug design and biomolecular engineering.[6]Because of their sizes, in the range of 30–40 amino acids, ACHTUNGTRENNUNGcyclotides are amenable both to recombinant production through bacterial expression and to chemical synthesis.[8] In both routes, two steps of post-synthetic processing—oxidation of six cysteines to form three disulfide bonds and head-to-tail cyclization—are required to obtain the final cyclic product. Although the processes by which cyclotide backbone cyclization occurs naturally are largely unknown,[9] two major strategies have been applied to generate synthetic macrocyclic CK peptides. The first approach relies on recombinant synthesis and makes use of modified protein splicing elements known as inteins to form a C-terminal thioester that reacts with the N terminus to result in macrocyclization.[10, 11] The second strategy is based on a solid-phase synthesis of the target peptide, followed by oxidation and cyclization. Fully deprotected peptides have successfully been “zipped” into macrocycles, followed by oxidation and cystine knot formation.[12] Here we present a strategy for the backbone cyclization of already folded miniproteins based on the formation of a stable hydrazone.[13] This method takes advantage of the combination of cheap and high-yielding recombinant production of linear peptide precursors that are already folded and oxidized. Chemical synthesis efficiently provides the artificial linkage of the termini, not interfering with the fold of the knotted motif stabilized by disulfide bonds. In a comparison of linear and cyclized derivatives, an increased efficiency in tryptase inhibition is reported for a representative iminocyclotide; this also indicates