Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
Chembiochem. 2012 May 7;13(7):1032-8. doi: 10.1002/cbic.201200049. Epub 2012 Apr 11.
Combinatorial libraries of structurally diverse peptide macrocycles offer a rich source for the development of high-affinity ligands to targets of interest. In this work we have developed linkers for the generation of genetically encoded bicyclic peptides and tested whether the peptides cyclised by them have significant variations in their backbone conformations. Two new cyclisation reagents, each containing three thiol-reactive groups, efficiently and selectively cyclised linear peptides containing three cysteine moieties. When the mesitylene linker of the bicyclic peptide PK15, a potent inhibitor of plasma kallikrein (K(i)=2 nM), was replaced by the new linkers, its inhibitory activity dropped by a factor of more than 1000, suggesting that the linkers impose different conformations on the peptide. Indeed, structural analysis by solution-state NMR revealed different NOE constraints in the three bicyclic peptides, indicating that these relatively small linkers at the centres of bicyclic peptide structures significantly influence the conformations of the peptides. These results demonstrate the prominent structural role of linkers in peptide macrocycles and suggest that application of different cyclisation linkers in a combinatorial fashion could be an attractive means to generate topologically diverse macrocycle libraries.
结构多样的肽大环组合文库为开发对目标具有高亲和力的配体提供了丰富的来源。在这项工作中，我们开发了用于生成遗传编码双环肽的连接子，并测试了由它们环化的肽在其骨架构象上是否有显著变化。两种新的环化试剂，每个试剂都含有三个硫醇反应基团，能够有效地、选择性地环化含有三个半胱氨酸残基的线性肽。当 PK15 双环肽（一种有效的血浆激肽释放酶抑制剂，K(i)=2 nM）的间二甲苯连接子被新的连接子取代时，其抑制活性下降了 1000 多倍，这表明连接子对肽施加了不同的构象。实际上，通过溶液态 NMR 的结构分析揭示了三种双环肽中的不同 NOE 约束，表明这些位于双环肽结构中心的相对较小的连接子显著影响了肽的构象。这些结果表明连接子在肽大环中的突出结构作用，并表明以组合方式应用不同的环化连接子可能是生成拓扑多样的大环文库的一种有吸引力的方法。
