University Chemical Laboratory, University of Cambridge, Cambridge, UK.
Nat Protoc. 2015 Apr;10(4):585-94. doi: 10.1038/nprot.2015.033. Epub 2015 Mar 12.
Peptide cyclization is a useful strategy for the stabilization of short flexible peptides into well-defined bioactive conformations, thereby enhancing their ability to interact with proteins and other important biomolecules. We present an optimized procedure for the stabilization of linear diazido peptides in an α-helical conformation upon reaction with dialkynyl linkers under Cu(I) catalysis. As this procedure generates side chain-cyclized peptides bearing a bis-triazole linkage, it is referred to as 'double-click' stapling. Double-click stapling can enhance the binding affinity, proteolytic stability and cellular activity of a peptide inhibitor. A distinguishing feature of double-click stapling is the efficiency with which peptides bearing different staple linkages can be synthesized, thus allowing for modular control over peptide bioactivity. This protocol describes the double-click reaction between a 1,3-dialkynylbenzene linker and peptides that contain azidoornithine. Subsequent peptide purification and confirmation steps are also described. The entire double-click stapling protocol can be completed in ∼48 h, including two overnight lyophilization steps.
肽环化是一种将短而灵活的肽稳定成明确的生物活性构象的有用策略，从而增强它们与蛋白质和其他重要生物分子相互作用的能力。我们提出了一种优化的程序，用于在 Cu(I)催化下，通过与二炔基接头反应，将线性叠氮肽稳定在α-螺旋构象中。由于该程序生成带有双三唑键的侧链环肽，因此称为“双点击”订书钉。双点击订书钉可以提高肽抑制剂的结合亲和力、蛋白水解稳定性和细胞活性。双点击订书钉的一个显著特点是可以高效合成带有不同订书钉连接的肽，从而实现对肽生物活性的模块化控制。本协议描述了 1,3-二炔基苯接头与含有叠氮鸟氨酸的肽之间的双点击反应。随后还描述了肽的纯化和确认步骤。整个双点击订书钉方案可以在约 48 小时内完成，包括两个过夜冻干步骤。
