Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, Illinois, USA.
Nat Protoc. 2010 Feb;5(2):294-302. doi: 10.1038/nprot.2009.223. Epub 2010 Jan 28.
This protocol describes the gram-scale solution-phase synthesis of the colorimetric caspase-3/7 substrate Ac-DEVD-pNA. The caspase enzymes are integral to cellular inflammation and apoptotic cascades, and are commonly studied by cell biologists, medicinal chemists and chemical biologists. In particular, the assessment of caspase enzymatic activity is a standard method to evaluate cell death pathways and new apoptosis-modulating agents. Caspase enzymatic activity can be conveniently monitored with peptidic chromogenic or fluorogenic substrates, with certain peptide sequences imparting selectivity for certain caspases. The synthesis of these peptide substrates is typically carried out by solid-phase synthesis, a method that is not ideal for production of the gram quantities needed for high-throughput screening. Described herein is a facile method for the synthesis of the Ac-DEVD-pNA caspase-3/7 substrate using solution-phase peptide synthesis. This protocol, involving iterative PyBOP-mediated couplings and Fmoc deprotections, is rapid (about 5 d), operationally simple and can be used to generate over 1 g of product at a fraction of the cost of the commercial substrate.
本方案描述了比色 caspase-3/7 底物 Ac-DEVD-pNA 的克级规模溶液相合成。半胱天冬酶酶在细胞炎症和细胞凋亡级联反应中起着重要作用，通常受到细胞生物学家、药物化学家及化学生物学家的研究。特别是，评估半胱天冬酶酶活性是评估细胞死亡途径和新的凋亡调节因子的标准方法。可以通过肽类生色或荧光底物方便地监测半胱天冬酶酶活性，某些肽序列为某些半胱天冬酶提供选择性。这些肽底物的合成通常通过固相合成进行，该方法不适合生产高通量筛选所需的克级数量。本文介绍了使用溶液相肽合成来简便地合成 Ac-DEVD-pNA caspase-3/7 底物的方法。该方案涉及迭代的 PyBOP 介导偶联和 Fmoc 脱保护，反应快速（约 5 天），操作简单，并且可以以商业底物成本的一小部分生成超过 1 g 的产物。
