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Preparation of the caspase-3/7 substrate Ac-DEVD-pNA by solution-phase peptide synthesis

Nature Protocols volume 5, pages 294–302 (2010)Cite this article

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Abstract

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.

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Figure 1: Pathways of caspase activation.
Figure 2
Figure 3
Figure 4: Evaluation of Ac-DEVD-pNA.

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Acknowledgements

We are grateful to the National Institutes of Health (R01-CA120439) for the support of this work. Q.P.P. was partially supported by a Chemistry–Biology Interface Training Grant from the National Institutes of Health (Ruth L. Kirschstein National Research Service Award 1 T32 GM070421 from the National Institute of General Medical Sciences) and by a predoctoral fellowship from the ACS Division of Medicinal Chemistry. D.C.W. was partially supported by Ruth L. Kirschstein National Research Service Award 3F31CA130138-01S1.

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Authors and Affiliations

  1. Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, Illinois, USA

    Quinn P Peterson & Paul J Hergenrother

  2. Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, Illinois, USA

    David R Goode, Diana C West, Rachel C Botham & Paul J Hergenrother

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  1. Quinn P Peterson
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All authors contributed extensively to the work presented in this paper.

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Correspondence to Paul J Hergenrother.

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NMR Spectrum for Ac-DEVD-pNA (PDF 123 kb)

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Peterson, Q., Goode, D., West, D. et al. Preparation of the caspase-3/7 substrate Ac-DEVD-pNA by solution-phase peptide synthesis. Nat Protoc 5, 294–302 (2010). https://doi.org/10.1038/nprot.2009.223

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