Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Nat Protoc. 2017 Oct;12(10):2189-2214. doi: 10.1038/nprot.2017.091. Epub 2017 Sep 21.


Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).

MeSH terms

  • Biotechnology
  • Caspases / analysis
  • Caspases / chemistry
  • Caspases / metabolism
  • Combinatorial Chemistry Techniques / methods*
  • Coumarins / chemistry
  • Cysteine Endopeptidases / analysis
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / metabolism
  • Humans
  • Models, Molecular
  • Peptide Hydrolases* / analysis
  • Peptide Hydrolases* / chemistry
  • Peptide Hydrolases* / metabolism
  • Peptide Library*
  • Spectrometry, Fluorescence
  • Substrate Specificity / physiology*
  • Viral Proteins / analysis
  • Viral Proteins / chemistry
  • Viral Proteins / metabolism


  • 7-amino-4-carbamoylmethylcoumarin
  • Coumarins
  • Peptide Library
  • Viral Proteins
  • Peptide Hydrolases
  • Caspases
  • Cysteine Endopeptidases
  • asparaginylendopeptidase