Native Chemical Ligation via N-Acylurea Thioester Surrogates Obtained by Fmoc Solid-Phase Peptide Synthesis

  • Judith Palà-Pujadas
  • Juan B. Blanco-CanosaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2133)


Native chemical ligation (NCL) enables the direct chemical synthesis and semisynthesis of proteins of different sizes and compositions, streamlining the access to proteins containing posttranslational modifications (PTMs). NCL assembles peptide fragments through the chemoselective reaction of a C-terminal α-thioester peptide, prepared either by chemical synthesis or via intein-splicing technology, and a recombinant or synthetic peptide containing an N-terminal Cys. Whereas the generation of C-terminal α-thioester proteins can be achieved via the recombinant fusion of the sequence of interest to an intein domain, chemical methods can also be used for synthetically accessible proteins. The use of Fmoc solid-phase peptide synthesis (Fmoc-SPPS) to obtain α-thioester peptides requires the development of novel strategies to overcome the lability of the thioester bond toward piperidine Fmoc-removal conditions. These new synthetic methods enable the easy introduction of PTMs in the thioester fragment. In this chapter, we describe an approach for the synthesis and use of C-terminal α-N-acylbenzimidazolinone (Nbz) and α-N-acyl-N′-methylbenzimidazolinone (MeNbz) peptides in NCL. Following stepwise peptide elongation, acylation with p-nitrophenylchloroformate and cyclization affords the Nbz/MeNbz peptides. The optimization of the coupling conditions allows the chemoselective incorporation of the C-terminal amino acid (aa) on the 3,4-diaminobenzoyl (Dbz) and prevents undesired diacylations of the resulting o-aminoanilide. Following synthesis, these Nbz/MeNbz peptides undergo NCL straightforwardly at neutral pH catalyzed by the presence of arylthiols. Herein, we apply the Nbz technology solid phase synthesis, NCL-mediated cyclization and folding of the heterodimeric RTD-1 defensin, an antimicrobial peptide isolated from the rhesus macaque leukocytes.

Key words

Native chemical ligation (NCL) Peptide-thioesters N-acylurea peptides Peptide o-aminoanilides 



This work was supported by the Spanish Ministerio de Economía y Competitividad (grants CTQ2012-31197 and RYC-2011-09001). J.P.-P. acknowledges an FPI scholarship (BES-2013-065237).


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Authors and Affiliations

  1. 1.Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST)BarcelonaSpain
  2. 2.Department of Biological ChemistryInstitute for Advanced Chemistry of Catalonia – CSIC (IQAC-CSIC)BarcelonaSpain

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