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

Abstract

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.