Synthesis of Cysteine-Rich Peptides by Native Chemical Ligation without Use of Exogenous Thiols

Native chemical ligation (NCL) performed without resorting to the use of thiol additives was demonstrated to be an efficient and effective procedure for synthesizing Cys-rich peptides. This method using tris­(2-carboxyethyl)­phosphine (TCEP) as a reducing agent facilitates the ligation reaction even at the Thr-Cys or Ile-Cys site and enables one-pot synthesis of Cys-rich peptides throughout NCL and oxidative folding

<i>N</i>‑Sulfanylethyl­aminooxy­butyramide (SEAoxy): A Crypto-Thioester Compatible with Fmoc Solid-Phase Peptide Synthesis

An <i>N</i>-sulfanylethyl­amino­oxy­butyramide (SEAoxy) has been developed as a novel thioester equivalent for native chemical ligation. SEAoxy peptide was straightforwardly synthesized by conventional Fmoc solid-phase peptide synthesis without a problem. Moreover, SEAoxy peptide could be directly applied to native chemical ligation owing to the intramolecular <i>N</i>-to-<i>S</i> acyl shift that releases the peptide-thioester <i>in situ</i>. This methodology was successfully applied to the synthesis of two bioactive peptides

Synthetic Procedure for <i>N</i>‑Fmoc Amino Acyl‑<i>N</i>‑Sulfanylethylaniline Linker as Crypto-Peptide Thioester Precursor with Application to Native Chemical Ligation

<i>N</i>-Sulfanylethylanilide (SEAlide) peptides <b>1</b>, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides <b>2</b> can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of <i>N</i>-Fmoc amino acyl-<i>N</i>-sulfanylethylaniline linkers <b>4</b>, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the <i>N</i>-sulfanylethylaniline linker <b>5</b>. <i>N</i>-Fmoc amino acyl aniline linkers <b>4</b> of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl₃- or SOCl₂-activated Fmoc amino acid derivatives with sodium anilide species <b>6</b>, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides <b>7</b> possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry