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

Development of an Anilide-Type Scaffold for the Thioester Precursor <i>N</i>‑Sulfanylethylcoumarinyl Amide

<i>N</i>-Sulfanylethylcoumarinyl amide (SECmide) peptide, which was initially developed for use in the fluorescence-guided detection of promoters of N–S acyl transfer, was successfully applied to a facile and side reaction-free protocol for N–S acyl-transfer-mediated synthesis of peptide thioesters. Additionally, 4-mercaptobenzylphosphonic acid (MBPA) was proven to be a useful catalyst for the SECmide or <i>N</i>-sulfanylethylanilide (SEAlide)-mediated NCL reaction

Molecular Complex Composed of β‑Cyclodextrin-Grafted Chitosan and pH-Sensitive Amphipathic Peptide for Enhancing Cellular Cholesterol Efflux under Acidic pH

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of β-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to β-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (<i>K</i> = 3.7 × 10⁷ at pH 5.5) compared to that at pH 7.4 (<i>K</i> = 7.9 × 10⁴). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis