Macrocyclization and Labeling of Helix-Loop-Helix peptide with intramolecular bis-thioether linkage
Toshio Nishihara, Hidekazu Kitada, Daisuke Fujiwara, Ikuo Fujii
Conformationally constrained peptide has developed as an inhibitor for protein-protein interactions (PPIs), and we have de novo designed cyclized helix-loop-helix (cHLH) peptide with a disulfide bond consisting of 40 amino acids, to generate molecular-targeting peptides. However, synthesis of long peptide has sometimes resulted in low yield according to the respective amino acid sequences. Here we developed a method for efficient synthesis and labeling for cHLH peptides. First, we synthesized two peptide fragments and connected them by copper-mediated alkyne and azide cycloaddition (CuAAC) reaction. Cyclization was performed by bis-thioether linkage using 1,3-dibromomethyl-5-propargyloxybenzene (DBMPB), and subsequently the cHLH peptide was labeled with azide-labeled probe. Finally, we designed and synthesized peptide inhibitor for p53-HDM2 interaction by structure-guided design, and successfully labeled it with fluorescent probe or a functional peptide respectively by click chemistry. This macrocyclization and labeling method for cHLH peptide would facilitate to discover de novo bioactive ligands and therapeutic leads. This article is protected by copyright. All rights reserved.
Circular dichroism, Secondary structure, Protein folding, Biochemistry