An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations

Amide-forming ligation reactions allow the chemical synthesis of proteins by the union of unprotected peptide segments, and enable the preparation of protein derivatives not accessible by expression or bioengineering approaches. The native chemical ligation (NCL) of thioesters and N-terminal cysteines is unquestionably the most successful approach, but is not ideal for all synthetic targets. Here we describe the synthesis of an Fmoc-protected oxazetidine amino acid for use in the α-ketoacid–hydroxylamine (KAHA) amide ligation. When incorporated at the N-terminus of a peptide segment, this four-membered cyclic hydroxylamine can be used for rapid serine-forming ligations with peptide α-ketoacids. This ligation operates at low concentration (100 μM–5 mM) and mild temperatures (20–25 °C). The utility of the reaction was demonstrated by the synthesis of S100A4, a 12 kDa calcium-binding protein not easily accessible by NCL or other amide-forming reactions due to its primary sequence and properties.ISSN:1755-4349ISSN:1755-433

The Problem of Aspartimide Formation During Protein Chemical Synthesis Using SEA-Mediated Ligation

International audienceAspartimide formation often complicates the solid-phase synthesis of peptides. Much less discussed is the potential occurrence of this side reaction during the coupling of peptide segments using chemoselective peptide bond-forming reactions such as the native chemical ligation and extended methods. Here we describe how to manage this problem using bis(2-sulfenylethyl)amido (SEA)-mediated ligation

The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila

The FYVE-domain protein Rush hour (Rush) binds directly to phosphatidylinositol 3-phosphate and to guanosine diphosphate dissociation inhibitor. It colocalizes with Rab7 and Hrs, and a rush null mutation interacts genetically with mutations in Rab5, Gdi, hrs, and carnation, the fly Vps33 homologue. Rush overexpression blocks the transition from late endosomes to lysosomes, pointing to a function for Rush in regulation of Rabs