title: Chemical Protein Synthesis Using a Second-Generation N-Acylurea Linker for the Preparation of Peptide-Thioester Precursors.
abstract: The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C-terminal peptide-thioesters, key intermediates in NCL. We have developed an N-acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into N-acylurea peptides. Here, we introduce a new N-acylurea linker bearing an o-amino(methyl)aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired N-acyl-N'-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer.
authors.forename: Juan B
authors.initials: JB
authors.lastname: Blanco-Canosa
authors.affiliation: ‡Departments of Chemistry and Biology, University of Salerno, Via Giovanni Paolo II 132, Fisciano 84084, Italy.
authors.forename: Brunello
authors.initials: B
authors.lastname: Nardone
authors.affiliation: §Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, Barcelona 08028, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Albericio
authors.affiliation: ∥Department of Chemistry, The Scripps Research Institute (TSRI), 10550 N. Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Dawson
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004952:Esters; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
chemical_list: D004952:Esters; D010455:Peptides; D011506:Proteins; D014508:Urea