title: A reversible protection strategy to improve Fmoc-SPPS of peptide thioesters by the N-Acylurea approach.
abstract: C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully or semisynthetic proteins. However, the efficient generation of C-terminal thioesters by Fmoc solid-phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on the deactivation of one amine of 3,4-diaminobenzoic acid (Dbz) during Fmoc SPPS. Here, we demonstrate that this approach results in the formation of side products through the over-acylation of Dbz, particularly when applied to Gly-rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc-Dbz(Alloc) base resin that may be directly used for synthesis with most C-terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N-acyl-benzimidazolinone (Nbz) species, which can be purified and converted in situ to thioester under ligation conditions. This method is compatible with the automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves the synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.
authors: Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors: Santosh K
authors: SK
authors: Mahto
authors: Cecil J
authors: CJ
authors: Howard
authors: John C
authors: JC
authors: Shimko
authors: Jennifer J
authors: JJ
authors: Ottesen
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D062365:Aminobenzoates / Q000737:chemistry; D004952:Esters; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
chemical_list: D062365:Aminobenzoates; D004952:Esters; D010455:Peptides; D013438:Sulfhydryl Compounds; C041192:3,4-diaminobenzoic acid; D014508:Urea