title: Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.
abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Gross
authors.forename: D
authors.initials: D
authors.lastname: Lelièvre
authors.forename: C K
authors.initials: CK
authors.lastname: Woodward
authors.forename: G
authors.initials: G
authors.lastname: Barany
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000138:chemical synthesis* / Q000737:chemistry*; D007611:Aprotinin / Q000138:chemical synthesis / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D005449:Fluorenes / Q000138:chemical synthesis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry*; D013457:Sulfur Compounds / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D000596:Amino Acids; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010446:Peptide Fragments; D013457:Sulfur Compounds; D007611:Aprotinin; D003545:Cysteine