Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: Considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine

C. M. Gross, D. Lelièvre, C. K. Woodward, G. Barany

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31 Scopus citations

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 Nα-9- fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use Nα-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 Nα-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 Nα-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon addolytic cleavage; and (iii) incorporation of Nα-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. Copyright Blackwell Munksgaard, 2005.

Original languageEnglish (US)
Pages (from-to)395-410
Number of pages16
JournalJournal of Peptide Research
Volume65
Issue number3
DOIs
StatePublished - Mar 1 2005

Keywords

  • 9-fluorenylmethoxycarbonyl (Fmoc) strategy
  • Backbone amide linker (BAL)
  • Bovine pancreatic trypsin inhibitor (BPTI)
  • C-terminal thioester intermediates
  • Carbamoyl disulfide (Snm) protection for cysteine
  • Native chemical ligation

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