Side-Chain Anchoring Strategy for Solid-Phase Synthesis of Peptid Acids with C-Terminal Cysteine

George Barany, Yongxin Han, Balazs Hargittai, Rong Qiang Liu, Jaya T. Varkey

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Many naturally occurring peptide acids, e.g., somatostatins, conotoxins, and defensins, contain a cysteine residue at the C-terminus. Furthermore, installation of C-terminal cysteine onto epitopic peptide sequences as a preliminary to conjugating such structures to carrier proteins is a valuable tactic for antibody preparation. Anchoring of Nα-Fmoc, S-protected C-terminal cysteine as an ester onto the support for solid-phase peptide synthesis is known to sometimes occur in low yields, has attendant risks of racemization, and may also result in conversion to a C-terminal 3-(1-piperidinyl)alanine residue as the peptide chain grows by Fmoc chemistry. These problems are documented for several current strategies, but can be circumvented by the title anchoring strategy, which features the following: (a) conversion of the eventual C-terminal cysteine residue, with Fmoc for N α-amino protection and tert-butyl for Cα -carboxyl protection, to a corresponding S-xanthenyl (2XAL 4) preformed handle derivative; and (b) attachment of the resultant preformed handle to amino-containing supports. This approach uses key intermediates that are similar to previously reported Fmoc-XAL handles, and builds on earlier experience with Xan and related protection for cysteine. Implementation of this strategy is documented here with syntheses of three small model peptides, as well as the tetradecapeptide somatostatin. Anchoring occurs without racemization, and the absence of 3-(1-piperidinyl)alanine formation is inferred by retention of chains on the support throughout the cycles of Fmoc chemistry. Fully deprotected peptides, including free sulfhydryl peptides, are released from the support in excellent yield by using cocktails containing a high concentration (i.e., 80-90%) of TFA plus appropriate thiols or silanes as scavengers. High-yield release of partially protected peptides is achieved by treatment with cocktails containing a low concentration (i.e., 1-5%) of TFA. In peptides with two cysteine residues, the corresponding intramolecular disulfide-bridged peptide is obtained by either (a) oxidation, in solution, of the dithiol product released by acid; (b) simultaneous acidolytic cleavage and disulfide formation, achieved by addition of the mild oxidant DMSO to the cleavage cocktail; or (c) concomitant cleavage/cooxidation (involving a downstream S-Xan protected cysteine), using reagents such as iodine or thallium tris(trifluoroacetate) in acetic acid.

Original languageEnglish (US)
Pages (from-to)652-666
Number of pages15
JournalBiopolymers - Peptide Science Section
Volume71
Issue number6
DOIs
StatePublished - 2003

Keywords

  • C-terminal cysteine
  • Cocktail X
  • Partially protected peptide
  • Piperidinyl adduct
  • Racemization-free anchoring
  • Side-chain anchoring
  • Solid-phase peptide synthesis
  • Xanthenyl handle

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