t-boc synthesis of huwentoxin-i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy

Parashar Thapa, Chino C. Cabalteja, Edwin E. Philips, Michael J. Espiritu, Steve Peigneur, Bea G. Mille, Jan Tytgat, Theodore R. Cummins, Jon Paul Bingham

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3 Scopus citations

Abstract

Tert-butyloxycarbonyl (t-Boc)-based native chemical ligation (NCL) techniques commonly employ hydrogen fluoride (HF) to create the thioester fragment required for the ligation process. Our research aimed to assess the replacement of HF with Trifluoromethanesulfonic acid (TFMSA). Here we examined a 33 amino acid test peptide, Huwentoxin-I (HwTx-I) as a novel candidate for our TFMSA cleavage protocol. Structurally HwTx-I has an X-Cys16-Cys17-X sequence mid-region, which makes it an ideal candidate for NCL. Experiments determined that the best yields (16.8%) obtained for 50 mg of a thioester support resin were achieved with a TFMSA volume of 100 μL with a 0.5-h incubation on ice, followed by 2.0 h at room temperature. RP-HPLC/UV and mass spectra indicated the appropriate parent mass and retention of the cleaved HwTx-I N-terminal thioester fragment (Ala1-Cys16), which was used in preparation for NCL. The resulting chemically ligated HwTx-I was oxidized/folded, purified, and then assessed for pharmacological target selectivity. Native-like HwTx-I produced by this method yielded an EC50 value of 340.5 ± 26.8 nM for Nav1.2 and an EC50 value of 504.1 ± 81.3 nM for Nav1.3, this being similar to previous literature results using native material. This article represents the first NCL based synthesis of this potent sodium channel blocker. Our illustrated approach removes potential restrictions in the advancement of NCL as a common peptide laboratory technique with minimal investment, and removes the hazards associated with HF usage.

Original languageEnglish (US)
Pages (from-to)737-745
Number of pages9
JournalBiopolymers
DOIs
StatePublished - Sep 1 2016

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Keywords

  • native chemical ligation
  • peptide cleavage
  • peptide toxin
  • sodium channels
  • trifluoromethanesulfonic acid

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

Cite this

Thapa, P., Cabalteja, C. C., Philips, E. E., Espiritu, M. J., Peigneur, S., Mille, B. G., Tytgat, J., Cummins, T. R., & Bingham, J. P. (2016). t-boc synthesis of huwentoxin-i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy. Biopolymers, 737-745. https://doi.org/10.1002/bip.22887