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t‐boc synthesis of huwentoxin‐i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy
Author(s) -
Thapa Parashar,
Cabalteja Chino C.,
Philips Edwin E.,
Espiritu Michael J.,
Peigneur Steve,
Mille Bea G.,
Tytgat Jan,
Cummins Theodore R.,
Bingham JonPaul
Publication year - 2016
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.22887
Subject(s) - native chemical ligation , thioester , chemistry , peptide , cysteine , stereochemistry , cleavage (geology) , amino acid , combinatorial chemistry , organic chemistry , biochemistry , enzyme , geotechnical engineering , fracture (geology) , engineering
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‐Cys 16 ‐Cys 17 ‐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 (Ala 1 ‐Cys 16 ), 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 EC 50 value of 340.5 ± 26.8 nM for Na v 1.2 and an EC 50 value of 504.1 ± 81.3 nM for Na v 1.3, 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. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 737–745, 2016.