t-boc synthesis of huwentoxin-i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy.
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 EC50 value of 340.5 ± 26.8 nM for Nav 1.2 and an EC50 value of 504.1 ± 81.3 nM for Nav 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.
Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822.
Parashar
P
Thapa
Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822.
Chino C
CC
Cabalteja
Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822.
Edwin E
EE
Philips
Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822.
Michael J
MJ
Espiritu
Department of Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N II, Leuven, 3000, Belgium.
Steve
S
Peigneur
Department of Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N II, Leuven, 3000, Belgium.
Bea G
BG
Mille
Department of Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N II, Leuven, 3000, Belgium.
Jan
J
Tytgat
Department of Pharmacology and Toxicology, Indiana University, Indianapolis, IN.|Department of Pharmacology and Toxicology, Stark Neurosciences Research Institute, 320 West 25th Street, NB-414F, Indianapolis, IN, 46202-2266.
Theodore R
TR
Cummins
Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822. jbingham@hawaii.edu.
Jon-Paul
JP
Bingham
D060326:Chemistry Techniques, Synthetic / Q000379:methods*; D008698:Mesylates / Q000737:chemistry*; D030162:Reptilian Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D013111:Spider Venoms / Q000138:chemical synthesis* / Q000737:chemistry
D008698:Mesylates; D030162:Reptilian Proteins; D013111:Spider Venoms; C083624:huwentoxin I; C012077:trifluoromethanesulfonic acid
native chemical ligation; peptide cleavage; peptide toxin; sodium channels; trifluoromethanesulfonic acid