期刊
BIOPOLYMERS
卷 106, 期 5, 页码 737-745出版社
WILEY-BLACKWELL
DOI: 10.1002/bip.22887
关键词
peptide cleavage; native chemical ligation; trifluoromethanesulfonic acid; peptide toxin; sodium channels
资金
- National Institutes of Health [NS054642]
- National Centre for Research Resources [5 G12 RR003061-26]
- National Institute on Minority Health and Health Disparities [8 G12 MD007601-26]
- USDA NIFA [2011-37610-31182]
- USDA HATCH [HAW00595-R]
- Inter-University Attraction Poles Program, Belgian State, Belgian Science Policy [IUAP 7/10, OT/12/081]
- [G.0433.12]
- [GOE3414N]
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 mu L 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 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. (C) 2016 Wiley Periodicals, Inc.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据