Thapa Parashar, Cabalteja Chino C, Philips Edwin E, Espiritu Michael J, Peigneur Steve, Mille Bea G, Tytgat Jan, Cummins Theodore R, Bingham Jon-Paul
Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822.
Department of Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N II, Leuven, 3000, Belgium.
Biopolymers. 2016 Sep;106(5):737-45. doi: 10.1002/bip.22887.
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.
基于叔丁氧羰基（t-Boc）的天然化学连接（NCL）技术通常使用氟化氢（HF）来生成连接过程所需的硫酯片段。我们的研究旨在评估用三氟甲磺酸（TFMSA）替代HF。在此，我们研究了一种33个氨基酸的测试肽——虎纹毒素-I（HwTx-I），作为我们TFMSA裂解方案的新候选物。从结构上看，HwTx-I在中间区域有一个X-Cys(16)-Cys(17)-X序列，这使其成为NCL的理想候选物。实验确定，对于50 mg硫酯支持树脂，在100 μL TFMSA、冰上孵育0.5小时、然后室温孵育2.0小时的条件下，获得了最佳产率（16.8%）。反相高效液相色谱/紫外（RP-HPLC/UV）和质谱表明，裂解后的HwTx-I N端硫酯片段（Ala(1)-Cys(16)）具有合适的母离子质量和保留时间，该片段用于NCL制备。所得化学连接的HwTx-I经过氧化/折叠、纯化，然后评估其对药理学靶点的选择性。用这种方法产生的类似天然的HwTx-I对Nav 1.2的半数有效浓度（EC50）值为340.5±26.8 nM，对Nav 1.3的EC50值为504.1±81.3 nM，这与之前使用天然材料的文献结果相似。本文代表了基于NCL首次合成这种强效钠通道阻滞剂。我们阐述的方法消除了NCL作为一种常见肽实验室技术发展中的潜在限制，只需最少的投入，并消除了与使用HF相关的危害。© 2016威利期刊公司。生物聚合物（肽科学）106: 737 - 745，2016。
