Synthetic study of peptide toxins for the elucidation of functions of ion channels

1.研究了ω-芋螺毒素TxVII（一种L-型钙通道阻断剂）和δ-芋螺毒素TxVIA（一种减缓钠通道失活的肽毒素）的两种嵌合类似物（δω和ωδ）的合成。8 w的线性前体的空气氧化以良好的产率得到所需的类似物。而ωδ则是二硫键异构体的复杂混合物。两步选择性二硫键形成策略也未能得到正确的产物，表明δω缺乏形成三维结构所必需的残基。采用核磁共振和模拟退火方法对δ-芋螺毒素TxVIA的三维结构进行了分析。δ-芋螺毒素TxVIA在分子的一侧显示了一个不寻常的疏水性斑块，这可能在钠通道结合中起重要作用。2.通过将C端的Ala 22分别替换为酸性Glu（A22 E）和碱性Lys（A22 K），合成了两个选择性钠通道阻断剂μ-芋螺毒素GIIIA类似物。A22 E的活性比天然μ GIIIA低90倍，但对钠通道的E765 K突变体显示出高亲和力，表明μGIIIA的C端部分与钠通道的结构域II密切相关。A22 K的活性也低于天然μGIIIA，说明赖氨酸残基的大侧链可能会干扰结合。3.化学合成了从黄足异蝎（Heterometrus fulvipes）毒中分离的两种肽毒素κ-hefutoxin 1和2，并证实其与天然毒素一致。通过酶消化测定二硫键配对。核磁共振研究表明，κ-hefutoxin 1是一种独特的三维折叠结构，由两个平行的螺旋通过两个二硫键连接而没有任何β-折叠。κ-Hefutoxin 1不仅阻断电压门控钾通道Kv1.3和Kv1.2，还减慢Kv1.3电流的激活动力学。

1.Syntheses of two chimeric analogs (δω and ωδ) of ω-conotoxin TxVII, an L-type calcium channel blocker, and δ-conotoxin TxVIA, a peptide toxin that slows the inactivation of sodium channels were examined. Air oxidation of a linear precursor of 8w afforded desired analog in good yield. However, that of ωδ gave complex mixture of disulfide bond isomers. Two-step selective disulfide bond formation strategy also failed to give correct product, suggesting that δω lack the residues essential for the 3D structure formation. 3D structure of δ-conotoxin TxVIA was analyzed by NMR and simulated annealing calculations. δ-Conotoxin TxVIA showed an unusual hydrophobic patch on one side of the molecule, which may play am important role in, the sodium channel binding.2.Two analogs of μ-conotoxin GIIIA, a selective blocker of muscle sodium channels, were synthesized by replacing C-terminal Ala22 with acidic Glu (A22E) and basic Lys (A22K) residues. A22E was 90-fold less active than native μGJIIA, however it showed high affinity against E765K mutant of sodium channel, indicating that C-terminal part of μGIIIA closely associates with domain II of sodium channels. A22K was also less active than native μGIIIA, suggesting that the large side chain of Lys residue may interfere the binding.3.Two peptide toxins, κ-hefutoxin 1 and 2, isolated from the venom of the scorpion Heterometrus fulvipes were chemically synthesized and confirmed to be identical to native toxins. The disulfide bond pairings were determined by enzymatic digestion. NMR studies indicated that κ-hefutoxin 1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any β-sheets. κ-Hefutoxin 1 not only blocks the voltage-gated potassium channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents.