Structural Studies of Copper(I) Complexes of Amyloid-β Peptide Fragments: Formation of Two-Coordinate Bis(histidine) Complexes
Structural Studies of Copper(I) Complexes of Amyloid-β Peptide Fragments: Formation of Two-Coordinate Bis(histidine) Complexes
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DOI：
10.1002/anie.200803908
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Karlin, Kenneth D.
中科院分区：
文献类型：
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作者：
Himes, Richard A.;Park, Ga Young;Karlin, Kenneth D.
大量证据表明，氧化应激是阿尔茨海默病（AD）发病机制和加重的关键事件。[1]过渡金属（如Zn、Fe和Cu）在AD脑沉积物中的浓度升高，主要由40或42聚体淀粉样β（Aβ）肽组成。具有氧化还原活性的铜（II）离子与Aβ的非结构化亲水性N端结合;[1g，2]并且铜通过与Aβ相互作用促进活性氧（ROS）形成并导致神经元死亡的能力已在体外得到证实。[1a，c，3，4] ROS的形成是通过还原态CuI-Aβ与O2或H2 O2的相互作用而发生的。然而，很少有直接研究CuI与Aβ肽或片段的结合或反应性的报道。[5，6]我们研究了Aβ肽的亲水性N-末端区域与CuI的相互作用。在未阐明还原（活性）铜（I）-肽复合物的结构/功能关系的情况下，对导致ROS形成和氧化应激（即，导致与AD发作相关的事件）的Cu-Aβ的完整氧化还原能力的理解是不完整的。我们在此报告了CuI离子与小部分Aβ肽相互作用的研究，这些Aβ肽包含特异性金属结合（His 6、His 13、His 14）或潜在氧化还原活性（Tyr 10）残基（图1）。相当感兴趣的是连续的His 13和His 14残基。我们以前曾报道过对修饰的（通过封端和/或区域特异性Nε-或Nδ-烷基化）His-His二肽的CuI络合物的研究，这些二肽显著地采用了双配位、近线性的NHis-Cu-NHis环境。[6]在这份报告中，我们证明了较长的Aβ肽片段的CuI复合物在固态和水溶液中采用相同的表观二配位结构。本文描述的初步反应性研究表明，His 13-CuI-His 14部分是结构的活性部分，负责铜-A β反应性。合成了一系列肽（图1），并通过反相（RP）HPLC纯化至单峰。通过ESI质谱法确认其身份和纯度。将肽以冻干粉末形式或以双蒸去离子水中的储备溶液形式储存，两者均在80 ℃下储存。[7]在不存在还原剂的情况下，直接从CuI起始材料制备铜（I）-肽复合物，并使用ESI-MS确认其配方。通过光谱技术获得固体和溶液状态的结构信息（见下文）。CuI-Aβ（6-14）和CuI-Aβ（10-14）的固体样品通过将化学计量量的相应肽与[CuI（CH 3CN）4]+盐在DMF中孵育制备，并通过用乙醚沉淀、过滤和减压干燥进行分离。使用ESI-MS确认其配方。[8]处理不当的样品变成深蓝色，表明氧化成CuII，而CuI复合物在排除空气时保持白色至灰色，表明金属-肽复合物减少。对于这些固体样品，X射线吸收光谱法（XAS）被用作测定衍生金属络合物中的氧化态、配位环境和键长的强大（尚未开发，在Cu-Aβ络合物的情况下）工具。[9，10]对于Aβ（6-14）和Aβ（10-14）复合物，1 s！在8983-84 eV处的4p跃迁（图2）明确表明，铜是
Extensive evidence points to oxidative stress as a key event in the pathogenesis and exacerbation of Alzheimer s Disease (AD).[1] Transition metals, such as Zn, Fe, and Cu, are present in elevated concentrations in AD brain deposits, composed primarily of 40-or 42-mer amyloid beta (Aβ) peptides. The redox-active copper (II) ion binds to the unstructured, hydrophilic N terminus of Aβ;[1g, 2] and the ability of copper to promote the formation of reactive oxygen species (ROS) and cause neuronal death by interaction with Aβ has been demonstrated in vitro.[1a, c, 3, 4] ROS formation is proposed to occur by interaction of reduced CuI–Aβ with O2 or H2O2. However, few direct studies of CuI binding or reactivity with Aβ peptides or fragments have been reported.[5, 6] We have studied the interactions of the hydrophilic N-terminal region of the Aβ peptide with CuI. An understanding of the full redox competency of Cu–Aβ, leading to ROS formation and oxidative stress (that is, to cause events associated with the onset of AD), is incomplete without elucidation of the structure/function relationships of the reduced (active) copper (I)–peptide complexes. We report herein studies on the interaction of CuI ions with small portions of the Aβ peptide incorporating specific metalbinding (His6, His13, His14) or potentially redox-active (Tyr10) residues (Figure 1). Of considerable interest are the contiguous His13 and His14 residues. We have previously reported studies on CuI complexes of modified (by endcapping and/or regiospecific Nε-or Nδ-alkylation) His–His dipeptides which, significantly, adopt a two-coordinate, nearlinear NHisÀCuIÀNHis environment.[6] In this report, we demonstrate that CuI complexes of longer Aβ peptide fragments adopt the same apparent two-coordinate structure in the solid state and aqueous solution. Preliminary reactivity investigations, described here, indicate that the His13–CuI–His14 moiety is the active part of the structure, responsible for copper-Aβ reactivity.A range of peptides (Figure1) were synthesized and purified by reverse-phase (RP) HPLC to a single peak. Their identity and purity were confirmed by ESI mass spectrometry. The peptides were stored either as lyophilized powders or as stock solutions in doubly distilled deionized water, both at À808C.[7] Copper (I)–peptide complexes were prepared directly from CuI starting materials in the absence of reductants, and their formulation confirmed using ESI-MS. Structural information was obtained by spectroscopic techniques for both solid and solution states (see below). Solid samples of CuI–Aβ (6–14) and CuI–Aβ (10–14) were prepared by incubating stoichiometric amounts of the respective peptides with a [CuI (CH3CN) 4]+ salt in DMF and isolated by precipitation with diethyl ether, filtration, and drying under reduced pressure. Their formulation was confirmed using ESI-MS.[8] Mishandled samples turned deep blue, indicating oxidation to CuII, whereas the CuI complexes remained white-to-gray when air was excluded, indicating reduced metal–peptide complexes. For these solid samples, X-ray absorption spectroscopy (XAS) was used as a powerful (yet unexploited, in the case of Cu–Aβ complexes) tool for the determination of oxidation state, coordination environment, and bond lengths in the derived metal complexes.[9, 10] For both Aβ (6–14) and Aβ (10–14) complexes, the occurrence of the 1s! 4p transition at 8983–84 eV (Figure 2) definitively indicated that copper was