有机-无机杂化的生物金属仿肽设计与Trp/His芳香环配对的阳离子-pi作用之构效与功效

Metal-site Trp/His (W/H) interactions are crucial to the functions of diverse metalloproteins such as prions and SOD, and the W/H pair is also key to the viable influenza A virus proton channel. Yet the physicochemical basis of functional and mechanistic versatility conferred by a W/H interplay has long been elusive..Design of metal-binding sites is a central step in the "bottom-up" approach to engineering a functional metalloprotein. Though it is well recognized that the basic requirements for a metal-binding site in a metalloprotein focus on the choice of metal ligands with appropriate spatial arrangement surrounding a metal center in order to achieve function, nevertheless, efforts dedicated to understanding how metal-ligand pi interactions (in second coordination sphere) tune active-site properties, and thereby contribute to reactivity of metal center as well as structure / function of the whole metalloprotein, are difficult tasks and currently at an infant research stage. .Since the carboxyl and imidazole group are the prevalent metal-coordinating ligands in metalloproteins, and noncovalent interactions between a Trp indole unit and an imidazole ring is expected to be metal-ion dependent, we envisage that an "organic-inorganic hybrid" strategy can be taken in mimicking metal-binding sites in metalloproteins, and this aggressive approach shall be instrumental in interrogating the structural nature and functional role of a noncovalent interactive W/H pair within a metal-binding site. And therefore we propose to use metal-binding peptoids featuring a W/H pair, constructed on the basis of multi-aminocarboxylate scaffold (such as EDTA chelator),in yielding novel insights into the physical organic chemistry basis of functional and mechanistic versatility conferred by a metal-site W/H interplay.Accordingly,an investigation platform using "M-W/H"-type metallpeptoids, formed with 7 synthetic W/H-containing peptoids together with 5 type of biometal ions (Cu2+, Ni2+, Mn2+, Co2+, Zn2+), will be employed for a systematic and intensive study in this project. .Due to the non C-2 symmetric nature inherent in both the heterocyclic indole and imdazole rings, and a preference of high coordination number of metal ions bound with chelator-based peptoids, metal binding is expected to control the orientation of W/H side chains, and as a consequence, multiple modes of inter-planar orientation of W/H side chain groups can be displayed, allowing the multifaceted physicochemical property of the W/H interactive couple to be captured and resolved. .It is anticipated that such metal-binding-assisted "secondary-structure mimic" will demonstrate a coordination-sphere/geometry dependence of the W/H side-chain interplay, and thus helps understand how aromatic rings around metal sites have unique capabilities through the accurate control of the spatiotemporal distribution of noncovalent interaction elements to achieve diverse functionality.

色氨酸基与组氨酸基（W/H）配对，是多个重要金属蛋白和感冒病毒质子通道中离子结合位点的关键基团，其构效、功效的化学原理不明。金属结合位点是金属蛋白"自下而上"设计的基点。除了对金属配位原子基团的选择，金属配体的阳离子-pi作用如何调节离子选择性、反应性及分子结构和功能，是当前的一个重要难题。项目以探索W/H作用特征为切入点,由氨羧基骨架合成含W/H的仿肽,获得金属仿肽"M-W/H"研究体系。由于羧基、咪唑基是天然结合位点的主要配位基团，吲哚与咪唑基是结构性质复杂的杂环，其非共价互相作用依赖于离子的存在，因而，随着金属的配位控制了支链基团构象，氨羧基的高配位产生的立体专一性与杂环的C-2不对称性,导致两个环之间多种形式的立体选择性趋向与互相作用,从而揭示W/H配对具有多种功效的化学本质。该"有机-无机杂化"的二级结构模拟对金属附近W/H互相作用、构效与功效的探索，具有基础与潜在应用研究意义。

天然氨基酸功能基团提供的非共价作用力、及其在金属蛋白活性中心、离子识别中的构效和功效，是本项目的研究内容。项目以亲水、疏水的有机、无机小分子作连接模板，合成含芳香氨基酸（组氨酸H、色氨酸W、酪氨酸Y）的仿肽，以多光谱实验技术（ESI-HRMS、13C NMR、荧光、园二色散、顺磁共振EPR、XPS），系统研究仿肽对金属阳离子、阴离子的识别原理，及功能基团的非共价作用。仿肽结合金属离子，获得模拟金属蛋白的金属仿肽“M—W/H”和“M—Y/H”，以探索这些结构中的“W/H”、“Y/H”配对非共价作用与构效、功效的关联。12个仿肽之化学结构式为X—（A，A’）系列，其中，氨羧基分子骨架X = EDTA、DTPA、EGTA、TMDTA；氨基酸A，A’= 分别是氨基酸W，H，Y的甲酯。金属离子为Cu2+、Ni2+、Zn2+、Co2+、Ca2+。结果分4个方面：（一）NMR、ESI-HRMS、荧光滴定，确立金属仿肽的稳定性、仿肽—金属离子的作用新模式。（二）通过改变仿肽中的氨羧基分子骨架的种类、长短、含有氨羧基基团的数量，深入认识组氨酸基(H) 在该类仿肽X- W/H结合过渡金属离子后的配位特点、结构特征， W的吲哚环与H的咪唑基之间的作用强度。（三）在多个氨羧基、组氨基酸基存在下的金属仿肽“M—H/H”中的“H/H”配对作用。确立了双组氨酸仿肽X—(H)2系列（X = EDTA、TMDTA、DTPA、EGTA)与Cu2+的配合物的脱羧反应机理。建立了以ESI-HRMS技术测定该类金属—仿肽配合物中的铜离子价态的方法平台 — 碱金属离子亲和法，外界配位体竞争法。（四）深入研究了金属仿肽中的“Y/H”配对作用与构效。以碱金属离子亲和法，重点确立了仿肽的单核、双核铜离子配合物的结构。仿肽与离子的配位导致W/H、Y/H 双环之间多种形式的立体选择性趋向与互相作用,从而体现出多种功效的化学本质。W/H 、Y/H在金属酶中心的功能实现、离子识别、分子组装中均发挥非共价作用基础上的多种特殊功效。该“有机—无机”相结合的结构模拟对W/H 、Y/H互相作用的探索，具有多方面的化学和化学生物学意义。