Surface Chemistries Enabling Photoinduced Uncoupling/Desorption of Covalently Tethered Biomolecules.
Surface Chemistries Enabling Photoinduced Uncoupling/Desorption of Covalently Tethered Biomolecules.
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表面化学能够实现共价束缚生物分子的光诱导解偶联/解吸附。
DOI：
10.1021/jo960534b
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发表时间：
1996
期刊：
影响因子：
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通讯作者：
T. Hutchens
中科院分区：
文献类型：
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作者：
J. Ching;K. Voivodov;T. Hutchens
研究和利用表面结合分子的分子识别 1, 2 的机会的扩大，推动了大分子与明确的无机表面结构的共价但可逆附着的新化学的发展。 “永久”束缚在探针表面的感兴趣分子可能会暴露于各种环境或受到物理/化学扰动，这些扰动会解离通过非共价相互作用简单吸附的分子。这项研究的总体目标是更好地定义构建纳米级探针表面结构所需的化学物质，其中分子装置可根据需要在指定区域结合和释放共价束缚分子。我们重点研究了利用相干光来控制分子释放功能。我们还需要一种机制来直接研究参与蛋白质和其他大分子共价固定到这些表面的表面残基的数量、类型和化学性质。目的是探索涉及偶氮苯衍生物的键的光解效率和光化学反应均匀性，以设计光不稳定的系链，使其在激光照射下能够同时解偶联/解吸。为构建该装置而选择的光解成分 4, 4'-偶氮二苯胺 (ADA) 被合并在相对较短的间隔臂末端，该间隔臂以羰基咪唑作为合适的离去基团，用于分子（例如肽）与反应性亲核试剂的共价连接（图 1）。所选的模型肽代表了富含组氨酸糖蛋白 (HRG) 的蛋白质中金属结合结构域的一个有趣部分。 4 肽序列由重复四次的五残基富含组氨酸排列 (GHHPH) 定义。将该肽引入二氧化硅探针表面上的一系列 ADA 系链探针装置中，以评估激光诱导的解偶联。共价偶联后进行广泛的表面清洗程序，以去除所有非共价结合的材料。随后的激光照射揭示了几种不同的光解事件。在每种情况下，通过用单个激光脉冲（3 ns）照射，实现了共价束缚生物分子的解偶联以及解吸。在大多数情况下，系链生物分子的光依赖性释放的特征是解偶联/解吸生物分子质量的增加，这允许直接验证系链探针装置内的光解裂解位点（图2）。图 2A 所示的结果是典型的；由标记为 (1) 和 (5) 的峰代表的光解反应产物是最主要的 (n) 12)。在一些实验中，光解裂解产物的总数和主要种类似乎有所不同，峰 (6) 成为主要种类（图 2B，顶部）。虽然通常仅观察到两种主要的光解反应产物（即峰 1 与峰 5 或 6 一起），但在某些情况下 (n) 5)，仅观察到单个光解裂解产物（图 2B，底部）。最有可能影响光诱导解偶联时观察到的解吸产物类型的因素包括激光功率、照射波长和用于促进解吸/电离的基质制备类型。对这些因素的详细调查正在进行中。与解离的生物分子共价连接的系链化学残余物提供了与探针表面共价连接的直接证据。完整的系绳探针装置的详细化学结构是……
The development of new chemistries for the covalent but reversible attachment of macromolecules to welldefined inorganic surface structures is being driven by the expanded realization of opportunities to investigate and exploit molecular recognition1, 2 of the surface-bound molecules. Molecules of interest that are “permanently” tethered to a probe surface may be exposed to a wide variety of environments or subjected to physical/chemical perturbations that would dissociate molecules simply adsorbed through noncovalent interactions. The overall goal of this investigation was to better define the chemistries needed to build nanoscale probe surface architectures with molecular devices functioning to bind and release covalently tethered molecules in defined areas on demand. We have focused on the use of coherent light to control the molecular release function. We also desired a mechanism to investigate directly the number, type, and chemical nature of surface residues involved in the covalent immobilization of proteins and other macromolecules to these surfaces. An objective was to explore the photolytic efficiency and photochemical reaction homogeneity of bonds involving azobenzene derivatives3 for the purpose of designing photolabile tethers that would enable simultaneous uncoupling/desorption upon laser irradiation. The photolytic component chosen for the construction of this device, 4, 4′-azodianiline (ADA), was incorporated at the end of a relatively short spacer arm that was terminated with carbonylimidazole as a suitable leaving group for covalent attachment of molecules (eg, peptides) with reactive nucleophile (s)(Figure 1). The model peptide chosen represents an interesting portion of the metal-binding domain in a protein referred to as histidine-rich glycoprotein (HRG). 4 The peptide sequence is defined by a five-residue histidine-rich arrangement (GHHPH) repeated four times. The peptide was introduced to an array of ADA tetherprobe devices on a silica-based probe surface for evaluation of laser-induced uncoupling. An extensive surface wash procedure followed covalent coupling to remove all noncovalently bound material. Subsequent laser irradiation revealed several different photolytic events. In each case, uncoupling of the covalently tethered biomolecule was achieved, together with desorption, by irradiation with a single laser pulse (3 ns). The light-dependent release of the tethered biomolecules was characterized, in most cases, by an increase in mass of the uncoupled/desorbed biomolecule that allowed direct verification of the photolytic cleavage site within the tether-probe device (Figure 2). Results such as those shown in Figure 2A were typical; photolytic reaction products represented by peaks labeled (1) and (5) were most predominant (n) 12). In some experiments, the total number and predominant species of photolytic cleavage products appeared to vary, with peak (6) becoming the predominant species (Figure 2B, top). Although only two major photolytic reactions products were typically observed (ie, peak 1 together with either peak 5 or 6), in some cases (n) 5), only a single photolytic cleavage product was observed (Figure 2B, bottom). Factors most likely to affect the type of desorbed product observed upon photoninduced uncoupling include laser power, irradiation wavelength, and type of matrix preparation used to promote desorption/ionization. A detailed investigation of these factors is underway. The chemical remnants of tether left covalently attached to the dissociated biomolecule provide direct evidence of covalent linkage to the probe surface. The detailed chemical structure of the intact tether-probe device is …