Preparation of Artificial Metalloenzymes
Preparation of Artificial Metalloenzymes
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DOI：
10.1002/9783527804085.ch1
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发表时间：
2018-03
期刊：
影响因子：
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通讯作者：
Jared C. Lewis;Ken Ellis-Guardiola
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文献类型：
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作者：
Jared C. Lewis;Ken Ellis-Guardiola
人工金属酶（ArM）有可能将过渡金属催化剂的关键优势（特别是催化各种具有挑战性的转化的能力）与酶的优势（包括其进化性和分子（即底物）识别能力）结合起来[1]。这些主题和更多的详细讨论，在本卷的其他地方，但他们的追求需要强大的方法为ArM的形成。这种方法本身的发展是相当具有挑战性的。需要位点特异性金属掺入以确保可以获得单位点催化剂。与各种金属和支架的相容性是期望的，以最大化可以探索的化学范围。与水性（理想的是需氧的）反应条件和广泛的官能团（包括在细胞环境中发现的那些）的相容性也是重要的。由预形成的催化剂产生的ArM面临额外的合成挑战，因为这些固有的反应性分子必须首先连接到支架锚定部分以产生ArM辅因子。ArMs的混合性质也使其表征复杂化，因为传统上使用不同的方法来分析过渡金属络合物和蛋白质。各种光谱，包括UV/维斯和电子顺磁共振（EPR），可以提供对金属初级配位球的一些了解[2]，而二色性光谱（CD）和荧光光谱可以提供关于支架折叠的信息[3-5]。在某些情况下，也可以使用NMR光谱法，但其实用性通常受到许多支架蛋白质的高分子量的限制[6]。电感耦合等离子体质谱法（ICP-MS）可用于确定支架：金属化学计量，但不确定支架内的金属位置[6]。高分辨率MALDI和ESI MS通常也可用于确定辅因子掺入和支架修饰的程度[4]。当然，X射线晶体学仍然是明确表征ArMs内金属位置和配位环境的最佳选择，但这种技术通常会因引入金属中心的构象灵活性和可变占用而变得复杂[7]。
Artificial metalloenzymes (ArMs) have the potential to merge key benefits of transition metal catalysts, particularly their ability to catalyze a wide range of challenging transformations, with those of enzymes, including their evolvability and capacity for molecular (ie, substrate) recognition [1]. These topics and more are discussed in detail elsewhere in this volume, but their pursuit requires robust methods for ArM formation. Such methods are in and of themselves quite challenging to develop. Site-specific metal incorporation is required to ensure that single-site catalysts can be obtained. Compatibility with a wide range of metals and scaffolds is desirable to maximize the range of chemistries that can be explored. Compatibility with aqueous, ideally aerobic, reaction conditions and a wide range of functional groups, including those found in cellular milieu, are also important. An additional synthetic challenge is faced for ArMs generated from preformed catalysts, since these inherently reactive molecules must first be linked to scaffold anchoring moieties to generate ArM cofactors. The hybrid nature of ArMs also complicates their characterization since distinct methods have conventionally been used for analysis of transition metal complexes and proteins. Various spectroscopies, including UV/Vis and electron paramagnetic resonance (EPR), can provide some insight into the metal primary coordination sphere [2], while dichroism spectrum (CD) and fluorescence spectroscopies can provide information on scaffold folding [3–5]. In some cases, NMR spectroscopy can also be used, but its utility is often limited by the high molecular weight of many scaffold proteins [6]. Inductively coupled plasma-mass spectrometry (ICP-MS) can be used to determine scaffold: metal stoichiometry, but not metal location within the scaffold [6]. High resolution MALDI and ESI MS can also be used to determine extent of cofactor incorporation and scaffold modification in general [4]. Of course, X-ray crystallography remains the best option for unambiguously charactering metal location and coordination environment within ArMs, but this technique is often complicated by conformational flexibility and variable occupancy of introduced metal centers [7].