A Genetically Encoded Norbornene Amino Acid for the Mild and Selective Modification of Proteins in a Copper-Free Click Reaction
A Genetically Encoded Norbornene Amino Acid for the Mild and Selective Modification of Proteins in a Copper-Free Click Reaction
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DOI：
10.1002/anie.201109252
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Carell, Thomas
中科院分区：
文献类型：
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作者：
Kaya, Emine;Vrabel, Milan;Carell, Thomas
目前，对蛋白质进行位点特异性化学修饰的方法对于合成用于制药和诊断目的的蛋白质杂化化合物具有重要意义大多数方法依赖于游离蛋白硫醇与马来酰亚胺[2]或赖氨酸侧链与活性酯[3]的反应这些方法只提供有限的特异性，这促使研究人员开发替代策略，包括将特殊的非天然氨基酸结合到蛋白质中，以实现位点特异性的生物正交功能化在已开发的方法中，含有炔基氨基酸的蛋白质与叠氮化合物的催化反应是研究最彻底的技术。[5,6]然而，对CuI盐的需求可能会损害蛋白质结构，限制了该技术的发展这激发了目前对开发与脆弱蛋白质结构兼容的无铜偶联反应的兴趣在这里，我们表明，这些要求可以满足特殊编码的降冰片烯氨基酸，选择性地与腈亚胺反应。为了将降冰片烯氨基酸插入到蛋白质中，我们采用了基于Methanosarcina mazei.[9]中pyrlyyl -tRNA合成酶（tRNAPyl/PylRS）对的琥珀色抑制技术该项目的主要任务是进化丙酮赖氨酸合成酶，使其接受合成的降冰片烯氨基酸1（方案1a）装载到丙酮赖氨酸- trna上。在这项研究中，我们用现成的原料，分七个步骤合成了含有降冰片烯的Pyl类似物1（见支持信息）。为了测试1在多大程度上被野生型（wt） PylRS接受，我们使用大肠杆菌细胞编码完整的tRNAPyl/PylRS对和含有一个帧内TAG停止密码子的修饰黄色荧光蛋白（YFP）在该系统中，只有当相应的Pyl类似物被PylRS接受并成功加载到tRNAPyl上并随后在琥珀色停止密码子位点插入蛋白质时，才能产生全长的荧光YFP。所制备的大肠杆菌细胞在含有5mm1的培养基中生长。除了野生型PylRS外，我们还测试了Yanagisawa及其同事先前使用的PylRS突变体（Y384F）。[4f]当使用突变体PylRS （Y384F）时，这些最初的实验只提供微弱的荧光。在野生型PylRS存在的情况下，不产生荧光，因此不产生全长YFP。为了提高PylRS的活性，我们使用Reetz和同事开发的迭代饱和诱变（ISM）来进化蛋白质。基于PylRS与腺苷化pyrysine （PDB 2Q7H）配合物的共晶结构，我们选择了wt-PylRS底物结合口袋中的5个残基进行实验。将基于质粒的PylRS文库转化为大肠杆菌后，在添加1的液体培养基中培养单个菌落。通过细胞的YFP荧光强度监测掺入情况。然后对最有效的PylRS变异体进行测序并用于下一轮饱和诱变。在
Methods for the site-specific chemical modification of proteins are currently of immense importance for the synthesis of protein–hybrid compounds for pharmaceutical and diagnostic purposes.[1] Most of the methods rely on the reaction of free protein thiols with maleimides [2] or the reaction of lysine side chains with activated esters.[3] These methods provide only limited specificity, which is prompting researchers to develop alternative strategies that involve the incorporation of special unnatural amino acid into proteins to enable site-specific bioorthogonal functionalization.[4] Among the developed methods, the CuI-catalyzed reaction of a protein containing an alkyne amino acid with azides stands out as the most thoroughly investigated technology.[5, 6] However, the need for CuI salts, which may harm the protein structure, limits the technology.[7] This fuels current interest to develop copperfree coupling reactions that are compatible with fragile protein structures.[8] Here we show that these requirments can be met with a specially encoded norbornene amino acid which reacts selectively with nitrile imines. In order to insert a norbornene amino acid into a protein we used the amber suppression technique based on the pyrrolysyl tRNA/pyrrolysyl-tRNA synthetase (tRNAPyl/PylRS) pair from Methanosarcina mazei.[9] The main task of the project was to evolve the pyrrolysine synthetase so that it accepts the synthetic norbornene amino acid 1 (Scheme 1 a) for loading onto the pyrrolysyl-tRNA. For the study we synthesized the norbornene-containing Pyl analogue 1 in seven steps from readily available starting materials (see the Supporting Information). To test to what extent 1 is accepted by wild-type (wt) PylRS, we used E. coli cells encoding the full tRNAPyl/PylRS pair and a modified yellow fluorescent protein (YFP) containing one in-frame TAG stop codon.[10] In this system the full-length and hence fluorescent YFP can only be produced when the corresponding Pyl analogue is accepted by the PylRS and successfully loaded onto the tRNAPyl for subsequent incorporation into the protein at the amber stop codon site. The so-prepared E. coli cells were grown in a medium containing 5 mm 1. In addition to the wild-type PylRS we also tested a PylRS mutant (Y384F) previously used by Yanagisawa and co-workers.[4f] These initial experiments provided a just faint fluorescence when the mutant PylRS (Y384F) was used. No flurescence and hence no full-length YFP was generated in the presence of wild-type PylRS. In order to increase the PylRS activity we evolved the protein using iterative saturation mutagenesis (ISM) developed by Reetz and co-workers.[11]Based on the co-crystal structure of PylRS in complex with adenylated pyrrolysine (PDB 2Q7H) we selected five residues in the substrate-binding pocket of wt-PylRS for the experiments. After transformation of the plasmid-based PylRS library into E. coli, single colonies were grown in liquid cultures supplemented with 1. The incorporation was monitored by means of the YFP fluorescence intensity of the cells. The most efficient PylRS variants were then sequenced and used in the next round of the saturation mutagenesis. In