A Genetically Encoded Boronate-Containing Amino Acid
A Genetically Encoded Boronate-Containing Amino Acid
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DOI：
10.1002/anie.200803240
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
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作者：
Brustad, Eric;Bushey, Mark L.;Schultz, Peter G.
有机硼酸盐作为铃木交偶联反应的合成中间体，[1,2]铜催化的杂原子烷基化反应，[3]不对称还原[4]diols - alder反应，[5]和各种其他转化引起了相当大的兴趣。硼酸还能与二醇、[6]氨基醇、[7]氨基酸、[8,9]烷氧化物和羟肟酸形成可逆的共价配合物后一种性质已被用于合成选择性识别糖的配体[12-14]和开发有效的丝氨酸蛋白酶抑制剂。[15-18]此外，硼酸盐还被用作硼中子捕获剂来杀死肿瘤细胞尽管具有新颖的化学性质，但人们并不知道硼酸在多肽中自然存在，无论是作为翻译后修饰还是作为辅助因子。因此，基因编码这一功能基团的能力将为生物分子识别和蛋白质的选择性化学修饰提供有用的工具。在这里，我们描述了在大肠杆菌中直接将对硼苯丙氨酸(1)特异位点结合到蛋白质中的一般方法（图1A）。我们还证明了该系统在硼酸盐介导的蛋白质修饰以及蛋白质的单步无疤痕纯化中的实用性。我们之前已经描述了在大肠杆菌、酵母、[21]和哺乳动物细胞[22]的遗传密码中添加非天然氨基酸的方法。该方法基于正交tRNA/氨基酰基-tRNA合成酶（aaRS）对的产生，该对允许新氨基酸选择性地结合到蛋白质中，以响应独特的无义和移码密码子。为了选择性地将对硼苯丙氨酸结合到大肠杆菌的蛋白质中，我们使用了一种从jannaschii甲烷球菌（Methanococcus jannaschii, Mj）衍生的琥珀色抑制因子酪氨酸基tRNA
Organoborates have attracted considerable interest as synthetic intermediates for Suzuki crosscoupling reactions,[1, 2] copper catalyzed heteroatom alkylation reactions,[3] asymmetric reductions [4] Diels-Alder reactions,[5] and a variety of other transformations. Boronic acids are also known to form reversible covalent complexes with diols,[6] aminoalcohols,[7] aminoacids,[8, 9] alkoxides [10] and hydroxamic acids.[11] This latter property has been exploited in the synthesis of ligands for the selective recognition of sugars [12–14] and for the development of potent serine protease inhibitors.[15–18] In addition, boronates are finding utility as boron neutron capture agents to kill tumor cells.[19] Despite their novel chemical properties, boronic acids are not known to occur naturally in polypeptides, either as posttranslational modifications or as cofactors. Therefore, the ability to genetically encode this functional group would provide useful tools for biomolecular recognition and for the selective chemical modification of proteins. Herein, we describe a general methodology for the sitespecific incorporation of p-boronophenylalanine (1) into proteins directly in E. coli (Figure 1A). We also demonstrate the utility of this system for boronate mediated protein modification as well as the single step scar-less purification of proteins.We have previously described methodology for the addition of unnatural amino acids to the genetic code of E. coli,[20] yeast,[21] and mammalian cells [22]. This method is based on the generation of an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair that allows the siteselective incorporation of novel amino acids into proteins in response to unique nonsense and frameshift codons. To selectively incorporate p-boronophenylalanine into proteins in E. coli, we have used a Methanococcus jannaschii (Mj) derived amber suppressor tyrosyl tRNA