Dadová Jitka, Wu Kuan-Jung, Isenegger Patrick G, Errey James C, Bernardes Gonçalo J L, Chalker Justin M, Raich Lluís, Rovira Carme, Davis Benjamin G
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
Departament de Química Inorgànica i Orgànica (secció de Química Orgànica) & Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain.
ACS Cent Sci. 2017 Nov 22;3(11):1168-1173. doi: 10.1021/acscentsci.7b00341. Epub 2017 Nov 13.
Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, His) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-His. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from and pantothenate synthetase.
仿生学有助于人们了解重现生物功能所需的基本化学成分，这一点很有价值。然而，评估氨基酸在蛋白质中作用的直接策略可能会受到获取合适的、经过细微改变的非天然变体的限制。在此，我们描述了一种策略，利用前所未有的化学翻译后侧链β,γ C-N键形成来剖析组氨酸残基在酶活性位点中的作用。引入脱氢丙氨酸（作为“标签”）能够在“氮杂迈克尔”1,4-加成反应（用于“修饰”）中测试氮共轭亲核试剂。这使得能够创建一种通过其前Nπ原子连接而非天然通过C4连接的组氨酸区域异构体（异组氨酸，His），以及一种氮杂改变的变体氮杂组氨酸。这些非天然氨基酸的位点选择性生成成功应用于探究组氨酸残基对来自嗜热栖热菌的枯草杆菌蛋白酶和泛酸合成酶等模型酶活性的贡献作用（例如大小、氢键作用）。
