Posttranslational, site-directed photochemical fluorine editing of protein sidechains to probe residue oxidation state via 19F-nuclear magnetic resonance.
Posttranslational, site-directed photochemical fluorine editing of protein sidechains to probe residue oxidation state via 19F-nuclear magnetic resonance.
复制标题
对蛋白质侧链进行翻译后定点光化学氟编辑，通过 19F 核磁共振探测残基氧化态。
DOI：
10.1038/s41596-022-00800-9
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发表时间：
2023
期刊：
影响因子：
14.8
通讯作者：
Isenegger PG
中科院分区：
文献类型：
--
作者：
Isenegger PG
氨基酸残基的重排代表了一种近电子等排的改变，具有报道生物学途径的潜力，但将复杂生物分子中的碳-氢（C-H）键定点编辑为碳-氟（C-F）键具有挑战性，导致其开发有限。在这里，我们描述了一个协议的翻译后和定点改变天然γ CH 2到γ CF 2的蛋白质侧链。这种改变允许在天然和修饰状态下安装蛋白质氨基酸的二氟化侧链类似物。这种化学编辑是稳健的，温和的，快速的和高效的，利用光化学和自由基介导的C-C键接枝到容易获得的半胱氨酸衍生的含脱氢丙氨酸的蛋白质作为起始材料。产生安装侧链的关键碳中心C·自由基所需的杂芳基磺酰基试剂可以从市售前体通过两到六个步骤合成。这种工作流程允许非专家在24小时内从相应的纯化的含半胱氨酸蛋白质前体开始创建氟化蛋白质，而不需要定制的生物系统。作为一个例子，我们很容易地引入三个γCF2-含有甲硫氨酸在所有三个渐进氧化态（硫化物、亚砜和砜）asd-/l-在位点4形成组蛋白eH3.1（一个相关的赖氨酸到甲硫氨酸的突变位点），并且每一个都可以通过γ CF 2基团的19 F-核磁共振检测到，以及亚砜的两种非对映异构体，即使是在这三种蛋白质的复杂混合物中发现的。C-H→C-F的定点编辑使得γ CF2可以作为蛋白质侧链中的高灵敏度、“零尺寸零背景”标记物，其可以通过基于19 F的检测方法用于探测生物现象、蛋白质结构和/或蛋白质-配体相互作用。
The fluorination of amino acid residues represents a near-isosteric alteration with the potential to report on biological pathways, yet the site-directed editing of carbon–hydrogen (C–H) bonds in complex biomolecules to carbon–fluorine (C–F) bonds is challenging, resulting in its limited exploitation. Here, we describe a protocol for the posttranslational and site-directed alteration of native γCH2to γCF2in protein sidechains. This alteration allows the installation of difluorinated sidechain analogs of proteinogenic amino acids, in both native and modified states. This chemical editing is robust, mild, fast and highly efficient, exploiting photochemical- and radical-mediated C–C bonds grafted onto easy-to-access cysteine-derived dehydroalanine-containing proteins as starting materials. The heteroaryl–sulfonyl reagent required for generating the key carbon-centered C• radicals that install the sidechain can be synthesized in two to six steps from commercially available precursors. This workflow allows the nonexpert to create fluorinated proteins within 24 h, starting from a corresponding purified cysteine-containing protein precursor, without the need for bespoke biological systems. As an example, we readily introduce three γCF2-containing methionines in all three progressive oxidation states (sulfide, sulfoxide and sulfone) asd-/l- forms into histone eH3.1 at site 4 (a relevant lysine to methionine oncomutation site), and each can be detected by19F-nuclear magnetic resonance of the γCF2group, as well as the two diastereomers of the sulfoxide, even when found in a complex protein mixture of all three. The site-directed editing of C–H→C–F enables the use of γCF2as a highly sensitive, ‘zero-size-zero-background’ label in protein sidechains, which may be used to probe biological phenomena, protein structures and/or protein–ligand interactions by19F-based detection methods.