Synergic "Click" Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells
Synergic "Click" Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells
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DOI：
10.1021/jacs.7b08693
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发表时间：
2017-10-11
影响因子：
15
通讯作者：
Hall, Dennis G.
中科院分区：
文献类型：
--
作者：
Akgun, Burcin;Li, Caishun;Hall, Dennis G.
快速，高产，和选择性的生物正交的“点击”反应，采用无毒的试剂是在高需求的巨大效用，在活细胞中的生物分子的共轭。尽管为此目的开发了许多点击反应，但许多都与缺点和限制相关，这些缺点和限制证明了开发用于单标记或双标记应用的替代系统的合理性。最近的报道强调了硼酸酯形成作为非生物硼酸和二醇之间的生物正交点击反应的潜力。硼酸酯的形成是一个快速的脱水过程;然而，它在水性介质中本质上是可逆的。我们设计并优化了一个基于两个双功能试剂的协同体系，一个氨基硫脲官能化的诺泊二醇和一个邻乙酰基芳基硼酸。两种试剂均显示在高达50 μ M的浓度下化学稳定且对HEK 293 T细胞无毒。所得的硼酸酯/缩氨基硫脲加合物是一个中等大小的环，在中性缓冲液中，在低μ M浓度下，在没有任何催化剂的情况下快速且不可逆地形成，通过NMR光谱法测量的速率常数为9 M-1 s（-1）。在竞争性硼酸存在下的对照实验显示没有交叉副产物，并证实了硼酸盐/缩氨基硫脲缀合物的稳定性和不可逆性。缀合物的形成不受生物二醇如果糖、葡萄糖和儿茶酚的存在的影响，并且氨基硫脲官能化的nopoldiol对在蛋白质结合的glycopyryl单元上发现的醛亲电体是惰性的。使用SNAP-标签方法将硼酸试剂安装到HEK 293 T细胞中β-2肾上腺素能受体的细胞外结构域上，然后与用荧光素染料标记的最佳氨基硫脲官能化的nopoldiol试剂孵育，证明了该系统在活细胞的细胞表面标记中的适用性。成功的可视化荧光显微镜是可能的试剂浓度低至10 μ M，从而证实了该系统在生物应用中的潜力。
Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as SO mu M. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low mu M concentrations, in neutral buffer, with a rate constant of 9 M-1 s(-1) as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 mu M, thus confirming the potential of this system in biological applications.