Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, USA.
Acc Chem Res. 2011 Sep 20;44(9):828-39. doi: 10.1021/ar200021p. Epub 2011 May 20.
Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used to label proteins rapidly (within ∼1 min) both in vitro and in E. coli . To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.
生物学中的可视化已经极大地受益于荧光蛋白作为细胞内探针的应用。编码这些波长可调蛋白的基因可以很容易地与感兴趣蛋白的 DNA 融合，从而能够实时直接监测活细胞内天然蛋白。然而，尽管荧光蛋白取得了成功，但它们仍存在一些局限性，这些局限性仅在过去十年中通过生物正交化学的发展才开始得到解决。在这种方法中，一个非常小的生物正交标签被嵌入到细胞的基本构建块中，然后可以选择性地将各种外部分子与这些预标记的生物分子缀合。其结果是为研究人员提供了一个真正的生物物理探针调色板可供选择。在本综述中，我们回顾了我们在开发光诱导的生物正交四唑-烯烃环加成反应（“光点击化学”）方面的进展，并将其应用于探测活细胞中蛋白质的动力学和功能。这里描述的工作总结了四唑的合成、结构和反应性研究，包括它们在生物学应用中的优化。基于早期报告中的关键见解，我们对该反应的初步研究揭示了完全的水兼容性、高光致活量子产率、可调谐的光致活波长以及广泛的底物范围；一个额外的好处是形成荧光环加成产物。随后的研究表明，反应具有快速的动力学（高达 11.0 M(-1) s(-1)），其速率取决于腈亚胺偶极子的 HOMO 能量以及烯烃双亲体的 LUMO 能量。此外，通过使用光结晶学，我们观察到光生成的腈亚胺在固态中采用弯曲的几何形状。这一观察结果导致了合成反应性的、大环四唑的产生，该四唑在两个侧翼的苯基环之间含有一个短“桥”。这种光点击化学已被用于快速标记蛋白质（在体外和大肠杆菌中约 1 分钟内）。为了与生物学建立有效的接口，我们分别鉴定了一种可代谢掺入的烯丙基氨基酸，即 homoallylglycine，和一种可遗传编码的四唑氨基酸，p-(2-四唑基)phenylalanine。我们分别展示了这两个部分在新合成蛋白质的时空控制成像和蛋白质的定点标记中的应用。此外，我们还展示了光点击化学在改变哺乳动物细胞中荧光蛋白定位方面的应用。
