Real-time bioluminescence imaging of glycans on live cells.
Real-time bioluminescence imaging of glycans on live cells.
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DOI：
10.1021/ja101766r
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发表时间：
2010-06-30
影响因子：
15
通讯作者：
Bertozzi CR
中科院分区：
文献类型：
--
作者：
Cohen AS;Dubikovskaya EA;Rush JS;Bertozzi CR
细胞表面聚糖是分子成像的有吸引力的靶标，因为它们反映了与发育和疾病进展相关的细胞过程。在本文中，我们描述了一种新的膦探针的设计，合成和生物应用的实时成像细胞表面聚糖使用生物发光。为了实现这一目标，我们利用了生物正交化学报告技术。该策略使用两步标记程序，其中含有功能手柄的非天然糖类似物（1）通过细胞自身的生物合成机制掺入含糖蛋白质中，然后（2）用外源添加的探针检测。我们设计了膦酰亚胺试剂1来激活生物发光，以响应与叠氮化物标记的聚糖的施陶丁格连接。我们选择使用膦探针，因为尽管它们的反应动力学缓慢，但它们仍然是标记小鼠叠氮糖的最佳试剂。考虑到生物发光成像（BLI）提供的灵敏度和可忽略的背景，我们推断1可能能够克服荧光膦探针遇到的一些限制。在这项工作中，我们合成了第一个用于实时BLI的膦-双硫代磷酸探针，并证明叠氮化物标记的细胞表面聚糖可以用1成像，使用浓度低至个位数纳摩尔，时间短至5分钟，这是任何以前的荧光膦探针都无法比拟的。尽管我们仅证明了其在可视化聚糖中的用途，但可以设想，该探针也可以用于任何含叠氮化物的生物分子（如蛋白质和脂质）的生物发光成像，因为叠氮化物先前已被掺入这些分子中。因此，膦-精氨酸探针在细胞和整个动物的实时成像中有许多应用。这些研究目前正在我们的实验室进行。
Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell’s own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine−luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine−luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine−luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.
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影响因子：
16.6
作者：
Ning, Xinghai;Guo, Jun;Boons, Geert-Jan
通讯作者：
Boons, Geert-Jan
DOI：
10.1073/pnas.0811481106
发表时间：
2009-01-06
影响因子：
11.1
作者：
Laughlin, Scott T.;Bertozzi, Carolyn R.
通讯作者：
Bertozzi, Carolyn R.
影响因子：
1.8
作者：
Tsujikawa, K.;Kuwayama, K.;Inoue, H.
通讯作者：
Inoue, H.
DOI：
10.1038/nri2417
发表时间：
2008-11
期刊：
Nature reviews. Immunology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1073/pnas.0506855103
发表时间：
2006-03-28
影响因子：
11.1
作者：
Dube, DH;Prescher, JA;Bertozzi, CR
通讯作者：
Bertozzi, CR