在1919年首次描述的“施陶丁格反应”（Staudinger reaction）中，一个叠氮化物和一个三氢化磷发生反应，形成一个Phosphazo化合物，名叫Aza-ylide。来自伯克利的Carolyn Bertozzi小组将经典有机化学中的这一反应重新命名为 “Staudinger ligation”，它在细胞表面工程领域已成为著名反应。2000年3月 Science杂志上的一篇论文描述了怎样用这种化学方法在不伤害细胞的情况下来对活细胞的表面低聚糖进行标记。现在，这项工作被延伸到了下一个也会让人非常激动的高度：用化学方法改造活体细胞表面。研究人员先用糖硅铝酸的一种叠氮模拟物来标记活小鼠的细胞和组织，然后通过注射一种适合“Staudinger ligation” 的氢化磷试剂来进行活体化学改造。这种能够在细胞表面原始环境中对其进行共价标记的能力，对于活体成像、蛋白组学和治疗目标的锁定等等都是有意义的。
Chemical remodelling of cell surfaces in living animals
Cell surfaces are endowed with biological functionality designed to mediate extracellular communication. The cell-surface repertoire can be expanded to include abiotic functionality through the biosynthetic introduction of unnatural sugars into cellular glycans, a process termed metabolic oligosaccharide engineering1, 2. This technique has been exploited in fundamental studies of glycan-dependent cell–cell and virus–cell interactions3-5 and also provides an avenue for the chemical remodelling of living cells6-8. Unique chemical functional groups can be delivered to cell-surface glycans by metabolism of the corresponding unnatural precursor sugars. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The exquisite chemical selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiology7, 9. Here we demonstrate that the Staudinger ligation can be executed in living animals, enabling the chemical modification of cells within their native environment. The ability to tag cell-surface glycans in vivo may enable therapeutic targeting and non-invasive imaging of changes in glycosylation during disease progression.
Figure 1 The Staudinger ligation and metabolic oligosaccharide engineering. a, The Staudinger ligation of an azide and functionalized phosphine results in the formation of an amide bond. b, Azides can be delivered to cell-surface glycoconjugates by metabolism of ManNAz to SiaNAz. c, Experimental overview for investigating the metabolic conversion of Ac4ManNAz in vivo. Splenocytes from mice treated with the azido sugar were collected and probed for the presence of cell-surface azides using Phos–Flag. Labelled cells were treated with FITC–anti-Flag and analysed by flow cytometry.
Figure 2 Ac4ManNAz is metabolized in vivo. a, Flow cytometry analysis of splenocytes from Ac4ManNAz-treated mice. b, Mean fluorescence intensity (MFI) of the cells from a as a function of azido-sugar dose (circles). Assay controls included unlabelled splenocytes from Ac4ManNAz-treated mice (squares), splenocytes from Ac4ManNAz-treated mice incubated with Phos–Flag followed by a class-matched control monoclonal antibody (diamonds), and splenocytes from Ac4ManNAz-treated mice incubated with FITC–anti-Flag only (triangles). c, MFI of splenocytes from Ac4ManNAz- and ManNAz-treated Es1e/Es1e mice. d, MFI of splenocytes from Es1e/Es1e mice (triangles) or wild-type B6D2F1 mice (circles, males; squares, females) treated with Ac4ManNAz. Error bars represent the standard deviation of the mean for three replicate Staudinger ligation reactions. For a–d similar results were obtained in two replicate experiments.
Figure 3 Analysis of SiaNAz on cells and in tissues. a, Western blot analysis of tissue lysates from Es1e/Es1e mice administered Ac4ManNAz (+ ) or vehicle alone (- ). The same patterns of labelling were apparent in several experiments. b, Splenocytes from B6D2F1 mice treated with Ac4ManNAz or vehicle were treated with A. ureafaciens sialidase (black bars) or left untreated (grey bars). The cells were analysed by flow cytometry; error bars represent the standard deviation of the mean for three replicate Staudinger ligation reactions. c, Western blot analysis of sialidase-treated (+ ) or untreated (- ) serum samples from mice administered Ac4ManNAz or vehicle. The samples were incubated with active or heat-killed (HK) sialidase. Total protein loading was confirmed by Coomassie-stained protein gel (not shown).
Figure 4 The Staudinger ligation proceeds in vivo. Mice were administered Ac4ManNAz or vehicle once daily for 7 days. On the eighth day, the mice were administered Phos–Flag (16 µmol in 200 µl PBS) or an equal volume of vehicle. After 1.5 h, splenocytes were treated with FITC–anti-Flag and analysed by flow cytometry (grey bars). A portion of the isolated splenocytes was further reacted with Phos–Flag and analysed as in Fig. 2 (black bars). Error bars represent the standard deviation of the mean for three replicate FITC–anti-Flag labelling reactions or Staudinger ligation reactions.
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