在1919年首次描述的“施陶丁格反應(yīng)”（Staudinger reaction）中,，一個(gè)疊氮化物和一個(gè)三氫化磷發(fā)生反應(yīng)，形成一個(gè)Phosphazo化合物,，名叫Aza-ylide,。來(lái)自伯克利的Carolyn Bertozzi小組將經(jīng)典有機(jī)化學(xué)中的這一反應(yīng)重新命名為 “Staudinger ligation”，它在細(xì)胞表面工程領(lǐng)域已成為著名反應(yīng),。2000年3月 Science雜志上的一篇論文描述了怎樣用這種化學(xué)方法在不傷害細(xì)胞的情況下來(lái)對(duì)活細(xì)胞的表面低聚糖進(jìn)行標(biāo)記?，F(xiàn)在，這項(xiàng)工作被延伸到了下一個(gè)也會(huì)讓人非常激動(dòng)的高度：用化學(xué)方法改造活體細(xì)胞表面,。研究人員先用糖硅鋁酸的一種疊氮模擬物來(lái)標(biāo)記活小鼠的細(xì)胞和組織,，然后通過(guò)注射一種適合“Staudinger ligation” 的氫化磷試劑來(lái)進(jìn)行活體化學(xué)改造。這種能夠在細(xì)胞表面原始環(huán)境中對(duì)其進(jìn)行共價(jià)標(biāo)記的能力,，對(duì)于活體成像,、蛋白組學(xué)和治療目標(biāo)的鎖定等等都是有意義的。

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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