Exploiting Diels-Alder Reactions with Inverse Electron Demand for Bioorthogonal Labeling of Alkene-Substituted Fucosylated Glycoproteins in Cells

糖基化是一种普遍存在的翻译后修饰形式，据估计，超过50%的真核细胞蛋白质发生糖基化修饰。在脊椎动物糖蛋白中发现的9种单糖中，岩藻糖的区别在于它存在于与细胞-细胞相互作用和蛋白质功能调节相关的众多表位中。岩藻糖也包含在许多肿瘤相关抗原中，这些抗原在癌症组织中上调，因此被认为是癌症的生物标记物。分析岩藻糖化糖蛋白是检测这些生物标志物的一种可能的策略。该项目的目的是开发和应用能够检测活细胞岩藻糖化糖蛋白的方法。代谢糖工程(MGE)将被用来将岩藻糖衍生物结合到细胞糖链中。连接到岩藻糖衍生物的是可以在生物正交连接反应中反应的化学报告基团，从而使得能够连接可检测的探针(荧光染料、生物素)。在本项目的第一个资助期，我们合成了一系列含末端烯烃的岩藻糖衍生物，并将其应用于MGE。烯烃的标记是通过与四嗪的反电子需求Diels-Alder(DAinv)反应实现的，这是一个生物正交配反应的例子，该反应在没有金属催化的情况下以高产率进行，并且与点击化学正交。为了提高岩藻糖衍生物的检测效率，我们现在将探索岩藻糖从头合成途径受阻的细胞系以及交替连接反应。岩藻糖化的蛋白质特异性检测将通过生物正交标记和免疫沉淀相结合的方法实现，并将用于研究已建立的癌症生物标志物α-1-酸性糖蛋白的岩藻糖基化。在第一个资助期，我们证明了DAinv反应可以与点击化学相结合，同时检测两种不同的碳水化合物。我们现在将使用荧光共振能量转移(FRET)来检测同一糖中两种不同碳水化合物的接近程度，以此作为某些肿瘤相关抗原发生的指示。

Glycosylation is a ubiquitous form of posttranslational modification that has been estimated to occur on more than 50% of the proteins from eukaryotic cells. Among the nine monosaccharides found in the glycoproteins of vertebrates, fucose is distinguished by its occurrence in numerous epitopes associated with cell-cell interactions and regulation of protein function. Fucose is also contained in a number of tumor-associated antigens that are upregulated in cancerous tissues and, therefore, have been suggested to act as cancer biomarkers. Profiling fucosylated glycoproteins is a possible strategy for the detection of these biomarkers. The aim of this project is to develop and apply methods that allow the detection of fucosylated glycoproteins of living cells. Metabolic glycoengineering (MGE) will be used to incorporate fucose derivatives into cellular glycans. Connected to the fucose derivatives are chemical reporter groups that can react in a bioorthogonal ligation reaction, thus, enabling the attachment of a detectable probe (fluorescence dye, biotin). In the first funding period of this project we synthesized a series of fucose derivatives containing terminal alkenes and applied them to MGE. Labeling of the alkenes was achieved by an inverse-electron-demand Diels-Alder (DAinv) reaction with tetrazines which is an example of a bioorthogonal ligation reaction that proceeds in high yields without metal catalysis and that is orthogonal to click chemistry. To improve the detection efficiency of the fucose derivatives we will now explore cell lines with blocked fucose de novo synthesis pathway as well as alternative ligation reactions. Protein-specific detection of fucosylation will be achieved by a combination of bioorthogonal labeling and immunoprecipitation and will be applied to study fucosylation of alpha-1-acid glycoprotein, an established cancer biomarker. In the first funding period we proved that the DAinv reaction can be combined with click chemistry for the simultaneous detection of two different carbohydrates. We will now use fluorescence resonance energy transfer (FRET) to detect the proximity of two different carbohydrates within the same glycan as indication for the occurrence of certain tumor-associated antigens.