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，据估计发生在来自真核细胞的超过50%的蛋白质上。在脊椎动物糖蛋白中发现的九种单糖中，岩藻糖的特征在于其存在于与细胞-细胞相互作用和蛋白质功能调节相关的许多表位中。岩藻糖也包含在许多肿瘤相关抗原中，这些抗原在癌组织中上调，因此被认为是癌症生物标志物。分析岩藻糖基化糖蛋白是检测这些生物标志物的可能策略。该项目的目的是开发和应用检测活细胞岩藻糖基化糖蛋白的方法。代谢糖工程（MGE）将用于将岩藻糖衍生物掺入细胞聚糖中。与岩藻糖衍生物连接的是化学报告基团，其可以在生物正交连接反应中反应，因此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。通过与四嗪的逆电子需求狄尔斯-阿尔德（DAinv）反应实现烯烃的标记，这是生物正交连接反应的一个实例，其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，我们现在将探索具有阻断的岩藻糖从头合成途径以及替代连接反应的细胞系。岩藻糖基化的蛋白质特异性检测将通过生物正交标记和免疫沉淀的组合来实现，并将应用于研究α-1-酸性糖蛋白（一种已确立的癌症生物标志物）的岩藻糖基化。在第一个资助期内，我们证明了DAinv反应可以与点击化学结合，用于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）来检测同一聚糖内两种不同碳水化合物的接近程度，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.