Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEG-linker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii) fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI) studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.

中文翻译：

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由未保护的碳水化合物制备糖结合物，用于蛋白质结合研究。

糖生物学，尤其是糖-蛋白质相互作用及其后继事件的研究，已成为近几十年来的重要研究重点。为了研究这些相互作用，许多测定需要适量的均质糖缀合物。它们的合成是糖生物学的方法学挑战之一。在这里，我们描述了一种通用的，三阶段的方案，用于从未保护的碳水化合物（包括从天然来源纯化的碳水化合物）中形成糖缀合物，如此处所列举的根瘤菌Nod因子和胞外多糖片段。第一步是通过苯胺催化的肟形成，将具有末端三苯基甲硫醇端基的寡聚乙二醇连接基（OEG-linker）添加到寡糖的还原端。然后从接头除去三苯甲基（三苯甲基）标签以暴露硫醇（阶段2）以允许在硫醇基上的缀合反应（阶段3）。取决于所需的应用，有许多可能的共轭反应。该协议中显示的示例如下：（i）将寡糖偶联至表面等离振子共振（SPR）研究的载体，（ii）微型热泳（MST）或生物成像的荧光标记，以及（iii）生物素化用于生物层干涉术（BLI）研究。该方案从无保护的碳水化合物开始，仅在2天内即可提供毫克量的糖结合物。（i）将寡糖偶联至表面等离振子共振（SPR）研究的支持物；（ii）微型热泳（MST）或生物成像的荧光标记；以及（iii）生物素化用于生物层干涉术（BLI）的研究。该方案从无保护的碳水化合物开始，仅在2天内即可提供毫克量的糖结合物。（i）将寡糖偶联至表面等离振子共振（SPR）研究的支持物；（ii）微型热泳（MST）或生物成像的荧光标记；以及（iii）生物素化用于生物层干涉术（BLI）的研究。该方案从无保护的碳水化合物开始，仅在2天内即可提供毫克量的糖结合物。