Department of Chemistry, Stanford University , Stanford, CA 94305, United States.
Bioconjug Chem. 2011 Sep 21;22(9):1811-23. doi: 10.1021/bc2002117. Epub 2011 Aug 25.
Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.
末端唾液酸残基通常介导细胞表面糖缀合物的相互作用。唾液酸依赖性相互作用通常表现出快速的解离速率，这使得传统的生物技术无法用于复杂的分离。为了稳定这些瞬时相互作用，我们采用了一种靶向光交联方法，其中通过代谢寡糖工程将叠氮化物光交联剂掺入细胞表面唾液酸化糖缀合物中。我们描述了三种叠氮化物修饰的 N-乙酰甘露糖胺（ManNAc）类似物，其中吡喃糖环和叠氮化物之间的连接链的长度有所不同。这些类似物都被代谢成它们各自的唾液酸类似物，然后添加到糖蛋白和糖脂中。叠氮化物修饰的唾液酸类似物可以掺入α2-3 和 α2-6 键中。暴露于紫外线照射下，叠氮化物修饰的糖缀合物与它们的相互作用伙伴发生共价交联。我们证明，所有三种叠氮化物修饰的类似物都能够与内源性唾液酸竞争，尽管程度不同。我们发现较大的类似物代谢效率较低，但仍能作为有效的交联剂发挥作用。值得注意的是，叠氮化物取代基的添加会干扰 ManNAc 类似物向除唾液酸以外的聚糖的代谢，从而提供选择性地将交联剂掺入唾液酸化分子的保真度。这些化合物无毒，在交联研究所需的浓度下仅显示出最小的生长抑制。本报告提供了有关使用光交联类似物捕获和研究短暂但至关重要的唾液酸介导相互作用的重要信息。
