Gray Taylor E, Labasan Kristin B, Daskhan Gour C, Bui Duong T, Joe Maju, Kumawat Dhanraj, Schmidt Edward N, Klassen John S, Macauley Matthew S
Department of Chemistry, University of Alberta Edmonton T6G 2G2 Canada.
Department of Medical Microbiology and Immunology, University of Alberta Edmonton T6G 2E1 Canada
RSC Chem Biol. 2025 Apr 17. doi: 10.1039/d5cb00030k.
An important approach for tracking and visualizing sialic acid-containing glycans involves using sialic acid reporters functionalized with bioorthogonal handles. More specifically, metabolic oligosaccharide engineering (MOE) commonly employs monosaccharides with an alkyne or azide handle for incorporation into cellular glycans, followed by a subsequent click reaction to elaborate with a biotin or fluorophore handle. For sialic acid, this has been carried out extensively, with an azide or alkyne appended to the C5 -acetamido group being the most common location for the handle. However, circumstances may require the handle to be at different positions and, to date, the C7 and C9 positions have been shown to work to varying degrees. Herein, we synthesized protected 4AzNeu5Ac that could be incorporated into cellular glycans nearly as efficiently as Neu5Az and targeted with DBCO-biotin through strain promoted azide-alkyne cycloaddition. Owing to the good incorporation of 4AzNeu5Ac into cellular glycans, we followed up this ability by first synthesizing the deprotected form of 4AzNeu5Ac, using a thioglycoside to lock the anomeric center during deprotection of the acetyl groups. Activation of 4AzNeu5Ac to CMP-4AzNeu5Ac then enabled the use of this donor by human sialyltransferase ST3GAL1 to transfer CMP-4AzNeu5Ac to β-Gal-(1→3)-α-GalNAc. With purified α-4AzNeu5Ac-(2→3)-β-Gal-(1→3)-α-GalNAc in hand, we tested it as a ligand for Siglec-7 and found that the C4-Az modification is tolerated, opening future possibilities to exploit this position to generate high affinity and selective ligands. These findings expand the repertoire of metabolic oligosaccharide engineering agents and show that azide modifications are tolerated at the C4 position of sialic acid.
一种用于追踪和可视化含唾液酸聚糖的重要方法涉及使用带有生物正交基团修饰的唾液酸报告分子。更具体地说，代谢性寡糖工程（MOE）通常采用带有炔烃或叠氮基团修饰的单糖，使其掺入细胞聚糖中，随后通过点击反应与生物素或荧光团基团进行进一步修饰。对于唾液酸而言，这一方法已被广泛应用，将叠氮基团或炔烃连接到C5 -乙酰氨基基团是最常见的修饰位置。然而，在某些情况下可能需要将修饰基团置于不同位置，迄今为止，C7和C9位置已被证明在不同程度上可行。在此，我们合成了受保护的4AzNeu5Ac，其掺入细胞聚糖的效率几乎与Neu5Az相同，并通过应变促进的叠氮-炔烃环加成反应与DBCO-生物素进行靶向反应。由于4AzNeu5Ac能很好地掺入细胞聚糖，我们通过首先合成4AzNeu5Ac的去保护形式来进一步研究这种能力，在乙酰基去保护过程中使用硫代糖苷锁定异头中心。将4AzNeu5Ac激活为CMP-4AzNeu5Ac后，人唾液酸转移酶ST3GAL1能够利用该供体将CMP-4AzNeu5Ac转移至β-Gal-(1→3)-α-GalNAc。获得纯化的α-4AzNeu5Ac-(2→3)-β-Gal-(1→3)-α-GalNAc后，我们将其作为Siglec-7的配体进行测试，发现C4-Az修饰是可耐受的，这为未来利用该位置生成高亲和力和选择性配体开辟了可能性。这些发现扩展了代谢性寡糖工程试剂的种类，并表明唾液酸的C4位置可耐受叠氮修饰。
