Ong Jun Yang, Alvarado-Melendez Erianna I, Maliepaard Joshua C L, Reiding Karli R, Wennekes Tom
Chemical Biology and Drug Discovery, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University Universiteitsweg 99 3584 CG Utrecht The Netherlands
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Netherlands Proteomics Center 3584 CH Utrecht The Netherlands.
Chem Sci. 2025 Jan 13;16(7):3336-3344. doi: 10.1039/d4sc07184k. eCollection 2025 Feb 12.
Sialyltransferases (ST) are key enzymes found in, among others, mammals and bacteria that are responsible for producing sialylated glycans, which play critical roles in human health and disease. However, chemical tools to study sialyltransferases have been limited to non-covalent inhibitors and probes that do not allow isolation and profiling of these important enzymes. Here we report a new class of covalent affinity-based probes (AfBP) for ST by using ligand-directed chemistry (LDchem). Our affinity-based probes are armed with a simple to synthesise but robust -nitrobenzoxadiazole (-NBD) warhead, which is a lysine-specific SAr electrophilic warhead with an advantageous turn-on fluorescence property. We chemoenzymatically synthesised a series of CMP-Neu5Ac based probes and demonstrated their high specificity in labelling a range of recombinant STs with submicromolar sensitivity. Importantly, with our LDchem ST probe, we successfully labelled the endogenous lipooligosaccharide ST (Lst) in live , a clinically relevant human pathogen. Our results demonstrated that this new class of covalent ST probes offer a robust platform for ST profiling and future studies of STs in their native environments.
唾液酸转移酶（ST）是在哺乳动物和细菌等生物中发现的关键酶，负责产生唾液酸化聚糖，这些聚糖在人类健康和疾病中起着关键作用。然而，用于研究唾液酸转移酶的化学工具仅限于非共价抑制剂和探针，这些工具无法对这些重要酶进行分离和分析。在此，我们报告了一类通过配体导向化学（LDchem）针对ST的新型基于共价亲和力的探针（AfBP）。我们基于亲和力的探针配备了一种易于合成但稳定的 -硝基苯并恶二唑（-NBD）弹头，它是一种赖氨酸特异性的SAr亲电弹头，具有有利的开启荧光特性。我们通过化学酶法合成了一系列基于CMP-Neu5Ac的探针，并证明它们在以亚微摩尔灵敏度标记一系列重组ST时具有高特异性。重要的是，使用我们的LDchem ST探针，我们成功地在活的、一种临床相关的人类病原体中标记了内源性脂寡糖ST（Lst）。我们的结果表明，这类新型的共价ST探针为ST分析以及在其天然环境中对ST进行未来研究提供了一个强大的平台。
