Sialic Acids, Sialyltransferases and How to Find Them: Isotopic and Affinity-Based Labelling Strategies with Chemical Probes

Abstract

Sialic acid, along with all other sugars, make up the intricate glycocalyx of cells. Chemical biology research is indispensable in providing tools that allow us to see what is happening to this sugar, and how it interacts with its environment. The impact of these interactions ranges from a healthy gut to disease-causing bacteria. Here we used two chemical strategies: Isotopic labelling of sialic acids, and affinity-based labelling of sialyltransferases. To study the biological role of natural sialic acids, Chapter 2 outlines a de novo synthesis of 13C-enriched sialic acids, which are identical to naturally occurring sialic acids apart from where the carbons are selectively enriched in heavy 13C isotopes. Starting from affordable 13C glucose, 13C enriched ManNAc and sialic acids could be synthesised on a gram scale. From that, an important component of human milk oligosaccharides, 6’-sialyl lactose was synthesised. An unusual bicyclic sialic acid found as a microbial metabolite, 2,7-anhydro sialic acid was also obtained by coupling with an additional recombinant enzyme RgNanH. We further reported the results from our preliminary SCFA fermentation and SIP experiments using these 13C-labelled sugars. In Chapter 3, the power of chemoenzymatic synthesis, click chemistry and ligand-directed chemistry (LDchem) were combined to yield a panel of ligand-directed affinity-based probes which were tested against various recombinant sialyltransferases as well as sialyltransferases present in live bacteria. Here, we showed that these LDchem sialyltransferase affinity-based probes were able to label recombinant sialyltransferases from different families, and the endogenous sialyltransferase Lst in Neisseria gonorrhoeae. In pursuit of a more efficient labelling of ST and as a venture into the broader field of protein labelling, Chapter 4 explores and validates a new generation of covalent sialyltransferase probes and inhibitors equipped with a new class of electrophilic warhead, chlorotriazine (ClTriZ) which was developed in-house. Here, we fully explored the power enabled by the concepts of proximity-induced chemistry and direct-to-biology (D2B) for the rapid synthesis and screening of probes and inhibitors. Using this platform, we succesfully synthesised a panel of sialyltranferase probes and inhibitors which have better efficiency and sensitivity than our first-generation LDchem probes.