Synthesis and Evaluation of Sialic Acid-based Molecular Tools to Probe and Perturb Viral Neuraminidase Activity

Compared with other glycosidases, sialidases have been less studied due to their unique catalytic mechanism. However, as enzymes that regulate numerous physiological and pathological processes, they warrant thorough investigation. Building on the rationale outlined above, this thesis focuses on the development of selective inhibitors and mechanism-based probes for GH34 sialidases, with a particular emphasis on influenza neuraminidase. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high-throughput approaches look at many enzymes at once. To make this diversity experimentally tractable, high-throughput approaches have emerged that allow many enzymes or inhibitors to be evaluated in parallel. Similarly, high-throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in industrial, laboratory, or medicinal settings. In Chapter 2, we provide an overview of how such approaches, including natural product and combinatorial library screening, phage, and mRNA display of (glyco)peptides, fluorescence-activated cell sorting, and metagenomics, can be applied to discover new enzyme targets and inhibitors. Chemical probes are powerful tools in studying carbohydrate-active enzymes. To explore the active time points of IAV neuraminidase, two mechanism-based DFSA probes were developed from a selective influenza neuraminidase inactivator, 4-amino-DFSA. The design, synthesis, and interactions of these probes with NA are detailed in Chapter 3, in which they function both as inhibitors and as active labeling probes of NA, enabling the detection of recombinant and viral forms in an activity-dependent manner. Because 2,3-fluorinated sialic acids are prone to elimination of the C2-fluorine, which reduces covalent inhibition and labeling efficiency, we designed a novel scaffold: C3-difluoro sialic acid. This structure represents a new class of neuraminidase inhibitors and a potential probe framework. In Chapter 4, we report the exploration of multiple synthetic strategies towards this scaffold and highlight the challenges encountered due to the presence of a C5-nitrogen substituent in the precursor and the CF2 group in the addition product. The same concept was applied to KDN, which avoids the synthetic complications of introducing a nitrogen atom at C5. Chapter 5 reports the successful synthesis of C3-difluoro KDN in a protected ester form starting from commercially available methyl α-d-mannose, with an overall yield of 3.7% over 12 steps. This represents a first step towards the development of fluorinated probes and inhibitors targeting KDN-related enzymatic pathways. In Chapter 6, practical synthetic methods for the preparation of sialylgalactose–based fluorogenic probes are presented. These probes allow detection of both the activity and specificity of influenza neuraminidase and paramyxoviral HN proteins. In addition, the HN inhibitor BCX2798 was synthesized, and its virological application is under investigation. Finally, Chapter 7 provides a general discussion of the research chapters in this thesis, together with some unpublished findings and perspectives. Based on these outcomes, future research directions are proposed for the development of high-affinity chemical probes to study sialidases.