Exploring Nature at the Molecular Level through Solid-State NMR and Chemical Biology: Unraveling the Supramolecular Architecture of Diatom and Fungal Cell Walls and the Development of Heparanase-targeting Chemical Probes

Understanding the composition and architecture of microbial cell walls remains a significant scientific challenge due to their complex, heterogeneous nature. In organisms such as fungi and diatoms, the cell wall—and in some cases, the extracellular matrix (ECM), which can be structurally and functionally integrated with the wall—plays a critical role in maintaining cellular integrity, mediating communication, and enabling interactions with the surrounding environment. These intricate biological systems are not only vital for the survival and adaptation of the organisms but also offer great potential as sustainable biomaterials. To realize this potential, a detailed understanding of their molecular structure and assembly is essential. Conventional analytical methods rely on harsh chemical treatments that can disrupt native architecture, limiting their utility. Solid-state nuclear magnetic resonance (NMR) spectroscopy presents a powerful alternative. It is a non-destructive technique capable of providing detailed atomic-level insights into molecular structure, composition, and dynamics while preserving the native state of biological samples. Over the past decade, solid-state NMR has proven valuable in the study of microbial cell walls from bacteria, plants, fungi, and algae. In this thesis, solid-state NMR is used to study the cell walls of two model organisms: Schizophyllum commune, a basidiomycete fungus, and Thalassiosira pseudonana, a representative diatom. These organisms provide distinct structural features and serve as promising systems for the development of bio-based materials. In addition to structural studies, this thesis addresses the development of selective and potent enzyme inhibitors, with a particular focus on heparanase (HPSE), a retaining β-D-glucuronidase. Heparanase is a medically relevant target due to its involvement in a wide range of pathological conditions, including aggressive cancers, chronic inflammation, fibrosis, diabetes, and viral infections. Current inhibitors, mainly structural analogues of the natural substrate heparan sulfate (HS), face significant limitations. These include high molecular weight, poor bioavailability, structural heterogeneity, and undesirable anticoagulant effects, all of which hinder clinical translation. Cyclophellitol derivatives have emerged as potent inhibitors of glycosidases by irreversibly trapping the enzyme–substrate complex through transition-state mimicry. They offer high selectivity and potency but are still limited by suboptimal pharmacological properties in vivo. To overcome these issues, the second part of this thesis focuses on the synthesis of glucurono-cyclophellitol derivatives equipped with bioorthogonal click handles. These handles enable conjugation with an in vitro–generated cyclic peptide library, creating a hybrid inhibitor platform that combines small-molecule potency with peptide-based targeting capabilities. This hybrid library can be screened against immobilized heparanase using the RaPID (Random non-standard Peptides Integrated Discovery) system, a high-throughput platform designed to identify high-affinity binders. The goal is to discover new classes of heparanase inhibitors with improved drug-like characteristics, such as enhanced specificity, reduced off-target effects, and greater therapeutic potential. Together, these investigations contribute both to a deeper understanding of microbial cell wall and ECM structures and to the advancement of rational drug design targeting glycosidases, with broader implications in biotechnology and therapeutic development.