Modified multivalent poly-N-acetyllactosamine glycans as novel ligands of human galectin-3

  • Modifizierte multivalente Poly-N-Acetyllaktosamin Glykane als neuartige Liganden von humanem Galektin-3

Laaf, Dominic; Elling, Lothar (Thesis advisor); Kren, Vladimir (Thesis advisor)

Aachen (2018)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


The carbohydrate moieties displayed by glycoconjugates are of significant importance for a homeostatic state of each living organism. The reversible interactions of carbohydrate binding proteins, e.g. galectins, define the cellular crosstalk which is often deregulated in disease states such as cancer. Galectin-3 (Gal-3) exclusively appears as oligomeric lectin and is involved in decisive regulatory processes. Gal-3 currently attracts special attention since identified as druggable target and biomarker for cancer and fibrotic diseases. In this work, main objective was the generation and evaluation of a glycan library to enable a selective attraction of Gal-3. For this purpose, we extended the repertoire of available glycan species by combinatorial biocatalysis using recombinant glycosyltransferases and methods for chemical modification. As a result, we obtained derivatives of the most abundant natural β-galactoside, (poly-)N-acetyllactosamine (LacNAc), which comprised a set of diverse glycosylation motifs, e.g. the N’,N’’-diacetyllactosamine (LacdiNAc). High-performance liquid chromatography (HPLC) in the normal phase (NP) or reversed-phase (RP) mode was a suitable technique for glycan analysis and purification in the (semi-)preparative scale. Electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy were used for structure confirmation. Selected glycan species were loaded on non-glycosylated bovine serum albumin (BSA) for a multivalent ligand presentation with the aim to address the ‘cluster glycoside effect’ during galectin interaction. And indeed, the screening of both univalent and multivalent glycan libraries in solid-phase binding assays confirmed a strengthening of galectin inhibition by several orders of magnitude. Their outstanding high affinity and selectivity made certain neo-glycoconjugates to effective agents for the capture and detection of Gal 3. After having identified most promising candidates, nature served as the model for the design of biomimetic chitin oligomers as inert spacers, which essentially ameliorated glycan accessibility and led to an elevated galectin interaction. Beyond that, we produced neo-glycoproteins carrying di-LacNAc type 1 tetrasaccharides. Using them as galectin ligands, we provided the first evidence for the discrimination of Gal 3 and Gal 3∆ (a truncated and tumor-associated version) on the basis of carbohydrate-lectin interaction. In another investigation, the enzymatic introduction of branching points on an oligomeric LacNAc building block in synergy with chemical coupling gave novel structures, which mimic branched I-antigen structures occuring on human erythrocytes. We assayed them as competitive Gal 3 inhibitors and confirmed a decasaccharide with two LacdiNAc-LacNAc branches as the most effective monovalent poly-LacNAc inhibitor of Gal 3 within the scope of the synthetic work up to now. In summary, a glycan library consisting of a variety of oligomeric LacNAc derivatives and corresponding multivalent neo-glycoconjugates was synthesized by biocatalytic and chemical methods, characterized by diverse analytical tools and evaluated as ligands for human Gal 3. We thereby envisaged putative applications of prepared conjugates in the context of cancer related glyco-medicine.