Synthese und Charakterisierung von Glykanliganden und Galektinen für die Entwicklung von Biomaterialbeschichtungen
- Synthesis and characterisation of glycan ligands and galectins for the development of biomaterial coatings
Kupper, Christiane Elisabeth; Elling, Lothar (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2013)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2013
The PhD-thesis „synthesis and characterisation of glycan ligands and galectins for the development of biomaterial coatings“ deals with the provision and characterisation of galectins and glycans for subsequent preparation of biomaterial coatings. To do so, two general principles were applied. Either glycans or galectins were immobilised onto a surface. On this surface galectins, glycoproteins of the extracellular matrix (ECM) and/or cells can bind. Assemblies of different complexities can be established to mimick the natural cellular environment. First, different glycans basing on poly-LacNAc-linker-tBoc saccharides were prepared in preparative scale. Variations at the non-reducing end were achieved enzymatically or chemically. By further enzymatic elongation saccharides with internal modifications were prepared. The glycan derivatives were evaluated for their binding properties with different galectins. In this regard two promising ligands were discovered. The alpha,beta-unsaturated aldehydes of tetra- and hexasaccharide-linker-tBoc showed outstanding inhibitory potencies regarding galectin-1C2S-asialofetuin binding. Glycan-galectin binding was not dissolved under conditions of SDS-PAGE sample preparation, leading to the assumption of covalent galectin-glycan binding. 6-biotinylated saccharides showed reduced binding to galectin-1C2S but unchanged binding strength to galectin-3. Therefore these saccharides had specificity to galectin-3 in comparison to galectin-1C2S. For the assembly of biomaterial surfaces with directly immobilised galectins fusion proteins were constructed which have an additional SNAP-tag for simple oriented immobilisation and a fluorescent protein for controlling successful immobilisation. They are generally comparable with unfused galectins regarding their binding and oligomerisation properties. Effective oriented coupling was shown on different surfaces. The coupling via SNAP-tag was beneficial compared to non-oriented coupling strategies by NHS-chemistry as it was shown by threefold increase of binding capacities after oriented coupling. Positive effects of immobilised galectins for cell adhesion were shown in first experiments. Moreover pro-angiogenic effects of galectin-3 were proven which could be further evaluated in experimental settings with immobilised galectin-3. Components of an artificial ECM which have been produced and characterised in this work can now be applied for the preparation of biomaterial coatings.