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The aim of this work was to build up a modular, artificial extracellular matrix (ECM) with poly-LacNAc, the human galectins-1, -3 and -8 and ECM-glycoproteins on a potential biomaterial surface. First, the synthesis of poly-LacNAc was characterized, optimized and up-scaled. This included the optimization of the single steps from enzyme expression and purification over synthesis till product isolation. The characterization of the one-pot-synthesis of the poly-LacNAc mixture was prioritized. The ratio of the substrates and the enzymes as well as their amount was optimized. During characterization of one-pot-synthesis with different enzyme ratios, I observed an unexpected product distribution which could be explained by kinetic data with longer substrates: The enzyme beta3GlcNAcT has higher activity to the tetra-saccharide. The synthesis based on acceptor substrates of different length showed a limitation of chain length from nona-saccharid onwards. In semi-preparative synthesis, poly-LacNAc structures till hexadeca-saccharides were detected and poly-LacNAc till dodeca-saccharides were isolated for the first time. Second, the human galectins-1, -3 and -8 were characterized with respect to their use for building up a modular, biomimetic ECM. Furthermore, common proteinchemical questions like stability and influence of His-Tag were discussed. It was possible to cross-link the ECM-glycoproteins laminin and fibronectin with all three galectins (His6Gal-1C2S, His6Gal-3 und His6Gal-8) on immobilized poly-LacNAc structures. His6Gal-1C2S binds already on the di-saccharid LacNAc on hydrogels. In contrast, the galectins-3 and -8 as well as the model galectin CGL2 need longer chain length for optimal binding on poly-LacNAc on hydrogels. Consequently, the poly-LacNAc mixture is the optimum for the first construction without any specific epitopes. Third, a new screening system with hydrogel in the microtiterplate was developed. Consequently, the galectins could be characterized on the final biomaterial surface. Finally, the biomimetic surface was tested as proof-of-principle with fibroblasts. They showed very good adhesion and proliferation on the human galectins (bound on poly-LacNAc) and on the surface poly-LacNAc-galectin-fibronectin. Furthermore, the biomimetic ECM (with model galectin CGL2) is more adhesive in comparison to the standard surface TCPS (tissue culture plates poly styrene) and to immobilized fibronectin. I hypothesized that the already established ECM leads to an increase of adhesion and proliferation of fibroblasts because no build-up of ECM is necessary. Additionally, the biomimetic ECM is more flexible than immobilized fibronectin and can be rearranged by adherent cells.

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.

Bei der Ätiopathogenese der chronisch entzündlichen Darmerkrankungen (CED) spielt die Dysregulation des intestinalen Immunsystems eine entscheidende Rolle. Es kommt zu einer überschießenden Immunantwort und Anreicherung von aktivierten T-Zellen in der Lamina propria der Mukosa und somit zur Zerstörung der Epithelzellschicht. Galektine wurden in den letzten Jahren zunehmend als Regulatoren der Immun- und Entzündungsantwort charakterisiert. In in vivo Untersuchungen wurde bereits das anti-inflammatorische und dadurch therapeutische Potential von Galektinen belegt. Ziel dieser Arbeit war, die Expression der Galektine -1, -2 und -4 mit der Methode der Immunhistochemie in Proben aus humanem Dickdarm von Patienten mit CED in Bezug auf eine gesunde Kontrollgruppe, den klinischen Aktivitätsindex, den endoskopischen Entzündungsgrad und die Therapie mit einem TNF-α-Antikörper zu analysieren. Unsere Arbeit zeigt, dass Galektin-1 hauptsächlich im Bindegewebe, in Muskelzellen, aber auch in Immunzellen der Lamina propria expremiert wird, nicht jedoch von Epithelzellen. Galektin-2 und -4 zeigten im Gegensatz hierzu ein vorwiegend epitheliales Expressionsmuster. Es konnte weiterhin gezeigt werden, dass bei den Patienten mit CED verglichen zu einer gesunden Kontrollgruppe keine einheitliche epitheliale Expressionsstärke von Galektin-2 und -4 untersucht wurde. Ferner konnten bei dieser Untersuchung Galektin-1 und -2-exprimierende Immunzellen in der Lamina propria bei den Patienten mit Morbus Crohn oder Colitis ulcerosa detektiert werden. Diese waren in der Kontrollgruppe nicht zu finden. Unsere Untersuchung legt somit nahe, dass die Galektin-Expression während der Entzündung durch eine Veränderung in der Aktivität der Immunzellen ausgelöst wird. Die vorliegende Arbeit konnte erstmals zeigen, dass bei den Patienten mit einer Colitis ulcerosa während der klinischen Aktivität eine reduzierte epitheliale Galektin-4-Expression im Vergleich zur Remission detektiert werden konnte. Weitere Untersuchungen beschäftigten sich mit der Galektin-Expression der chronisch entzündlich Darmerkrankten in Assoziation mit dem endoskopischen Entzündungsgrad. Der Vergleich zeigt, dass bei mehr als der Hälfte der Patienten mit Colitis ulcerosa eine reduzierte epitheliale Galektin-4-Expression in der entzündeten Mukosa detektiert werden konnte. Gegenstand dieser Arbeit war zudem der Vergleich der Expressionen vor und nach der Therapie mit einem TNF-α-Antikörper. Unsere Untersuchung konnte dabei zeigen, dass eine erhöhte epitheliale Expression von Galektin-4 bei mehr als der Hälfte der Patienten nach der Therapie detektiert wurde. Dabei war vor der Therapie keine Expression zu verzeichnen, wohingegen unter der Therapie es zu einem Anstieg der Expression gekommen ist. Die in dieser Arbeit generierten Ergebnisse konnten die Hypothese, dass Galektine und ihr verändertes Expressionsprofil bei CED einen von vielen Erklärungsansätzen für die gestörte Homöostase des intestinalen Immunsystems darstellen, bestätigen. Unsere Untersuchungen unterstützen die Hypothese, dass Galektin-4 eine entscheidende Rolle in der Modulation der intestinalen Entzündung spielt., Inflammatory bowel diseases (IBD) are characterised by a multifactoriel aetiology, in which an uncontrolled immune system with an impaired T cell apoptosis plays an important role. This results in a persistent inflammation of the intestinal mucosa. Galectins are increasingly recognized as new regulators of inflammation. They belong to a family of supar-binding lectins which more and more emerged as major regulator of different signaling processes, including proliferation, cytokine secretion and apoptosis. In vivo studies could already demonstrate their anti-inflammatory potential. The aim of this thesis was to explore the expression of Galectin-1, -2 and -4 in the gatrointestinal tract by patients with an inflammatory bowel disease first in comparison to a healthy control group, then to the clinical activity index, to endoscopic disease activity and at last to treatment with an TNF-α-antibody. This was determined by immunochemistry. In our work, we could demonstrate, that Galectin-1 is mainly expressed by connective tissue, muscle cells and immune cells in the lamina propria. Galectin-2 und -4 could be detected in epithelial cells. In comparison to the control group there was no homogeneous epithelial expression of Galectin-2 and -4. Galectin-1 and -2 were expressed in immune cells in the lamina propria of patients with IBD, not in the control group. Patients with clinically active ulcerative colitis showed a reduced epithelial expression of Galectin-4, not Galectin-2, in comparison to remission. Furthermore, the Galectin-4-expression was decreased in endoskopic inflamed biopsies from IBD patients, compared to macroskopic normal ones. We explored the expression of galectins before and after treatment with an immunosuppresive agens like an TNF-α-antibody. After treatment, there was an increased Galectin-4-expression in epithelial cells. Our data provides evidence of a dysregulated expression of galectins in the gastrointestinal tract of patients with IBD which contributes to the inflammation. We could show, that Galectin-4 is a key player in the modulation of the mucosal immune system.

Aim of this work was to modularize the setup of a multivalent glycan presentation on biomaterial surfaces. Oligosaccharides should be anchored onto artificial surfaces via multifunctional coupling molecules with their subsequent characterisation. Different workpackages were combined to reach this goal. First, the LacNAc derivatives were synthesized, which had a functionalization at their reducing end. The synthesis step was performed and characterized with His6-propeptide-beta4GalT1, which proved to be particularly affine for hydrophobic functionalizations. LacNAc derivatives with azide-, amino-, and propargyl-functionalization were successfully synthezised in preparative scale, were isolated and their molecular structure was proven. An exception was the joint molecule of one azide saccharide and the bifunctional coupler. The LacNAc synthesis of this derivative did not show efficient kinetic velocities. Overall 300 mg (0,6 mmol) were synthezised of both, the LacNAc-(CH2)6-azide and the LacNAc-amino-tBoc derivatives. Furthermore, their corresponding GlcNAc substrates could be used for the synthesis of new LacDiNAc structures via catalysis with the mutant enzyme beta4GalT Y284L. Poly-LacNAc derivatives of GlcNAc-(CH2)6-azide and GlcNAc-amino-tBoc were synthezised with the combined use of up to three different enzymes and via two different synthesis strategies: Firstly, via the successive synthesis route, which is time consuming but has the advantage to be well organizable. Secondly, via the economic one-pot synthesis route. With this, yields of more than 90% could be reached in a preparative scale. Via this chemo-enzymatic preparative strategy the oligosaccharide derivatives poly-LacNAc-azide and -amino were achieved for the first time with up to 10 saccharide units. Of every oligosaccharide chain length were synthezised up to 50 mg (50 micromol) and prepared for further investigations. During the next step a variety of coupling methods was established to immobilize the poly-LacNAc oligosaccharides in microtiterplates. An acetylene equipped bifunctional coupler with a protected amino functional group was joined via click chemistry with azide bearing oligosaccharides and subsequently immobilized on aminoreactive surfaces. Alternatively it was possible to immobilize propargylamine at the surface and click it with the azide oligosaccharides. This system was established with commercially available microtiterplates and as well for the first time with NCO-sP(EO-stat-PO)-hydrogel layers (cooperation with Dr. J. Groll, DWI at ITMC of RWTH Aachen). The functional proof was given by the binding of either the model galectin His6CGL2, the human galectin His6Gal1, or of commercially available Gal- or GlcNAc-specific lectins. Qualitative differences of lectin binding could be detected between the different artificial surfaces. Surfaces equipped with hydrogel layers showed superior results compared to the standard reference surfaces. Further studies are necessary to optimize the coating procedure of the hydrogel layers. This building block system with its high variety is a new strategy to immobilize poly-LacNAc structures in a defined way. This system will be the basis for the establishment of multivalent oligosaccharide presentation via combination of different methods with the corresponding building blocks. In the future, biomaterial surfaces from multivalently functionalized hydrogels could interact specifically with galectins and further ECM glycoproteins. This will enable the adhesion of distinct cell types to biomaterial surfaces.