Synthese von Oligosaccharid-Polyetherbausteinen für den Aufbau von neuartigen ultradünnen biofunktionalisierten PEG-Stern-Polymerschichten

  • Synthesis of oligosaccharide-polyether building blocks for the setup of new ultra thin biofunctionalized PEG-star polymer layers

Adamiak, Kathrin; Elling, Lothar (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2012)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2011


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.