Investigations into enzymatic transglycosylation reactions for the synthesis of glycoconjugates and glyco-functionalized polyphenols

With a myriad of functions, glycans constitute the most abundant of the four fundamental biological macromolecules (DNA, RNA, proteins and glycans). Besides structural and metabolical importance, they provide an essential coding platform for the transfer of information within the cellular network. Despite the arrival of glycome-related fields in mainstream science throughout the last decades, much of the glyco code remains unexplored. This is largely due to a lack of defined sample material. Enzymatic synthesis of glycans via a diverse toolbox of natural and artificial catalysts can help close this gap. This thesis explores the glycosylation of aglycon acceptor substrates for use as effector molecules with increased bioactivity or as building blocks for the assembly of glycan-presenting macromolecules. Section one addresses the demand for tailored synthetic flavonoid glycosylation. Four enzymes with different regiospecificities of plant and bacterial origin are recombinantly produced and their respective reactions analyzed. During the process, a novel analytical method based on multiplexed capillary electrophoresis suitable for the analysis of flavonoid glycosylation and related polyphenolic small molecules in a 96-well formate is validated. For the macrolide glycosyltransferase SorF from Sorangium cellulosum, a substrate screening revealed an interesting spectrum of polyphenols that indicates a potential application of this enzyme as a tool in the glycodiversification of small molecules. Section two covers the application of the hyperthermophilic glycosidase PwGly from P. furiosus in the glycosylation of acrylates and amine-functionalized linker substrates. The enzyme’s remarkable tolerance towards synthesis conditions are analyzed. The gained insights are put into practice during the synthesis of galacto functionalized acrylates on a 100 mL scale. High production yields > 80% and an efficient purification procedure enable the subsequent assembly of the galactomonomers to galactose presenting polymers with a tunable amount of incorporated sugars. The resulting glycopolymers represent suitable multivalent binding scaffolds for the lectin RCA120. PwGly’s versatility regarding transglycosylation reactions is further expored in the glycosylation of amine functionalized alkanols. Finally, the glycosynthase mutant BgaC Glu233Gly is applied in the synthesis of the azide functionalized pan-carcinomic Thomsen-Friedenreich (TF ) antigen. Subsequent coupling of the disaccharide to BSA via Cu2+-catalyzed alkyne azide cycloaddition yield tailored TF antigen presenting neo-glycoproteins whose properties as novel Gal-3 binding partners are investigated. In conclusion, the presented results highlight the role of a diverse spectrum of enzyme catalysts in synthetic glycotechnology. While the first part represents a first step towards rational-driven effector enhancement through tailored glycosylation, the second section lays the foundation for the assembly of glycan-presenting scaffolds via an efficient supply of functionalized building blocks