En Route to Tailor-made Oligosaccharides - Chemo-Enzymatic Synthesis and Physiological Functions of Novel Carbohydrate Structures

It is known that eukaryotic cells have an envelope of carbohydrates – the glycocalyx – which plays a crucial role in cell-cell and cell-ligand interaction processes. Additionally, bacterial glycosylation patterns are in the spotlight. Carbohydrate structures influence inflammation, tomor formation and progression, infections and many other biological processes. Glycobiology is a science on the crossroads of chemistry and biology. Synthesis and analysis of carbohydrate structures demand the application of methods from both fields of science and beyond. Carbohydrates are on the one hand chemically similar, on the other hand its structure determination demands basic chemical methods like nuclear magnetic resonance (NMR) and mass spectroscopy (MS) or even their combination. Glycosyl transferases are carbohydrate-active enzymes and are of outstanding relevance in glycobiology. They are involved in the synthesis as well as the degradation and carbohydrate recognition. In this thesis novel oligosaccharides were synthesized by the consecutive action of two fructosyltransferases. The fructosyltransferases SacB from Bacillus megaterium transfers a fructosyl moiety to different acceptors (galactose, mannose, fucose and xylose). The fructosyltransferase Suc1 from Aspergillus niger synthesizes defined tri- and tetrasaccharides. These oligofructosides were examined concerning their immuno-stimulatory properties by a co-cultivation with human colorectal epithelial carcinoma cells (Caco-2). A test regarding the secretion of 25 different cytokines yielded a significantly high level of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1). The fructosyltransferase SacB from Bacillus megaterium was examined by a intensive mutagenesis study. By enzyme engineering, 27 variants were created on the basis of sequence alignments with fructosyltransferases from other organisms and the solved structure of the fructosyltransferase from Bacillus megaterium (in cooperation with Rebekka Biedendieck and Martin Gamer, Department of Microbiology, TU Braunschweig and Christian Strube, Helmholtz-Centre for Infection Research). The biochemical data of these variants proved their influence on the polysaccharide formation. The variants of the amino acids Y247, N252, K315 and K373 are not located in the active site, but have a clear impact on the poly- vs. oligosaccharide formation. In spite of their altered product spectrum, their kinetic data are similar to the wild-type SacB. On the basis of these examinations, the polymer vs. Oligosaccharide formation mechanism of glycosyltransferases became clearer. Furthermore, a sucrose isomerase from Protaminobacter rubrum was examined. Substrate engineering of this enzyme led to the synthesis of an isomaltulose analogue consisting of a galactose unit instead of glucose. The linkage was determined as alpha-(1,6) analogous to isomaltulose. Finally, chemically functionalized carbohydrates were synthesized and presented on the surface of eukaryotic cells by glycoengineering techniques. In this position they are specific targets on the surface of human larynx carcinoma cells (HEp-2) for further chemical modification. This was performed by the incorporation of azido-functionalized N-acetyl-glucosamine and alkyne-functionalized N-acetyl neuraminic acid. By “click chemistry” with alkynylated rhodamine or azido fluorescein, respectively, these modified carbohydrates were detected.