Sialic acids, a diverse family of acidic 9-carbon sugars, play important roles directly or indirectly in a variety of physiological and pathological processes. In order to understand the biological importance of the different sialic acids, sialoconjugates in nature and also to develop the novel therauptic targets, improvement in the methodology of enzymatic activation, sialyltransfer based on the novel, well accessible enzymes and accordingly application of perspective for the preparative synthesis of novel sialoconjugates are immensely important. We have synthesized different sialic acids by the combination of chemical flexibility and the high efficiency of the N-Acetylneuraminsäure-Aldolase (NeuA). The cytosine-5‘-monophosphate-acetylneuraminate synthetase (CSS) from Neisseria meningitides (Nm.) was used to active non-natural sialic acids. We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of CSS activity based on the pH change of a released proton. Using this method, kinetic data of the CSS-wildtype and 6 active site-mutants was determined for different structural variations of the sialic acids, which were used for the preparation of neo-sialoconjugate analogs of 2,3/8-Sialyllactose and 2,6-Sialyllactose in a one-pot two-enzyme cascade by CSS from Nm. together with Hilfe von α2,3-Sialyltransferase (α2,3-SiaT) from Campylobacter jejuni (CstII Δ32) oder α2,6-SiaT from Photobacterium Leiognathi JT-SHIZ-145 (Pl). Visualization of biological phenomena with the aid of fluorescent markers has emerged as an exciting challenge for chemical biologists. Thus, the synthesis of fluorescently labelled sialic acids and their transfer to biological structures are attractive targets. For this purpose, we have optimized the synthesis of the various CMP-activated fluorescent sialic acids. Furthermore, we report the first successful example of glycosyltransferase-catalyzed polymerization yielding structurally defined poly¬saccharides with internal sialic acid residues. We have found that the sialic acids with N-acyl-linked lactosyl units after efficient activation by the CSS from Nm. can be trans¬ferred repetitively by the α2,6-SiaT from Pl. to yield neo-glycopolysaccharides containing internal sialyllactoside repeating units, respectively, thus mimicking naturally occurring polysaccharides. | English |
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