Semisynthesis of N-glycoprotein libraries

糖蛋白的功能通常受其碳水化合物部分的调节。这些糖分显示出一种天然的微观异质性。由于均一的糖蛋白(糖形式)不能通过足够数量的纯化获得，所以微观异质性的后果还不能很好地理解。因此，通过天然化学连接和随后的重折叠提供均一糖蛋白的聚合合成方法被开发出来。在第一个资助期内，通往糖型的严格合成路线可以大大缩短，从而产生第一批天然糖型IL-6和皂苷D。所有合成的糖型都具有生物活性。对于溶酶体适配蛋白皂苷D，发现与膜重塑相关的几种生物活性是碳水化合物依赖的。在第二个资助期，我们的目标是进一步改进IL-6和皂苷糖形式的合成，利用化学酶方法合成N-糖链和N-糖肽。Pimary的目标是组装一个具有代表性的四种人皂素糖型文库。由于它们在结构和功能上的同源性，可以假设皂苷A-C也具有碳水化合物依赖的生物活性，这应该通过已建立的分析系统来揭示。皂苷在体内的作用与与脂类形成络合物的皂苷低聚体的形成有关。由于这些络合物只与非糖基化皂苷进行了研究，因此糖基化络合物将被特定地作为靶点并随后进行表征。

The function of glycoproteins is frequently regulated by their carbohydrate moieties. These sugar moieties show a natural microheterogeneity. The consequences of microheterogeneity are not well understood since homogenous glycoproteins (glycoforms) cannot be obtained by purification in sufficient quantities. Thus, convergent synthetic approaches were developed providing homogenous glycoproteins via native chemical ligation and subsequent refolding. The demanding synthetic routes towards glycoforms could be significantly shortened in the first funding period leading to the first libraries of natural glycoforms of IL-6 and saposin D. All synthetic glycoforms were biologically active. For the lysosomal adapter protein saposin D several bioactivities related to membrane remodelling were found to be carbohydrate-dependent.In the second funding period we aim to further improve the synthesis of glycoforms of IL-6 and saposins by using chemoenzymatic methods in the synthesis of N-glycans and N-glycopeptides. The pimary goal is the assembly of a representative library of glycoforms of all four human sapsins. Due to their structural and functional homology it can be assumed that also the saposins A-C have bioactivities with carbohydrate-dependency, which should be revealed by established assays systems. The in vivo functions of saposins are correlated with the formation of saposin oligomers complexed with lipids. Since these complexes have been investigated only with non-glycosylated saposins, the glycosylated complexes will be targeted specifically and characterized subsequently.