Ingrid 't Hart

22 Chapter 1 1 1.7 Scope and outline This thesis describes the chemical and chemoenzyma�c syntheses of biologically relevant GSLs and SGLs and analogs thereof. In Chapter 2 , the chemoenzyma�c synthesis of globo-series glycans Gb5, MSGb5 and DSGb5 is described. Chemical synthesis provided pentasaccharide Gb5 and sialic acids were a�ached by mammalian sialyltransferases ST3Gal1 and ST6GalNAc5. Gb5, MSGb5 and DSGb5 were equipped with an aminopentyl linker at their reducing ends, through which they were linked to NHS-ac�vated microarray slides. Binding studies with plant lec�ns SBA and WGA indicated correct prin�ng of all three oligosaccharides. Strong binding was observed between α2,8α2,3-disialylated ganglioside GD3 and Siglec-7, however no binding was seen between Siglec-7 and DSGb5. Chemical and chemoenzyma�c syntheses of globo-series glycosphingolipids Gb3-Cer, Gb4-Cer and Gb5-Cer are described in Chapter 3 . The first part of this chapter focusses on the chemical synthesis of protected Gb5-Sph. Overall, chemical glycosyla�on of saccharide donors with a sphingosine acceptor resulted in low yields. Whereas benzoyl ester protec�ng groups dras�cally reduced donor reac�vity, benzyl ether protec�ng groups proved difficult to remove by Birch reduc�on, which is required to maintain the unsaturated sphingosine residue. In the second part of this chapter a successful chemoenzyma�c strategy for glucosylceramides is described. Chemically synthesized Gb3-Sph is extended by the bifunc�onal bacterial enzyme LgtD to obtain Gb4-Sph and Gb5-Sph. Pep�de coupling with palmi�c acid provides Gb3-Cer, Gb4-Cer and Gb5-Cer. The chemical synthe�c target of Chapter 4 is the sulfated glycan “SM1-core3”. SM1- core3 is a hybrid of sulfoglycolipid SM1a and the core3 (GlcNAcβ1,3GalNAc) disaccharide found in O-glycans. This sulfated glycolipid is proposed to be a ligand for cancer- specific an�body HAE3 and thereby a possible epithelial tumor marker. An op�mized chemical synthe�c strategy provided pure protected SM1-core3 glycan. SM1-core3 will be globally deprotected and immobilized through its reducing aminopentyl linker. The results of a glycan microarray binding study will provide us insight in the SM1-core3 – HAE3 interac�on. This will promote the search for SM1-core3 and similar structures as epithelial cancer biomarkers. In Chapter 5 , the chemoenzyma�c synthesis of C. jejuni core oligosaccharide fragments is described. Chemical glycosyla�on, followed by a two-step deprotec�on provided the Galβ1,3Hepα(CH2)5NH2 disaccharide. Ganglioside mimics GM3-Hep, GM2-Hep and GM1-Hep were then synthesized by bacterial enzymes pmST1, CgtA and CgtB. The low stability of CgtA and quick loss of ac�vity could be overcome by using cell lysate and storage at -20 °C. All synthesized Hep-ganglioside mimics will be immobilized on NHS- ac�vated slides alongside their human ganglioside analogs. A glycan microarray binding study with serum an�bodies of GBS pa�ents will elucidate the effect of heptose on an�body binding.