Ingrid 't Hart

110 Chapter 6 6 Chapter 3 In theprevious chapter the focuswason thesynthesisof theglobo-seriesoligosaccharides. Recent literature studies on the importance of the lipid moiety of GSLs on protein binding and cellular organiza�on encouraged us to synthesize the full glycolipids of globo-series GSLs. Incorpora�on of a lipid moiety introduces new challenges, such as the introduc�on of new func�onal groups, the lower reac�vity of lipids for chemical glycosyla�on and altered solubility proper�es. A first fully chemical synthe�c strategy was designed and resulted in the fully protected formof globopentaosylsphingosine (Gb5-Sph). The removal of the benzyl ether protec�ng groups required a Birch reduc�on, to avoid reduc�on of the unsaturated sphingosine. However, removal of the benzyl ether protec�ng groups proved unsuccessful on a model glycolipid, which might be due to solubility issues. Next, a chemoenzyma�c approach was chosen, star�ng from chemically synthesized globotriaosylsphingosine (Gb3-Sph). A bifunc�onal bacterial enzyme, LgtD from Haemophilus Influenzae , was used to extend the glycan chain of Gb3-Sph to obtain Gb4- Sph. Gb4-Sph was then converted to Gb5-Sph by the same enzyme. Although a lower conversion was seen on the sphingosine-linked glycans compared to the free reducing oligosaccharides, both substrates could be converted to their respec�ve products. To obtain full conversion, mul�ple enzyma�c reac�ons were required. Gb3-Sph, Gb4-Sph and Gb5-Sph were coupled with palmi�c acid, under amide coupling condi�ons (HOBt/ EDC) and provided globotriaosylceramide (Gb3-Cer), Gb4-Cer and Gb5-Cer. We propose that engineered bacterial enzymes or mammalian enzymes β3GalNAc-T1 (for Gb4) and β3GalT5 (for Gb5) will show higher tolera�on of the reducing lipid residues. This way, the lower enzyma�c conversions of GSL substrates can be overcome. Chapter 4 Sulfoglycolipids (SGLs) are in many ways similar to gangliosides, since they both contain a nega�vely charged glycan residue and a reducing-end ceramide. Also, SGLs are o�en sulfated on the same posi�on as the sialic acid in a ganglioside, and a good example is found in the SGL SM1a and ganglioside GM1a. SGLs such as SM3 and SB1a have been associated with cancer, however li�le is known of SM1a. A microarray study from Ten Feizi et al . revealed that SM1a strongly binds to the cancer- specific an�-epiglycanin an�body HAE3. A surprise finding, since HAE3 is known to bind mucin-type O -glycans. O -glycan epitopes similar to SM1a were found on mucin glycoproteins, all having a core3 (GlcNAcβ1,3GalNAcα-Ser/Thr) glycan residue. Therefore, a hybrid glycan of SM1a and core3 was proposed, here named SM1-core3 (Galβ1,3GalNAcβ1,4Galβ1,3GlcNAcβ1,3GalNAcα). SM1-core3 is the synthe�c target of this chapter and the protected pentasaccharide was successfully obtained through chemical synthesis. First, all monosaccharide building blocks were synthesized with the appropriate orthogonal protec�ng groups and C-2 esters to selec�vely form all β-anomeric linkages. The reducing N -acetylgalactosamine was equipped with an α-anomeric aminopentyl linker, to resemble the stereochemistry in O -glycans and to provide a handle for immobiliza�on. Various GalN 3 donors were synthesized and tested to form the 1,2- cis α-linkage with an aminopentanol acceptor. The 3,4,6-tri- O -acetylated GalN 3 trichloroace�midoyl donor provided the best outcome (~1/1 α/β anomeric mixture). A�er hydrolysis of the acetyls and installa�on of a benzylidene acetal on C-4 and C-6, the anomeric mixture could be purified by silica column chromatography. All