Ingrid 't Hart

58 Chapter 3 3 While chemical and enzyma�c syntheses of the oligosaccharide moie�es of globosides have been described, there are only a few reports dealing with the synthesis of the intact glycolipid of GSLs. Glycosphingolipid synthesis adds a number of challenges and different approaches can be considered. 34 Few reports deal with the chemical glycosyla�on of a sphingosine or ceramide acceptor with the fully protected saccharide donor obtained by chemical synthesis. 35,36 Since sphingosine or ceramide acceptors are of low reac�vity, another strategy has been used where this key chemical glycosyla�on is performed in an earlier stage of GSL synthesis. 37 The la�er chemical synthe�c strategy is also called the “Glc-Cer casse�e strategy” and was used to synthesize GSL mimic(s), such as GQ1b and the hybrid ganglioside X2. 37,38 In addi�on to full chemical synthesis, chemoenzyma�c approaches have been developed. The first approach is the enzyma�c synthesis of an oligosaccharide moiety, which is protected by benzoyl esters and then converted into an imidate donor for chemical glycosyla�on with sphingosine. 39 A second strategy starts with the chemical synthesis of glycans having an anomeric α-fluoride, which can then be glycosylated with sphingosine or ceramide by the glycosidase mutant of EGC-II (EGC-E). 40 In this way, the less reac�ve acceptor is coupled more efficiently and without by-product forma�on. More recently, a chemoenzyma�c strategy to synthesize GSLs has been reported, where ini�ally lactosyl sphingosine is chemically synthesized and then extended by bacterial enzymes. 41 In this chapter, the chemical and chemoenzyma�c syntheses of various globo-series GSLs are described. Full chemical synthesis of protected Gb5-sphingosine (Gb5-Sph) was achieved, however benzyl ethers could not be cleaved by Birch reduc�on. Next to that, we explored a chemoenzyma�c approach, star�ng from chemically synthesized Gb3- Sph. Extension of Gb3-Sph by the enzyme LgtD from Haemophilus influenzae. provided Gb4-Sph and Gb5-Sph consecu�vely. Lower conversion rates of LgtD were observed on the sphingosine analogues of Gb3, Gb4 and Gb5 compared to the free reducing glycans. With a number of op�miza�ons, Gb3-, Gb4- and Gb5-Sph were successfully synthesized by this strategy. The full GSLs Gb3-Cer, Gb4-Cer and Gb5-Cer were obtained by coupling of the C-2 amine on sphingosine with palmi�c acid. Results and Discussion Chemical synthesis of globopentaosylsphingosine The chemical synthesis of the Gb5 pentasaccharide 10 is described in Chapter 2 and was envisaged as a good star�ng point for making the sphingolipid deriva�ve. 28 A�achment of a ceramide to compound 10 requires adap�on of the chemical synthesis. First, we searched for appropriate reac�on condi�ons to form the β-glycosidic bond with sphingosine in the absence of C-2 neigbouring group par�cipa�on. Compound 4 was chosen as a model substrate for the op�miza�on of the β-glycosyla�on in the absence of neighboring group par�cipa�on (Scheme 1A). Sphingosine acceptor 5 was synthesized from phytosphingosine in 7 steps as described before. 41 Lactosyl donor 4 and sphingosine acceptor 5 were glycosylated in the presence of BF 3 OEt 2 in DCM in the presence of 4 Åmolecular sieves at -40 °C. These condi�ons provided lactosylsphingosine