Ingrid 't Hart

64 Chapter 3 3 of Gb3-Sph. The bifunc�onal enzyme LgtD from Haemophilus influenzae was used to provide both Gb4-Sph and Gb5-Sph. Although the used enzymes showed higher ac�vity on the free reducing glycans than on the glycolipids, cycles of resubmission converted all substrate to product. Coupling with palmi�c acid provided glucosylceramides Gb3- Cer, Gb4-Cer and Gb5-Cer. We found that C4 SPE purifica�ons using MeOH/H 2 O as the eluent provided higher yields than earlier reported C18 SPE purifica�ons with CH 3 CN/ H 2 O. Future op�miza�ons of the chemoenzyma�c synthesis requires higher enzyme ac�vi�es. We suggest that expression of engineered bacterial enzymes or the use of mammalian enzymes βGalNAc-T1 and β3GalT5 will provide be�er conversion of GSL substrates. Combined with slight op�miza�ons in the purifica�on methods sufficient material may be provided to study binding with immunological or cancer-associated proteins. Experimental Sec�on Chemical synthesis NMR nomenclature The monosaccharides of glycosphingolipid Gb5-Cer have been labeled as shown in Fig. S1. Star�ng from the reducing end of the pentasaccharide core Gb5, these were labeled as Glc-I, Gal-II, Gal-III, GalNAc-IV, Gal-V. Globo-series glycans contain either sphingosine (Sph, R = H), or ceramide (Cer, R = C 16 H 33 ). Figure S1. Monosaccharide labeling system for globo-series glycosphingolipids having either sphingosine (Sph, R = H) or ceramide (Cer, R = COC 16 H 33 ). Experimental procedures 2,2,2-Trichloroace�midate 2,3-di- O -benzyl-4,6- O -benzylidene-β-D-galactopyranosyl- (1→4)-2,3,6-tri- O -benzyl-β-D-glucopyranoside (4). Compound 4 was synthesized as described before (Chapter 2; compound 26). 28 (2S,3R,4E)-2-Azido-3-O-benzoyloxy-octadec-4-ene-1-ol (5). Compound 5 was synthesized as described before . 41 O HO OH O AcHN O HO OH HO OH O HO OH O HO O O OH HO OH O O OH HO OH HN OH C 13 H 27 O R I II V IV III Sph : R = H Cer : R = COC 15 H 33 O O OBn BnO BnO O O O BnO BnO Ph O NH CCl 3 N 3 OBz C 13 H 27 HO