Ingrid 't Hart

109 Summary / Samenva�ng 6 Cer) is extended by bacterial glycosyltransferases (GTs). Stereo- and regioselec�vity are the key factors in carbohydrate synthesis. In chemical synthesis, regioselec�vity is usually obtained by careful choosing orthogonal protec�ng groups. This way, the acceptor can be designed in such a way that only one free hydroxyl can react as the nucleophile. A stereoselec�ve outcome is more challenging to obtain in chemical glycosyla�ons. The general approach to form 1,2- trans linkages (β in the case of Glc or Gal) is by neighboring group par�cipa�on of a C-2 ester. Forming a 1,2- cis linkage (α in the case of Glc and Gal) is more challenging, although some effec�ve ways have been designed where the β face is blocked. One way is the use of a chiral auxiliary between C-1 and C-2, which forms a trans -decalin intermediate, or a di- tert -butane-silane group on the C-4 and C-6 of galactose which creates a steric bulk. Enzyma�c synthesis provides both high stereo- and regioselec�vity, although not all enzymes have been iden�fied or expressed and sugar nucleo�de donors are o�en expensive, thereby limi�ng the scale. Enzymes from both mammalian and bacterial sources have been used to synthesize oligosaccharides. Mammalian enzymes are usually more selec�ve, but harder to express. Bacterial enzymes are easier to express, but also show less selec�vity, which can be an advantage or disadvantage. Bacterial enzymes have been iden�fied, expressed and used for the synthesis of both globo- and the ganglio-series GSLs. Chapter 2 The globo-series ganglioside, disialosyl globopentaosylceramide (DSGb5), is o�en expressedinrenalcellcarcinoma.DSGb5isproposedtobindSiglec-7;animmunoinhibitory receptor mainly found on NK-cells. Therefore, DSGb5 would aid cancer cells in escaping immune detec�on and immune-mediated clearance. We designed a chemoenzyma�c strategy to synthesize DSGb5 and study its binding interac�on with Siglec-7. The core pentasaccharide Gb5 was chemically synthesized by a 2 + 3 block approach. Four saccharide building blocks were protected to enable proper stereo- and regioselec�ve outcomes. The α1,4-linkage between galactose and lactose was obtained by the use of a bulky di- tert -butyl-silane group on the galactose donor, solely providing α-anomeric product. Other chemical glycosyla�ons provided β-linkages through neighboring group par�cipa�on of C-2 esters. Chemical glycosyla�on of the Galβ1,3GalNAc disaccharide donor with the Galα1,4Lac (Gb3) trisaccharide acceptor provided anomeric mixtures when the donor contained a benzylidene acetal. Replacement of the benzylidene acetal by C-4 and C-6 acetyls provided pure β-anomeric selec�vity. Gb5 was globally deprotected and mammalian sialyltransferases ST3Gal1 and ST6GalNAc5 installed the terminal α2,3- and internal α2,6-sialic acids. Glycan microarray binding studies indicated proper prin�ng of Gb5, MSGb5 and DSGb5, by the plant lec�ns Soybean Agglu�nin (SBA: binds to Gal or GalNAc residues) and Wheat Germ Agglu�nin (WGA: binds to GlcNAc or sialic acid residues). It was found that Siglec-7 does not recognize DSGb5, and preferen�ally binds Neu5Acα(2,8)Neu5Ac containing glycans.