Ingrid 't Hart

20 Chapter 1 1 Forming a 1,2- cis linkage has proved more challenging, although some effec�ve methods have been developed. Incorpora�on of a chiral auxiliary on C-2 can provide a trans -decalin intermediate and results 1,2- cis glycosidic bonds (Scheme 2B). Furthermore, a bulky di-tert-butylsilyl group on the C-4 and C-6 hydroxyls of galactose also provides 1,2-cis linkages,most likely by steric hindranceof theβ-face (Scheme2C). 87 The la�er cis -direc�ng strategy has been used to synthesizeGal(α1,4)Lac in the globo-series oligosaccharides. 98,99 Regioselec�ve outcomes are usually obtained by use of orthogonal protec�ng groups, keeping one hydroxyl free as the nucleophile. Another op�on to control regioselec�vity is by exploi�ng hydroxyl reac�vity. 100,101 As an example, in the synthesis of ganglio-series oligosaccharides, the lactose acceptor can have both 3- and 4-hydroxyls as possible sites for glycosyla�on. Due to the low reac�vity of the axial C-4 hydroxyl group, a (near) selec�ve glycosyla�on with C-3 can be obtained. 102,103 Furthermore, reac�vity of both donor and acceptor should be taken into account by choosing protec�ng groups. It has been observed that benzyl compounds are more reac�ve (“armed”) than the benzoylated or acetylated deriva�ves (“disarmed”). 104 This does not promote the sole use of benzyl ether protec�ng groups, but provides handles to increase reac�vity. Matching of donors, acceptors and suitable ac�vators is of key importance in all chemical glycosyla�ons. 105 Glycosyla�ons with sialic acid donors are a common challenge in the chemical synthesis of gangliosides. The anomeric center is crowded and contains an electron-withdrawing carboxylic acid. Furthermore, they lack a C-3 hydroxyl for neighboring group par�cipa�on. Different op�miza�ons, such as the oxazolidinone protec�ng group and phosphate leaving groups, have led to more reac�ve and α-selec�ve donors. 106,107,108 However, the availability of sialyltransferases, such as pmST1, provide a good alterna�ve method for sialyla�on. 109 Protected synthe�c glycans are usually purified by C18, silica column or size-exclusion chromatography (SEC) (e.g. LH-20, SX1), while deprotected synthe�c glycans are mainly purified by SEC (e.g. Bio-Gel P-2) or hydrophilic interac�on chromatography (HILIC). Next to various mass spectrometric methods (ESI, MADLI-TOF, ion mobility), 1D and 2D NMR analysis is highly relevant for the proper analysis of complex carbohydrates. 110,111 1.6 Enzyma�c synthesis of GSL oligosaccharides All oligosaccharides in nature are assembled and cleaved by enzymes and over 500,000 glycosyltransferase (GT) sequences have been iden�fied to far. 112 These natural catalysts provide an efficient regio- and stereoselec�ve way to synthesize oligosaccharides in the lab. Therefore, expression and isola�on of stable and ac�ve glycosyltransferases is of high importance. Next to GTs, (mutant) glycosyl hydrolases can be used for glycan synthesis as well under specific condi�ons. 87,92 The advantage of enzyma�c oligosaccharide synthesis is the high regio- and stereoselec�vity, which dras�cally reduces the amount of reac�on steps compared to chemical synthesis. However, enzymes are usually expressed in small quan��es and expensive sugar-nucleo�des are required, which limits the scale of the reac�on.