Ingrid 't Hart

18 Chapter 1 1 chemoenzyma�c synthesis of α-fluoride glycan donors (Scheme 1B). These donors can be coupled with ceramide or sphingosine acceptors, catalyzed by a mutant endoglycoceramidase II (EGCase): EGC-E. Enzyma�c coupling of the lipid overcomes the problem of lower reac�vity of sphingosine or ceramide acceptors. However, the fluoride donors are prone to hydrolysis, par�cularly under acidic condi�ons. Also, the enzyme is s�ll selec�ve towards certain donor substrates. 91,92 Scheme 1. Chemical and chemoenzyma�c strategies to synthesize GSL-ceramides, where the synthesis of Lac-Cer is used as an example for each strategy. ( A ) This chemoenzyma�c strategy starts with the chemical synthesis of Lac-Sph, followed by extension of the glycan moiety by bacterial glycosyltransferases (GTs). Coupling of an acyl chloride with the free amine provides the respec�ve GSL-ceramides. (a) i. extension of glycan chain by bacterial GTs; ii. coupling with acyl chloride.87 ( B) Another chemoenzyma�c strategy starts with chemically synthesized α-fluoride glycosyl donors. EGC-E, a mutant EGC-II enzyme, glycosylates the α-F glycans with sphingosine or ceramide. (b) EGC-E, pH 5.0.88,89 ( C ) Full chemical synthesis of GSLs by chemical glycosyla�on of a protected saccharide donor and sphingosine acceptor. (c) i. chemical glycosyla�on; ii. coupling with fa�y acid; iii. global deprotec�on.90,91 ( D ) Chemical synthesis of GSLs through the Glc-Cer casse�e approach. (d) i. chemical glycosyla�on; ii. global deprotec�on.92 R = alkyl chain of fa�y acid. Abbrevia�ons: PG = protec�ng group, LG = leaving group. Few chemical synthe�c strategies have been developed up �ll now. In the most straigh�orward approach, the lipid moiety is coupled to the glycan reducing end by chemical glycosyla�on (Scheme 1C). Since ceramide acceptors have poor reac�vity, a protected form of sphingosine is preferred as the acceptor substrate and coupling with a fa�y acid is performed in a later stage. 93,94 In a more recent “casse�e” approach, a glucosyldonor is chemically glycosylated with ceramide (Glc-Cer) (Scheme 1D). Reducing the size of the glycan donor increases reac�vity. The Glc-Cer acceptor is then glycosylated with a sugar donor to form the protected glycosylceramide. S�ll, the chemical synthesis of all building blocks requires a series of (de)protec�on steps and carefully chosen of orthogonal protec�ng groups. 95 O O OH HO OH O HO OH HO OH NH 2 OH (CH 2 ) 12 CH 3 O O O OH HO OH O HO OH HO OH HN OH (CH 2 ) 12 CH 3 O R O A O O OH HO HO O HO OH HO OH F b B C HN OH (CH 2 ) 12 CH 3 R O HO O O OPG PGO OPG O PGO OPG PGO OPG OLG N 3 OPG (CH 2 ) 12 CH 3 HO OLG O PGO OPG PGO OPG O HO OPG PGO OPG HN OH (CH 2 ) 12 CH 3 O R O d a D + c + +