Ingrid 't Hart

29 Chemoenzyma�c synthesis of DSGb5 2 Here, we report a strategy for the prepara�on of the oligosaccharide moiety of DSGb5 by chemically synthesizing the pentasaccharide Gb5, whichwas modified by themammalian glycosyltransferases ST3Gal1 and ST6GalNAc5. This compound and a number of glycans derived from other globosides and gangliosides were printed as a glycan microarray and their interac�on with Siglec-7 was inves�gated. A challenging aspect of the prepara�on of DSGb5 is the regio- and stereoselec�ve introduc�on of the α2,3- and α2,6-linked sialosides. 12 Recently, a number of microbial sialyltransferases have been described that make it possible to prepare gangliosides from the ganglio-, lacto-, and globo-series having α2,8-Neu5Ac-α2,3-Neu5Ac and/or α2,3-sialosides at the terminal galactose. 13 These enzymes (e.g. PmST1 and CstII) can readily be expressed in E. coli making it straigh�orward to install these sialosides. To date, no microbial sialyltransferase has been iden�fied that can selec�vely install an α2,6-linked sialoside at GalNAc of a Gal-β1,3-GalNAc epitope. Recently, considerable progress has been made in the expression of human sialyltransferases, 14 and therefore we were compelled to inves�gate whether DSGb5 can be prepared by human ST3Gal1 and ST6GalNAc6, which are enzymes that can install an α2,3- and α2,6- sialoside at the terminal Gal and internal GalNAc, respec�vely. 15 Results and Discussion Chemical synthesis of core pentasaccharide Gb5 It was envisaged that the oligosaccharide moiety of Gb5 ( 4a ) could be assembled by block coupling of disaccharide 7a with trisaccharide 8a , which in turn were expected to be available frombuilding blocks 9 , 10 , 11 and 12 (Scheme 1). We an�cipated that a chemical strategy to prepare Gb5 would be more a�rac�ve than reported chemoenzyma�c 16 or enzyma�c approaches using microbial enzymes because these give low conversions, requiring large quan��es of enzyme and can be promiscuous to give unwanted side products such as Gb5 modified by addi�onal β1,3-Gal that is difficult to remove. 13b , 17 Thus, a TMSOTf catalyzed glycosyla�on of trichloroace�midate 9 with galactosamine acceptor 10 gave disaccharide 13 in a yield of 56% as only the β-anomer. The anomeric TDS protec�ng group of 13 could easily be cleaved by treatment with HF-pyridine in pyridine to give a lactol, which was converted into trichloroace�midate 7a by reac�on with trichloroacetonitrile in the presence of Cs 2 CO 3 . The use of DBU as the base resulted in substan�ally lower yield due to hydrolysis of the base sensi�ve Troc protec�ng group. Trisaccharide acceptor 8a was prepared by a glycosyla�on of thioglycosyl donor 11 with lactosyl acceptor 12a using NIS/TMSOTf as the promoter system. The glycosyla�on proceeded with absolute α-anomeric selec�vity due to the presence of the bulky 4,6-di- O- tert -butyl-silane protec�ng group that sterically blocks the β-face of the acceptor. 18 The trisaccharide was isolated in a yield of 90% a�er purifica�on by silica column chromatography. Treatment of 15a with DDQ in a mixture of DCM and PBS (24/1 v/v) gave, a�er purifica�on by silica column chromatography, acceptor 8a in a yield of 52%.