Ingrid 't Hart

99 Chemoenzyma�c synthesis of heptose-ganglioside mimics 5 losses during P2 Bio-Gel purifica�on. The β1,4 linkage between GalNAc and lactose is catalyzed by the bacterial enzyme CgtA. However, ini�al a�empts on UDP-GalNAc extension by commercially available CgtA did not show any ac�vity on disaccharide 2 or 2,3-Neu5Aclactose (data not shown). In-house expressed and purified CgtA proved inac�ve as well. CgtA was expressed again and the crude lysate was directly used to successfully convert trisaccharide 2 to tetrasaccharide 3 . S�ll, CgtA lysate was quite labile and lost its ac�vity a�er overnight storage at 4 °C. Immediate storage at -20 °C was required to maintain enzyme ac�vity over a longer period of �me. 30 Hep-GM2 ( 3 ) was successfully extended by a β1,3-Gal moiety by treatment with CgtB in the presence of UDP-Gal to provide pentasaccharide 4 (Hep-GM1). Each enzyma�c conversion was monitored by LC-MS, and products were purified by Bio-Gel P-2 size exclusion chromatography and the structural iden�fied was confirmed by NMR spectroscopy (1D and 2D) and HRMS. Scheme 2 . Enzyma�c synthesis of LOS ganglioside mimics: Hep-GM3 ( 2 ), Hep-GM2 ( 3 ) and Hep- GM1 ( 4 ). Reagents and condi�ons: substrate (10 mM), sugar-nucleo�de (11 mM), Tris buffer pH 7.7 (100 mM), MgCl 2 (20 mM) and enzyme (pmST1, CgtA or CgtB) were shaken at 37 °C. Saccharides 1 - 4 will be immobilized on a NHS-ac�vated microarray slide, together with the oligosaccharide moie�es of the human gangliosides. Binding studies will be performed with various serum samples derived from GBS pa�ents. This study will elucidate the effect of the heptoside on an�ganglioside an�body binding. Conclusions Molecular mimicry by the lipooligosaccharides of C. jejuni has been iden�fied as a trigger for Guillain-Barré Syndrome. However, exact immune responses to these ganglioside mimics are not well understood. Our aim is to study the effect of bacterial saccharide components, such as heptose, on elici�ng an�ganglioside an�bodies. For this purpose, we designed a chemoenzyma�c strategy to obtain heptosyl containing analogs of the oligosaccharide moiety of the gangliosides GM3, GM2 and GM1. The disaccharide, Hepβ1,3Galα(CH 2 ) 5 NH 2 , was successfully obtained by chemical glycosyla�on of a galactosyl donor with a properly protected heptosyl acceptor. The deprotected disaccharide was then extended by the bacterial enzymes pmST1, CgtA and CgtB to 4, 92% UDP-Gal CgtB OH O O O OH O HO HO HO HO O O O HO HO HO OH NHAc HOOC O HO OH O AcHN O HO OH HO HO 3, 68% UDP-GalNAc CgtA OH O O O OH O HO HO HO HO O O O HO HO HO OH NHAc HOOC O HO OH HO AcHN 2, 50% CMP-Neu5Ac PmST1 OH O O HO OH O HO HO HO HO O O NH 2 O HO HO HO OH NHAc HOOC n = 5 NH 2 n = 5 NH 2 n = 5 1