Sialidase-catalyzed transsialylation using polymer-supported solution-phase techniquesElectronic supplementary information (ESI) available: Further experimental data. See http://www.rsc.org/suppdata/cc/b0/b003943h/Transsialylation assay using V. cholerae sialidase. Solutions of pNp-Neu5Ac (21 mg, 50 μmol) and acceptor (120–160 mg, 25 μmol, donor–acceptor ratio 2∶1) in degassed incubation buffer (150 μl, 0.1 M NaOAc, 0.5 mM CaCl2, pH5.5) are incubated with V. cholerae sialidase (1 U) at 30 °C. Compounds 5 (24 mg, 17%), 7 (21 mg, 15 %), 9 (17 mg, 12 %) and 11 (22 mg, 16%) were obtained as white powders. The amounts refer to the calculated absolute amounts of polymer-bound product, whereas the percentages are relative to polymer-bound acceptor units.Transsialylation assay using S. typhimurium sialidase. Solutions of pNp-Neu5Ac (21 mg, 50 μmol) and acceptor (110–150 mg, 20 μmol, donor–acceptor ratio 2.5∶1) in degassed incubation buffer (150 μl, 0.1 M NaOAc, pH 5.1) are incubated with S. typhimurium sialidase (1 U) at 30 °C. Compounds 6 (34 mg, 24%), 8 (23 mg, 16%), 10 (21 mg, 14%) and 12 (23 mg, 15%) were obtained as amorphous solids.General procedure. The course of the reaction is followed photometrically at 400 nm and by TLC (EtOH–1 M ammonium acetate (pH 7.4) 5∶1) using resorcinol spraying reagent for N-acetylneuraminic acid detection. The incubation is terminated by addition of 0.1 M Na2CO3 (1 ml)followed by ultrasonification for 3 min. The mixture is acidified to pH 6.8 with Dowex 50WX8 H+ cation exchanger and centrifuged. The supernatant is decanted and codistilled twice with toluene (10 ml). The residue is taken up in dry dichloromethane (10 ml) and precipitated with tert-butyl methyl ether (200 ml). The precipitate is filtered, rinsed with tert-butyl methyl ether (50 ml) and recrystallized from EtOH.Cleavage of the product from the resin exemplified for compound 3. 160 mg of polymer-bound trisaccharide 11 (0.032 mmol) is dissolved in dry dichloromethane (1.5 ml). Acetic anhydride (1.5 ml) and scandium(iii) trifluoromethanesulfonate [Sc(OTf)3, 10 mg, 0.02 mmol] are added sequentially. After stirring under argon for 6 h the mixture is cooled to 0 °C in an ice bath and tert-butyl methyl ether (150 ml) is added to precipitate the cleaved polymer. The filtrate is concentrated to dryness. The residue is suspended in dry MeOH (10 ml) and 100 mg cation exchange resin (DOWEX 50W-X8 H+) is added. After stirring for 6 h at rt the resin is filtered off. The filtrate is concentrated and purified by flash chromatography (petrol ether–ethyl acetate 3∶1 to 1∶1) to yield p-O-acetyl-[DOX]yl [methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d -glycero-α-d-galacto-2-nonulopyra nosyl)onate]-(2-3)-O-(2,3,4-tri-O-acetyl-β-d -galactopyranosyl)-(1-4)-O-2,3,6-tri-O-acetyl- β-d-glucopyranoside (1.8 mg, 14% relative to polymer bound 4, 88% relative to 11).

Abstract

By combination of glycosidase-catalyzed transglycosylation with polyethylene glycol ω-monomethyl ether (MPEG; mw 5000) based solution phase synthesis, various terminally sialylated di- and trisaccharides were synthesized.