296776-03-9

Two isomeric 4-methylumbelliferyl-α-d-N- acetylneuraminylgalactopyranosides (1 and 2) were synthesised. These compounds contain either the natural α-2,3 or α-2,6 sialyl-galactosyl linkages, as well as an attached 4-methylumbelliferone for convenient detection of their hydrolyses. These compounds were designed as natural sialoside analogues to be used in a continuous assay of sialidase activity, where the sialidase-catalysed reaction is coupled with an exo-β-galactosidase- catalysed hydrolysis of the released galactoside to give free 4-methylumbelliferone. The kinetic parameters for 1 and 2 were measured using the wild-type and nucleophilic mutant Y370G recombinant sialidase from Micromonospora viridifaciens. Kinetic parameters for these analogues measured using the new continuous assay were in good agreement with the parameters for the natural substrate, 3′-sialyl lactose. Given the selection of commercially available exo-β-galactosidases that possess a variety of pH optima, this new method was used to characterise the full pH profile of the wild-type sialidase with the natural sialoside analogue 1. Thus, use of these new substrates 1 and 2 in a continuous assay mode, which can be detected by UV/Vis or fluorescence spectroscopy, makes characterisation of sialidase activity with natural sialoside linkages much more facile. The Royal Society of Chemistry.

Stereoselective synthesis of α-L-Fucp-(1,2)- and -(1,3)-β-D-Galp(1)-4-methyllumbelliferone was accomplished using glycosyl donor substituted by propane-1,3-diyl phosphate as leaving group. The α-stereochemistry for the anomeric centre was assigned on the basis of the observed 13C-H coupling constant of 165.4 Hz for the bond. The results suggested that the bond-forming reaction intermediate was formed through participation of the second carbon acetoxy function or by an SN2-like process.