AI Article Synopsis
- Two isomeric compounds, 4-methylumbelliferyl-alpha-D-N-acetylneuraminylgalactopyranosides, were synthesized to study sialidase activity, featuring either alpha-2,3 or alpha-2,6 linkages.
- The compounds are designed for a continuous assay that couples sialidase-catalyzed reactions with exo-beta-galactosidase activity, allowing for easy detection of hydrolysis through 4-methylumbelliferone release.
- Kinetic parameters obtained match well with natural substrates, and the new method simplifies characterization of sialidase activity at various pH levels using UV/Vis or fluorescence spectroscopy.
Article Abstract
Two isomeric 4-methylumbelliferyl-alpha-D-N-acetylneuraminylgalactopyranosides (1 and 2) were synthesised. These compounds contain either the natural alpha-2,3 or alpha-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-beta-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-beta-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.
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| http://dx.doi.org/10.1039/b613909d | DOI Listing |
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