Synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 for use in the assay of alpha-glucosidase I activity.
The chemical synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp p-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 (9), a substrate specific for alpha-glucosidase I, is reported. This enzyme removes the terminal alpha-D-Glcp unit to produce alpha-D-Glcp-(1-->3)-alpha-D-Glcp-O-(CH2)8 COOCH3 (10). This is the first synthetic substrate described for glucosidase I that allows kinetic evaluation of substrates and inhibitors of this enzyme. Tetramethylrhodamine was coupled to 9 through an ethylenediamine linker to produce a brilliant red derivative. Addition of this fluorescent dye did not affect enzyme binding to the substrate, as determined by a comparison of the Km value (1.3 mM). The fluorescent label allows visual detection of 2-3 pmol of product by TLC.
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Department of Chemistry, University of Alberta, Edmonton, Canada.
Hindsgaul
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Srivastava
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D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D004187:Disaccharides / Q000138:chemical synthesis; D004952:Esters / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005960:Glucosides / Q000138:chemical synthesis / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D012235:Rhodamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D014312:Trisaccharides / Q000138:chemical synthesis* / Q000378:metabolism; D000520:alpha-Glucosidases / Q000032:analysis / Q000378:metabolism*
D004187:Disaccharides; D004952:Esters; D005456:Fluorescent Dyes; D005960:Glucosides; D012235:Rhodamines; D014312:Trisaccharides; C005358:tetramethylrhodamine; D000520:alpha-Glucosidases