O-oligosaccharidyl-1-amino-1-deoxyalditols as intermediates for fluorescent labelling of oligosaccharides |
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Authors: | Miller Janice G Farkas Vladimír Sharples Sandra C Fry Stephen C |
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Affiliation: | Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK. |
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Abstract: | Reducing monosaccharides were efficiently converted to stable 1-amino-1-deoxyalditols (=glycamines; distinguished from glycosylamines by mass-spectrometry) during incubation at 20 degrees C in saturated aqueous NH(4)HCO(3) containing NaCNBH(3). Potentially useful by-products included a novel, fully-reduced dimer (the corresponding secondary glycamine) and several relatively long-lived, unreduced products. With increasing incubation time, monomers exceeded dimers. Reducing disaccharides and oligosaccharides underwent similar reactions at their reducing termini; the yield of dimers decreased with increasing oligosaccharide M(r). The O-oligosaccharidyl-1-amino-1-deoxyalditols (OADs) obtained by reductive amination of oligosaccharides reacted readily with lissamine rhodamine sulfonyl chloride to yield OAD-sulforhodamine conjugates linked by a stable sulfonamide bond. Conditions for this reaction were optimised (borate buffer, pH9.0-9.5). The highly fluorescent OAD-sulforhodamine products were purified on a C(18) cartridge. They were electrophoretically immobile at pH2.0 and 6.5, and migrated towards the anode in borate buffer, pH9.4. The OAD-sulforhodamines were amenable to TLC and were excellent substrates for enzymic transglycosylation and for glycosylhydrolase action. |
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Keywords: | BAW, butan-1-ol/acetic acid/H2O (12:3:5, unless otherwise stated) BPW, butan-1-ol/pyridine/H2O (4:3:4) DMF, dimethylformamide DNP-Lys, 2,4-dinitrophenyl-lysine DP, degree of polymerisation EAW, ethyl acetate/acetic acid/H2O (10:5:6) LRSC, lissamine rhodamine sulfonyl chloride mGlcN, electrophoretic mobility relative to that of glucosamine (and corrected for electro-endo-osmosis) OAD, O-oligosaccharidyl-1-amino-1-deoxyalditol PC, paper chromatography PE, high-voltage paper electrophoresis PyAW/CB, 1% (v/v) pyridine, 1% (v/v) acetic acid, 0.5% (w/v) chlorobutanol, pH 4.7 SR, sulforhodamine XET, xyloglucan endotransglucosylase activity XGO, xyloglucan-derived oligosaccharide (structure not specified) XLLG, XXLG, XXXG, XXFG, etc., specific xyloglucan-derived oligosaccharides (for nomenclature, see Ref. 22) |
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