Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins

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Abstract
Aiming at the introduction of a fluorescent sialic acid into glycoconjugates, 5‐acetamido‐9‐(3‐fluoresceinylthioureido)‐3,5,9‐trideoxy‐2‐nonulosonic acid (9‐fluoresceinyl‐NeuAc) was synthesized which has an intact carbon chain. a) Despite the space‐filling substituent at C‐9 the fluorescent NeuAc analogue was activated to the corresponding CMP‐glycoside by CMP sialic acid synthase from bovine brain. Whereas the km value of the synthase was little affected by the modification (Km= 2.1 mM, for NeuAc Km= 1.4 mM), the V value decreased to 7.5%. b)CMP‐9‐fluoresceinyl‐NeuAc was synthesized on a preparative scale (17% overall yield), and characterized by analytical HPLC, absorption and fluorescence spectra. c) 9‐Fluoresceinyl‐NeuAc was transferred onto asialo‐α1‐acid glycoprotein by both Galβ1, 4GlcNAc α2, 6sialyltransferase and Galβ1,4(3)GlcNAc α2,3sialyltransferase (rat liver), and onto antifreeze glycoprotein by GalNAc α2,6‐sialyltransferase (porcine submaxillary glands). Using analytical HPLC, transfer was confirmed after release of the fluorescent sialic acid by Vibrio cholerae sialidase. d) Initital rate studies indicated a low Km value of Galβ‐1,4GlcNAc α2,6sialyltransferase, and GalNAc α2,6sialyltransferase (specific for O‐linked oligosaccharide chains) for CMP‐9‐fluoresceinyl‐NeuAc.

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