| mots-clés : Synthèse chimio-enzymatiqu0e – glycoconjugué – mucine – Ga1NAc-transférase – protéines recombinantes – analogues d'UDP-Ga1NAc – sondes de glycosyltransférases – STD-NMR |
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| DEVELOPMENT OF A REGENERATION SYSTEM FOR UDP-GAINAC FOR THE ENZYMATIC GLYCOSYLATION OF OLIGOSACCHARIDES AND PEPTIDES OF THERAPEUTIC INTEREST. |
| The sugar N-acetylgalactosamine (Ga1NAc) is a constituent of many glycoproteins, glycolipids and proteoglycans. It is the immunodominant sugar in blood group and oncofetal antigens which are potential therapeutic targets. However, the chemical synthesis of these antigens is time consuming and expensive. In vivo, GaINAc is incorporated into glycoconjugates by specific GaINAc transferases using UDPGa1NAc as the donor substrate. At présent, the chemo-enzymatic synthesis of glycans is hampered by the difficulties involved in the synthesis of large amounts of UDPGa1NAc. To overcome these limitations we have developed the chemo-enzymatic synthesis of Ga1NAc bearing glycoconjugates based on the simple substrates Ga1NAc, UTP and creatine-P and the use of four enzymes. In this method UDP-Ga1NAc is synthesized as an intermediate and the product UDP immediately recycled to UDP-Ga1NAc. With this technique an oligosaccharide and several mg of a small glycoprotein were synthesized and the immune response of mice to the synthesized glycoprotein investigated. The system developed to synthesize the UDP-GaINAc intermediate could be adapted to produce large amounts of UDP-Ga1NAc and we could show that it accepts also derivatives of Ga1NAc which allows the rapid synthesis of •UDP-Ga1NAc analogues. These molécules have been tested as probes for the active site of polypeptide aGa1NAc transferase Tl by kinetic studies and by STD-NMR. |
| mots-clés en anglais : Chemoenzymatic synthesis – glycoconjugate – mucin – Ga1NAc-transferase – recombinant proteins – UDP-Ga1NAc analogues – glycosyltransferase probes – STD-NMR |
