Analysis of saccharides that interact with the beta-D-galactopyranosyl binding site as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase

AGUSTI, R; MENDOZA, VM; GALLO-RODRIGUEZ, C; PARIS, G; RATIER, L; FRASCH, ACC; MUCHNIK DE LEDERKREMER, R

Glasgow, Escocia

Simposio; 22nd International Carbohydrate Symposium; 2004

Trypanosoma cruzi, the agent of American trypanosomiasis, expresses an unusual surface trans-sialidase that transfers sialic acid from the host glycoconjugates to parasite mucins. The trisaccharide 2,3-di-O-(b-D-galactopyranosyl)-D-galactose (1) is an external unit in the larger oligosaccharides of the mucins, and a site for sialylation.1 Trisaccharide 1 was previously synthesized in our laboratory.2 The last step of the synthesis was the hydrogenolysis of crystalline benzyl trisaccharide 2. In the present communication the acceptor substrate specificities of compounds 1 and 2 for the sialic acid transferred from sialyl-lactose was studied by high pH anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD). Taking into account that lactitol inhibited parasite mucins re-sialylation,3 we have now prepared the trisaccharide alditol 3 and tested it as a substrate and inhibitor of TcTS. Recombinant TcTS and sialyl-lactose as the donor of sialic acid were incubated with the substrate acceptors 1-3 for 15-30 min at 25 ºC. Transference values of more than 60% of sialic acid were determined for all the compounds. Two monosialylated derivatives, one of them predominant, were observed for the benzyl glycoside and the alditol, indicating that both terminal units could be sialylated. Only in the case of the trisaccharide alditol a disialylated compound was also obtained (10% of total transfer). We further determined that the three compounds efficiently inhibited TcTS activity towards the conventional substrate N-acetyllactosamine. Interestingly, and as predicted from the in vitro inhibition experiments, the benzyl glycoside (1 mM) prevented parasite sialylation (94% of inhibition). We previously found that lactitol, a good inhibitor of TcTS was however quickly eliminated from blood. For that reason, we chose the benzyl glycoside for the in vivo studies.  Current research is directed to the synthesis of other sugar derivatives that should interact with the b-D-galactopyranosyl binding site of TcTS.