Synthesis of a model trisaccharide for studying the interplay between the anti α-Gal antibody and the trans-sialidase reactions in Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal β-D-Galp unit in the host glycoconjugate to terminal β-D-Galp units in the parasite mucins to construct the D-NeuNAc(α2→3)β-D-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-D-Galp is only present in the infective stage whereas β-D-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-D-Galp nor D-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal β-D-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-D-Galp(1→3)-[β-D-Galp(1→6)]-D-Galp, the smallest structure containing both, the antigenic D-Galp(α1→3)-D-Galp unit and the sialic acid-acceptor β-D-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by β-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3′-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-D-NeuNAc(2→3)-β-D-Galp(1→6)-[α-D-Galp(1→3)]-β-D-Galp, was purified and characterized.