Chemoenzymatic Synthesis of Asymmetrical Multi-Antennary N-Glycans to Dissect Glycan-Mediated Interactions between...

Complex N-glycans of glycoproteins of the zona pellucida (ZP) of human oocytes have been implicated in the binding of spermatozoa. The termini of these unusual bi-, tri-, and tetra-antennary N-glycans consist of the tetrasaccharide sialyl-Lewis^x (SLe^x ), which was previously identified as the minimal epitope for sperm binding. We describe here the chemoenzymatic synthesis of highly complex triantennary N-glycans derived from ZP carrying SLe^x moieties at the C-2 and C-2' arm and a sialyl-Lewis^x -Lewis^x (SLe^x -Le^x ) residue at the C-6 antenna and two closely related analogues. The compounds were examined for their ability to inhibit the interaction of human sperm to ZP. It was found that the SLe^x -Le^x moiety is critical for inhibitory activity, whereas the other SLe^x moieties exerted minimal effect. Further studies with SLe^x -Le^x and SLe^x showed that the extended structure is the more potent inhibitor. In addition, trivalent SLe^x -Le^x and SLe^x were prepared which showed greater inhibitory activity compared to their monovalent counterparts. Our studies show that although SLe^x can inhibit the binding of spermatozoa, presenting this epitope in the context of a complex N-glycan results in a loss of inhibitory potential, and in this context only SLe^x -Le^x can make productive interactions. It is not the multivalent display of SLe^x on a multi-antennary glycan but the presentation of multiple SLe^x -Le^x on the various glycosylation sites of ZP that accounts for high avidity binding.