Chemoenzymatic Synthesis of Asymmetrical Multi‐Antennary N‐Glycans to Dissect Glycan‐Mediated Interactions between Human Sperm and Oocytes

Abstract

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 (SLe ), 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 moieties at the C-2 and C-2' arm and a sialyl-Lewis -Lewis (SLe -Le ) 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 -Le moiety is critical for inhibitory activity, whereas the other SLe moieties exerted minimal effect. Further studies with SLe -Le and SLe showed that the extended structure is the more potent inhibitor. In addition, trivalent SLe -Le and SLe were prepared which showed greater inhibitory activity compared to their monovalent counterparts. Our studies show that although SLe 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 -Le can make productive interactions. It is not the multivalent display of SLe on a multi-antennary glycan but the presentation of multiple SLe -Le on the various glycosylation sites of ZP that accounts for high avidity binding.

Human Zona Pellucida Glycoproteins: Binding Characteristics With Human Spermatozoa and Induction of Acrosome Reaction

“…In hemizona assay, sialyl-Lewis x tetrasaccharide as well as neoglycoproteins terminated by sialyl-Lewis x showed significant inhibition of human sperm-ZP binding ( Clark, 2013 ). By using chemoenzymatically synthesized highly complex triantennary N -glycans again confirmed that sialyl-Lewis x moiety is critical for human sperm-egg binding ( Chinoy et al, 2018 ). On the other hand, eggs obtained from transgenic mice expressing human ZP2, mouse ZP1, and mouse ZP3 that bind human sperm do not show expression of sialyl-Lewis x expression suggesting that it may not be critical for sperm-egg binding ( Avella et al, 2014 ).…”

Recent Progress in Chemo-Enzymatic Methods for the Synthesis of N-Glycans

“…One application of the above method was the assembly of some tri-antennary N-glycans of zona pellucida carrying sialyl Lewis X (SLe x ) moieties at the C-2 and C-2' arms and a sialyl Lewis X-Lewis X (SLe x -Le x ) residue at the C-6 antenna and another two analogs. The synthesized compounds were used to analyze the glycan-dependent interactions between human sperm and oocytes, indicating that the SLe x -Le x residue is essential for the inhibiting effect, while the other SLe x moieties showed much less effect (Chinoy et al, 2018).…”

Glycan Assembly Strategy: From Concept to Application

“…Boons and co‐workers also synthesized the complex N ‐glycans by installing different protecting groups on the core pentasaccharide 124 , including Lev, Fmoc, allyloxycarbonate (Alloc), and 2‐naphthylmethyl (Nap) groups (Scheme 26). [71] These protecting groups were orthogonally removed, which was followed by sequential glycosylation with donors 125 , 128 and 131 to afford the regioselective glycosylation product 132 . The yield of each coupling step is 91 %, 52 % and 75 %, respectively.…”