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The tetrasaccharide 1, a substructure of ganglioside GQ1b alpha, shows a remarkable affinity for the myelin-associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program. In our search for structurally simplified and pharmacokinetically improved mimics of 1, modifications of the core disaccharide, the alpha(2-->3)- and the alpha(2-->6)-linked sialic acid were synthesized. Biphenylmethyl and (S)-lactate were identified as suitable replacements for the alpha(2-->6)-linked sialic acid. Combined with a core modification and the earlier found aryl amide substituent in the 9-position of the alpha(2-->3)-linked sialic acid, high affinity MAG antagonists were identified. All mimics were tested in a competitive target-based binding assay, providing relative inhibitory potencies (rIP). Compared to the reference tetrasaccharide 1, the rIPs of the most potent antagonists 59 and 60 are enhanced nearly 400-fold. Their K(D)s determined in surface plasmon resonance experiments are in the low micromolar range. These results are in semiquantitative agreement with molecular modeling studies. This new class of glycomimetics will allow to validate the role of MAG in the axon regeneration process.

Siglecs are the largest family of sialic acid-recognizing lectins identified so far with 11 members in the human genome. Most of these siglecs are exclusively expressed by cells of the immune system. Comparison of different mammalian species has revealed differential and complex evolutionary paths for this protein family, even within the primate lineage. To understand the evolution of siglecs, in particular the origin of this family, we investigated the occurrence of corresponding genes in bony fish. Interestingly, only unambiguous orthologs of mammalian siglec-4, a cell adhesion molecule expressed exclusively in the nervous system, could be identified in the genomes of fugu and zebrafish, whereas no obvious orthologs of the other mammalian siglecs were found. As in mammals, fish siglec-4 expression is restricted to nervous tissues as demonstrated by northern blot. Expressed as recombinant protein, fish siglec-4 binds to sialic acids with a specificity similar to the mammalian orthologs. Relatively low sequence similarities in the cytoplasmic tail as well as an additional splice variant found in fish siglec-4 suggest alternative signaling pathways compared to mammalian species. Our observations suggest that this siglec occurs at least in the nervous system of all vertebrates.

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