Synthesis of D-mannosides with thiol-terminated aglycons and studies of their binding properties with mannose-binding lectins

Carbohydrates and proteins interactions are essential in viral and bacterial infection, the immune response, differentiation and development, and the progression of tumor cell metastasis. An understanding of carbohydrate-protein interactions at the molecular level would lead to a better insight into the biological process of living systems and assist the development of therapeutic and diagnostic strategies. To this end, derivatives of D-mannose (monosaccharide, 1,2-linked disaccharide- and 1,2,3-linked trisaccharide) with tethered –SH groups were synthesized. These molecules were constructed by glycosylation of appropriately protected glycosyl donors and acceptors, followed by free-radical addition to introduce the thiol terminals onto the aglycons. Subsequent deprotection afforded the corresponding free-OH saccharides. Standard robotic microarray printing technology was used to couple these thiol-terminated aglycons to epoxide-functionalized glass slides. Carbohydrate-protein binding studies were conducted with Rhodamine-labelled Conconavalin A and mannose-binding lectins. Although the printed concentrations of the disaccharide varied by up to 40-fold from 25 to 1000 µM, the calculated dissociation constants (K_D,surf) were narrowly distributed with an average of 2.11 µM from seven replicate experiments. The differences in binding constants seem to be greater at concentrations above 400 μM.  The monosaccharide had an average surface binding constant of 3.89 µM and 3.65 µM for the trisaccharide. Generally, as the concentration of the sugars increased the fluorescence intensities observed increased indicating stronger binding interactions with proteins. However, this was not necessarily the case when the protein concentrations increased. We are currently working to further optimize binding analysis and expand the toolbox of sugars and proteins used for studies.