(W1130-04-22) Conformational Analysis of Alkyl/Aryl-α-O-Glycosides Using Isothermal Calorimetry: Stereo-Electronic Effects vs Hydrophobic Effect

Purpose: The biological activity of carbohydrates extends far beyond their classical roles as energy source or structural building blocks. The molecular recognition of sugars by protein receptors can be found in many biological processes including cell-cell communication, immune response, infection, among many others. The success of protein-carbohydrate interactions is conditioned to the fulfillment of conformational requirements imposed by the recognition site in the protein over the, inherently flexible, carbohydrate ligand. Hence, the study of the conformational preferences of saccharides is fundamental for the understanding of protein-carbohydrate interactions. The flexibility of sugars is mainly dependent on the free rotation around the inter-glycosidic bond and the hydroxymethyl group (C5-C6). The rotation around C5-C6 is, in turn, determined by the configuration of the stereocenter C4, the type of substitution on hydroxyl C4 and C6, the configuration of the anomeric carbon, nature of substituent on anomeric position and the media polarity. Studies on protected alkyl β-O-glycosides have also demonstrated that the rotation around the C5-C6 bond can be modulated by the steric and electronic properties of the aglycone. From the study it was observed that as the size of aglycone substituent increases from smaller groups, like methyl, to a bulkier group, like tert-butyl, a conformational shift from gg conformer to a gt conformer is observed. Currently, there are no reports of this type of study on deprotected saccharides. Hence, we decided to explore the conformational properties of model alkyl/aryl β and α-O-glycosides to unveil their structure-conformation properties against Concanavalin A using Isothermal Calorimetry and NMR. Here, we report our findings in the conformational analysis of α-O-glycosides, a model scaffold to study the potential modulation of the aglycone over the interglycosidic linkage.

Methods: Towards a better understanding of the effects that the aglycone substituent has over the conformational properties around C5-C6 bond, we first designed and synthesized a series of model alkyl/aryl-α-O-glycoside. We changed the anomeric position of the glucose monosaccharide with various aglycone groups ranging from the smallest one being methyl and the bulkiest one being tert-butyl and p-Me-phenyl, p-F-phenyl, p-NO₂-phenyl and studied their conformational preferences first in solution by NMR. Using the coupling constant values between H5 and the pro-chiral protons H6R and H6S (³J_H5H6S, ³J_H5H6R), obtained from NMR, and using Crich equations (below), we will calculate the percent contribution of the gg, gt, and tg rotamers to the conformational equilibrium for each of the glycoside.
³J_H5,H6R = 1.0 P<sub>gg + 11.0 P_gt + 4.8 P_tg
³J_H5,H6S = 2.2 P_gg + 2.5 P_gt + 10.2 P_tg
1 = P_gg + P_gt + P_tg
Next, we focused on studying the relationship between flexibility and binding affinity using the mannose-binding lectin concanavalin A. From the conformational study, we expect to establish structure-conformation relationships that will help for a better understanding of carbohydrate recognition mechanisms, the stereo-electronic anomeric effect, the effect of hydrophobicity of the aglycone groups over the C5-C6 bond and could eventually assist in the design of conformationally tailored ligand candidates in the future.

Results: The ITC analysis of our synthesized monosaccharides against Concanavalin A was performed to study the effect of ligand’s conformation and hydrophobicity over the binding to the protein receptor. From the ITC analysis we found that as the size of the aglycone increased from methyl to tert-butyl the K_d value of the saccharide binding to receptor was found to decrease by 2-folds. The K_d value for the methyl glucoside was found to be 552.17uM which was seen to drastically decrease in case of tert-butyl glucoside with a K_d of 204.9uM. The drastic decrease in Kd for the alkyl-O-Glycosides can be attributed to two factors firstly, as reported earlier, due to conformational change afforded in the saccharide because of the stereoelectronic effects of the attached aglycone and secondly, due to increased bulkiness of the aglycone thus increasing the binding interactions of the molecule to the protein receptor. In case of aryl-glycosides the K_d value of the glycosides was found to be decreasing with change in group attached the phenyl ring at para position. We found that the K_d value dropped from 152.9uM in case of EDG p</em>-Me-phenyl to 118.3uM in p-F-phenyl to 100uM in EWG like p-NO₂-phenyl. This change in case of aryl-glucosides could be attributed to the stereoelectronic effects of the EDG and EWG attached on the ring which led to the increase/decrease in binding affinity of the glycoside to the receptor. However, further NMR studies are required to confirm the binding conformation of the molecule which will provide further insights about conformational change and the change in binding mechanism of the saccharides.

Conclusion: Based on the ITC results, the C5-C6 flexibility of alkyl/aryl-α-glycosides could be influenced by the nature of the aglycone. To further explore the effect of the anomeric configuration about the C5-C6 flexibility, we will continue this study focusing on the conformational properties of the respective glycosides in solution by NMR.

References: (1) Padrón, J. I.; Vázquez, J. T. Rotational Population Dependences of the Hydroxymethyl Group in Alkyl Glucopyranosides: Anomers Comparison. Chirality 1997, 9 (5–6), 626–637. https://doi.org/10.1002/(SICI)1520-636X(1997)9:5/63.0.CO;2-O.
(2) Sanhueza, C. A.; Dorta, R. L.; Vázquez, J. T. Conformational Properties of Glucosyl-Thioglucosides and Their S-Oxides in Solution. Tetrahedron: Asymmetry 2013, 24 (9–10), 582–593. https://doi.org/10.1016/j.tetasy.2013.03.020.
(3) Morales, E. Q.; Padron, J. I.; Trujillo, M.; Vazquez, J. T. CD and 1H NMR Study of the Rotational Population Dependence of the Hydroxymethyl Group in .Beta. Glucopyranosides on the Aglycon and Its Absolute Configuration. J. Org. Chem. 1995, 60 (8), 2537–2548. https://doi.org/10.1021/jo00113a038.

Acknowledgements: Department of Pharmaceutical Sciences, St. John's University, Jamaica, NY 11439

tg, gg, gt conformers in carbohydrates

Rotamer population calculation using coupling constants

ITC analysis of glycosides to determine binding affinity of glycosides against Concanavalin A