A New Combined Computational and NMR-Spectroscopical Strategy for the Identification of Additional Conformational Constraints of the Bound Ligand in an Aprotic Solvent

Abstract

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.

Calix[n]arene‐Based Glycoclusters: Bioactivity of Thiourea‐Linked Galactose/Lactose Moieties as Inhibitors of Binding of Medically Relevant Lectins to a Glycoprotein and Cell‐Surface Glycoconjugates and Selectivity among Human Adhesion/Growth‐Regulatory Galectins

“…Final concentrations of aprotic solvent was up to 4.4 % at 2.5 m M inhibitor concentration in solid‐phase assays and up to 6.8 % in cell assays. Parallel controls with the identical solvent concentration in the absence of glycocluster ascertained the lack of a significant solvent effect on lectin activity, as reported previously 33…”

Selective Recognition of β‐Mannosides by Synthetic Tripodal Receptors: A 3D View of the Recognition Mode by NMR

“…This study shows that the complexation mode of the investigated tripodal receptors involves the interaction of the faces of the sugars with aromatic surfaces,14 van der Waals interactions and hydrogen bonding through the sugar hydroxy groups,15 as frequently found in the recognition of saccharides by lectins 16. However, there is no evident direct parallel between the recognition of mannose by these tripodal receptors and by natural lectins, because cations (usually calcium ions) are frequently employed for mannose recognition by the biological binding site 17.…”

Efficient 1H-NMR Quantitation and Investigation of N-Acetyl-D-glucosamine (GlcNAc) and N,N'-Diacetylchitobiose (GlcNAc)2 from Chitin

“…From the 1 H-NMR study of (GlcNAc) 2 in pyridine- d 5 , the signal of the α-form acetyl group increases with reaction time; however, the signal of the β-form acetyl group decreases, implying opening and closing of the ring in (GlcNAc) 2 [17–19]. The signal magnitude of the β-form acetyl group changes to the signal magnitude of the α-form acetyl group gradually, suggesting that the intramolecular hydrogen bonds of (GlcNAc) 2 in pyridine- d 5 make α-form more stable than β-form.…”