Nanomolar E‑Selectin Antagonists with Prolonged Half-Lives by a Fragment-Based Approach

Selectins, a family of C-type lectins, play a key role in inflammatory diseases (e.g., asthma and arthritis). However, the only millimolar affinity of sialyl Lewis^x (sLe^x), which is the common tetrasaccharide epitope of all physiological selectin ligands, has been a major obstacle to the development of selectin antagonists for therapeutic applications. In a fragment-based approach guided by NMR, ligands binding to a second site in close proximity to a sLe^x mimic were identified. A library of antagonists obtained by connecting the sLe^x mimic to the best second-site ligand via triazole linkers of different lengths was evaluated by surface plasmon resonance. Detailed analysis of the five most promising candidates revealed antagonists with <i>K</i>_D values ranging from 30 to 89 nM. In contrast to carbohydrate–lectin complexes with typical half-lives (<i>t</i>_1/2) in the range of one second or even less, these fragment-based selectin antagonists show <i>t</i>_1/2 of several minutes. They exhibit a promising starting point for the development of novel anti-inflammatory drugs

Stabilization of Branched Oligosaccharides: Lewis^x Benefits from a Nonconventional C–H···O Hydrogen Bond

Although animal lectins usually show a high degree of specificity for glycan structures, their single-site binding affinities are typically weak, a drawback which is often compensated in biological systems by an oligovalent presentation of carbohydrate epitopes. For the design of monovalent glycomimetics, structural information regarding solution and bound conformation of the carbohydrate lead represents a valuable starting point. In this paper, we focus on the conformation of the trisaccharide Le^x (Gal­[Fucα(1–3)]­β(1–4)­Glc<i>N</i>Ac). Mainly because of the unfavorable tumbling regime, the elucidation of the solution conformation of Le^x by NMR has only been partially successful so far. Le^x was therefore attached to a ¹³C,¹⁵N-labeled protein. ¹³C,¹⁵N-filtered NOESY NMR techniques at ultrahigh field allowed increasing the maximal NOE enhancement, resulting in a high number of distance restraints per glycosidic bond and, consequently, a well-defined structure. In addition to the known contributors to the conformational restriction of the Le^x structure (exoanomeric effect, steric compression induced by the <i>N</i>HAc group adjacent to the linking position of l-fucose, and the hydrophobic interaction of l-fucose with the β-face of d-galactose), a nonconventional C–H···O hydrogen bond between H–C(5) of l-fucose and O(5) of d-galactose was identified. According to quantum mechanical calculations, this C–H···O hydrogen bond is the most prominent factor in stabilization, contributing 40% of the total stabilization energy. We therefore propose that the nonconventional hydrogen bond contributing to a reduction of the conformational flexibility of the Le^x core represents a novel element of the glycocode. Its relevance to the stabilization of related branched oligosaccharides is currently being studied