Recognition of carbohydrates (glycans) by lectins (carbohydrate-binding proteins different from antibodies and enzymes) is involved in numerous biological events. Selective inhibitors of lectins are not only indispensable research tools in glycobiology [1] but also potential therapeutic agents [2]. The use of endogenous carbohydrates as inhibitors for in vivo studies, or as drug candidates, is limited by their low affinity and selectivity to specific lectins, rapid metabolic degradation and high hydrophilicity [3]. Attachment of carbohydrates to multivalent carriers emulates their multivalent presentation on the cell surface [4] and improves affinity and selectivity to lectins.[5] However, these conjugates may suffer from poor drug-like properties [6]. Replacing a hydroxyl group with a fluorine atom (deoxyfluorination) can improve metabolic stability, lipophilicity and selectivity of carbohydrate lectin ligands [7]. Moreover, the resulting fluorinated glycans are invaluable probes for studying glycan-lectin interactions by NMR methods thanks to the favorable properties of the 19F nucleus [8]. Unfortunately, deoxyfluorinated sugars often display poorer affinity to lectins than endogenous carbohydrates. A significant increase in affinity to a lectin by a single-site deoxyfluorination of a monovalent glycan is an exceptionally rare phenomenon [9] although carbohydrate deoxyfluorination has been otherwise successful in the design of glycosidase inhibitors [10], and introducing fluorine into bioactive drug candidates has been tremendously successful in drug development [11]. The reported examples of interest are summarized in Fig. 1. An eight-fold enhancement in binding affinity to galectin-1 compared to parent disaccharide 1 was previously reported for methyl β-glycoside of 2′-fluoro-N-acetyllactosamine 2 (2′F-LacNAcβ1-OMe) [9]. The cluster glycoside effect can potentiate the effect of fluorine introduction, as documented for fluorinated trisaccharides 4 and 5 that showed improved binding to protozoan adhesive protein TgMIC1 compared to their non-fluorinated counterpart 3 only after immobilization to glass slides [12]. Strong binding to galectin-3 and -7 was also reported for 3′-fluoro-lacto-N-biose 7 immobilized on gold nanoparticles [7d]. However, immobilized multivalent fluoroglycomimetics cannot be readily utilized as 19F NMR probes, mainly due to the complexity of resulting NMR spectra, or as leading compounds in drug development owing to difficulties in formulation. Notably, the impact of deoxyfluorination on lectin binding can also be employed to deduce the role of the deoxyfluorinated hydroxyl group in carbohydrate-lectin interactions—an abrogated or strongly reduced binding affinity suggests that the hydroxyl is engaged in hydrogen bonding or cation complexation [13].

One of the most studied lectins is wheat germ agglutinin (WGA), a plant lectin abundant in the seeds of Triticum vulgare (common wheat). It is conveniently used as a model lectin to study the principles of glycan-lectin recognition [14]. WGA binds terminal and internal N-acetylglucosamine (GlcNAc), β-1,4-linked GlcNAc oligomers (chitooligosaccharides) [15] and terminal non-reducing N-acetylneuraminic acid using multiple binding sites [16]. It is routinely used to detect terminal GlcNAc residues in glycoconjugates [17] and has been studied as a potential therapeutic agent for hematological malignancies [18]. Disaccharide N,N′-diacetylchitobiose 8 (Fig. 3), the simplest chitooligosaccharide, binds WGA with about one-to-two-orders-of-magnitude higher affinity than GlcNAc [19]. WGA is a stable 36 kDa homodimer with a two-fold symmetry axis. At neutral pH, it is composed of two identical polypeptide chains associated in a head-to-tail manner, each chain consisting of four carbohydrate-binding hevein domains A–D (Fig. 2A) [20]. The homodimeric molecule of WGA has eight independent binding sites positioned at the interface of contacting domains A/D and B/C. However, only four of them, located at the interface of domains B and C and labeled B1C2, C2B1, B2C1, and C1B2, (Fig. 2B) are considered strong enough to bind a monovalent carbohydrate ligand in solution with detectable affinity [20]. Numbers 1 or 2 in the labels denote the polypeptide chain, to which a domain belongs. Due to the twofold symmetry axis of the protein, sites B1C2 and B2C1, and C1B2 and C2B1 are identical. In the nomenclature adopted from the literature [20], the first of the two contacting domains in the binding site label (for example, B1 in B1C2) contributes to the binding site with three aromatic and one serine residues, while the other domain in the label (for example, C2 in B1C2) contributes with two polar residues. Commercially available WGA is isolated from wheat as a mixture of three highly homologous isoforms designated WGA 1–3 with minor differences in the amino acid composition of the binding sites and their binding affinities (Table 1) [20].

Inspired by the above-mentioned instances of the increased binding to lectins after ligand monodeoxyfluorination, we decided to investigate the interaction between WGA and monodeoxyfluorinated analogs of two disaccharide ligands, N,N′-diacetylchitobiose 8 and N,N′-diacetyllactosamine (LacdiNAc) 9 (Fig. 3). While chitobiose 8 is the simplest of very thoroughly studied chitooligosaccharide WGA ligands, [14], [21] LacdiNAc 9 (Fig. 3), though recently reported as a WGA binder [16b], is a practically unexplored WGA ligand with unknown binding energetics, for which no glycomimetic study has ever been conducted. The LacdiNAc epitope caps the antennae of N- and O-glycans and is of clinical significance [22]. To fix the anomeric stereochemistry and facilitate synthesis, disaccharides 8 and 9 were prepared and studied as methyl glycosides in the β-configuration denoted herein CH and LDN, respectively (Fig. 3).

First, we estimated the relative binding free energies of fluorinated disaccharides by Alchemical free energy calculation (AFE) [23] that indicated increased binding after deoxyfluorination of some positions at the non-reducing-end pyranose. This motivated us to synthesize the complete series of monodeoxyfluorinated analogs of methyl chitobioside CH and LacdiNAcβ1-OMe LDN and determine the impact of deoxyfluorination of each non-anomeric hydroxyl position on WGA binding. We found unprecedented enhancements in the binding affinities of these monovalent lectin ligands caused by single-site deoxyfluorination. The profiling of ligand-WGA interactions by systematic deoxyfluorination also revealed the importance of conformational preorganization for binding to WGA. Finally, NMR investigation revealed which interactions significantly contribute to the affinity enhancement after deoxyfluorination.