It is now well established that carbohydrates and glycoconjugates mediate important biological processes, such as cell–cell communication and pathogen infection, through recognition events with a wide range of carbohydrate-binding biomolecules (proteins, carbohydrates and nucleic acids). As a result, considerable progress has been made in method development of carbohydrate-based diagnostic and/or therapeutic agents. For instance, the design of glycomimetics of higher affinity was developed as synthetic ligands to compete with carbohydrate-binding receptors present on pathogenic micro-organisms (bacteria, viruses). The shortest access to glycomimetics or (neo)glycoconjugates requires chemoselective ligations to the anomeric position of carbohydrates which display an aldehyde function under its opening form. In carbohydrate chemistry, the aldehyde-condensation reaction is traditionally performed by amination with primary amines or with more reactive α-nucleophiles such as oxyamine or hydrazine derivatives. The latter condensation reactions were preferred because of the efficiency, chemo- and stereo-selectivity and tolerance to various solvent including water. Interestingly, the Knoevenagel condensation using β-diketones is another very convenient method that leads to β-C-glycosides in one step directly from unprotected sugar. However, it has been less developed in spite of its chemical and enzymatic stability and ring integrity of the terminal reducing sugar.
The approach developed here offers a straightforward and efficient access to β-C-glycosyl barbiturate ligands, spanning from glycomimetics to multivalent C-neoglycoconjugates, with the aim of deciphering structural parameters impacting the binding to pathogenic lectins. We reinvestigated the Knoevenagel condensation of barbituratic acid on protecting-group free carbohydrates and successfully designed sodium and 5,5 disubstituted N,N-dimethyl barbiturate forms of D-galactose, L-fucose, melibiose, 2’-fucosyllactose, maltose and evaluated their binding affinity by isothermal titration calorimetry with LecA (galactose-binding lectin) and LecB (fucose-binding lectin) from Pseudomonas aeruginosa and RSL (fucose-binding lectin) from Ralstonia solanacearum. The barbiturate ring was shown detrimental for binding to LecA (Kd in mM range) and even more to LecB (non-interaction) while RSL is much more tolerant especially in presence of an aromatic group (Kd in μM range). However, distancing the barbiturate ring from the recognition carbohydrate residue by using oligosaccharides increased affinity up to low micromolar range. Extension of our convenient synthetic approach led in two-steps to melibiose-based C-glycosyl barbiturate cluster and C-glycosyl barbiturate glycopolymers exhibiting a dramatic enhancement of binding avidity for LecA.