Double affinity amplification of galectin-ligand interactions through arginine-arene interactions: synthetic, thermodynamic, and computational studies with aromatic diamido thiodigalactosides.

A series of aromatic mono- or diamido-thiodigalactoside derivatives were synthesized and studied as ligands for galectin-1, -3, -7, -8N terminal domain, and -9N terminal domain. The affinity determination in vitro with competitive fluorescence-polarization experiments and thermodynamic analysis by isothermal microcalorimetry provided a coherent picture of structural requirements for arginine-arene interactions in galectin-ligand binding. Computational studies were employed to explain binding preferences for the different galectins. Galectin-3 formed two almost ideal arene-arginine stacking interactions according to computer modeling and also had the highest affinity for the diamido-thiodigalactosides (K(d) below 50 nM). Site-directed mutagenesis of galectin-3 arginines involved in binding corroborated the importance of their interaction with the aromatic diamido-thiodigalactosides. Furthermore, the arginine mutants revealed distinct differences between free, flexible, and solvent-exposed arginine side chains and tightly ion-paired arginine side chains in interactions with aromatic systems.