Structural and Biophysical Characterization of C‐Glycosylic 1,2‐Thiodisaccharides Reveals Determinants of Selective Binding to Galectin‐7 and Galectin‐8N

Achieving isoform selectivity within the galectin family remains a central challenge in glycomimetic drug design due to the highly conserved architecture of their carbohydrate recognition domains. Here, we define the structural and thermodynamic basis of recognition of a series of C‐glycosylic 1,2‐thiodisaccharides targeting human galectin‐7 and the N‐terminal domain of galectin‐8 (galectin‐8N). Using an integrated approach combining fluorescence polarization, isothermal titration calorimetry, and high‐resolution X‐Ray crystallography, we establish a clear structure–activity relationship across the ligand series. Compound 17 emerges as the most potent galectin‐8N ligand ( K d = 13 μM), outperforming thiodigalactoside, while compound 13 shows preferential binding to galectin‐7, demonstrating tunable isoform bias. Structural analysis reveals a conserved anchoring mechanism in which the β‐galactoside unit (Gly‐1) drives affinity through a rigid hydrogen‐bonding and π‐stacking network, whereas the second sugar (Gly‐2) modulates potency by adopting distinct orientations in galectin‐specific extended binding sites. Notably, ligand binding converges on conserved motifs while leaving nonconserved regions unexploited, highlighting clear opportunities for structure‐guided optimization. Collectively, this work establishes C‐glycosylic thiodisaccharides as a robust platform for selective galectin targeting and provides actionable design principles for next‐generation inhibitors.