Atomistic Insights into the Hydration Structure of Stachyose: A Minimum-Distance Distribution Function Approach

Stachyose, a raffinose-family tetrasaccharide abundant in legumes, exhibits significant prebiotic and anti-nutritional activities that are intimately linked to its aqueous solvation behavior. Yet, the atomistic details of how water molecules organize around this non-globular, flexible tetrasaccharide remain poorly understood, largely because traditional radial distribution functions fail for molecules lacking a well-defined geometric center. Here, we employ molecular dynamics simulations (3 independent 300 ns replicas, GLYCAM06j/TIP3P) in conjunction with the minimum-distance distribution function (MDDF) framework to dissect the hydration structure of stachyose at multiple length- and time-scales. Our results reveal a densely populated first hydration shell (3.5 Å) comprising 69.5 ± 0.3 water molecules, with a pronounced MDDF peak at 1.86 Å characteristic of O–H···O hydrogen bonding. Direct hydrogen-bond analysis identifies an average of 21.1 ± 0.1 solute–water H-bonds per frame, while residence-time calculations show that 11.1 ± 0.1 water molecules remain within the 5.5 Å shell for longer than 200 ps, forming a dynamic hydration core. Notably, DBSCAN cluster analysis of first-shell waters reveals a percolated hydrogen-bonded network: the largest instantaneous cluster spans 53–58 of the ~70 first-shell waters, yet the network is compositionally fluid, with individual waters exchanging on a ~40 ps timescale and only ~16% of shell waters being isolated at any instant. Conformational clustering further resolves four major families (89.2% of all frames), with terminal residues (2CU and 0LA) exhibiting higher solvent exposure than internal ones. Surprisingly, the fructose terminus (0LA) shows a dynamic hydration contribution (29.0%) essentially equal to that of the galactose terminus (28.6%) despite a smaller hydrophilic fraction (65.9% vs. 70.0% for the galactose terminus), underscoring the limitation of geometry-only descriptors. These findings provide a quantitative, residue-resolved picture of stachyose solvation and establish a computational workflow applicable to complex carbohydrates in general.