Abstract
<jats:p>Rhamnogalacturonan II (RG-II) is a conserved pectic polysaccharide critical for plant cell wall integrity and growth, primarily through borate diester cross-linking of its side chain A. Despite its importance, the three-dimensional structure of this glycan remains unresolved due to its complex architecture and challenges in obtaining homogenous samples. We report the total chemical synthesis of RG-II side chain A by a convergent route culminating in a gold-catalyzed [4+5] glycosylation as the key fragment union. Structural elucidation of the resulting nonasaccharide via Nuclear Magnetic Resonance (NMR)-validated molecular dynamics (MD) simulations, which reproduce 99% of 90 experimentally observed proton-proton proximities, reveals that side chain A adopts a globular conformation, stabilized by a network of conventional and nonclassical hydrogen bonds. Variable-temperature NMR together with the MD simulations support the persistence of key intramolecular interactions. These findings establish the secondary structure of RG-II side chain A and provide a foundation for understanding the molecular basis of RG-II-mediated cell wall mechanics.</jats:p>