## 2 Evolution of NMR Methodologies
### Solution-State NMR for Polysaccharides
#### 2.1.2 Conformational Dynamics  
Besides static structural information, NMR provides dynamic insights into polysaccharide flexibility and conformational behavior. A main advantage of NMR lies in its ability to probe molecular motions across multiple timescales, providing a comprehensive picture of conformational changes and interactions. For example, [PERSON] et al. used ROESY and \(T_{1}/T_{2}\) relaxation measurements to study the conformational dynamics of extracellular polysaccharides in slow-growing rhizomes from _Bradyrhizobium_. They found out how molecular flexibility affects biological functions [22]. The paper emphasizes how the dynamic features of polysaccharides influence their ability to interact with other biomolecules like proteins and receptors. It shows the functional role of polysaccharides in biological  
Figure 1: Schematic illustration of rapid preparation and proton NMR fingerprinting of polysaccharides from _Radix Astragali._ Copyright (2025) Elsevier. systems. NMR-based technologies, like NOESY and relaxation time experiments, can also help us figure out transient conformations. These conformations play a part in biological activity. For instance, [PERSON] et al. demonstrated the synergy between NMR and MD by using MD simulations to validate NOESY results, offering dynamic insights into transient hydrogen bonds of scleroglucans. These simulations helped confirm the formation and stability of the hydrogen bonds observed in the NMR experiments. The MD simulations allowed the researchers to explore the flexibility and transient nature of these hydrogen bonds, which were difficult to capture in static NMR data alone. By simulating the molecular dynamics of glucan in different environments, the MD results complemented the NMR findings, giving a deeper understanding of the behavior of hard glucans under physiological conditions [23]. [PERSON] et al. compared the conformational dynamics of polysaccharides and their repeating oligosaccharidesudite units. They showed how molecular size affects elasticity and interactions [24]. This comparative method highlights that molecular structure is important in determining dynamic behavior. These studies give us basic insights into the conformation of polysaccharides. They also allow us to rationally design materials with specific mechanical or biological properties. Recent progress in computational modelling, when combined with NMR data, has further enhanced our ability to analyze conformational dynamics. For instance, researchers used a combination of molecular dynamics simulation and dipole coupling (RDC) measurement. They did this to improve the conformation model of polysaccharides. It also increased the accuracy of predicting the functional role of polysaccharides [6; 15]. This integration is a very promising way to study polysaccharides in more and more complex biological systems. [PERSON] et al. used a combination of molecular modeling and NMR spectroscopy to investigate the conformations of pentose fragments derived from the O-specific polysaccharide of _Shigella flexneri_ 5a. The NMR study, utilizing \({}^{1}\)H and \({}^{13}\)C chemical shifts, inter-residue distances, and heteronuclear coupling constants, revealed that one of the pentasaccharides closely mimics the conformation of the native polysaccharide in solution. Molecular modeling, carried out using the CICADA algorithm and MM3 force field, allowed for a detailed conformational search, which was validated by the NMR data. These findings demonstrated functional and structural correlations, particularly showing how specific structural features of the pentasaccharides relate to their biological recognition by monoclonal antibodies targeting _Shigella flexneri_ 5a [25]. [PERSON] et al. utilized MD simulations to refine the conformation of an octasaccharide molecule by integrating RDC data. The RDC data provided long-range orientational restraints, which were then incorporated into the MD simulations.