Assessing glycosidic linkage flexibility: conformational analysis of the repeating trisaccharide unit of Aeromonas salmonicida.

A detailed conformational analysis was performed for the synthetic branched trisaccharide beta-D-ManNAc-(1-->4)-[alpha-D-Glc-(1-->3)]-L-Rha 1 which represents the repeating unit of the O-antigenic polysaccharide of Aeromonas salmonicida. The study was based on 26 experimental NOE curves from 1D transient NOE experiments, employing Gaussian-shaped inversion pulses at 600 MHz. Eight of the NOE curves were interglycosidic and thus useful for an analysis of glycosidic linkage orientations. Metropolis Monte Carlo (MMC) simulations and minimum-energy calculations with the program GEGOP were used to obtain theoretical NOE curves which were compared to the experimental ones. MMC simulations with different temperature parameters of 310, 600, 900 and 2000 K allowed identification of NOEs which are sensitive towards different conformation distributions--not only different conformations--at both glycosidic linkages in 1. A comparison of trisaccharide 1 with the constituent disaccharides beta-D-ManNAc-(1-->4)-L-Rha 2 and alpha-D-Glc-(1-->3)-L-Rha 3 revealed effects of branching on glycosidic linkage flexibility. A quantitative evaluation was facilitated by the introduction of entropy-related flexibility parameters. Our study indicates a notable restriction of flexibility, especially at the (1-->3) linkage in 1. Although overall flexibility in 1 is reduced as compared to the constituent disaccharides 2 and 3, it cannot be neglected altogether. In summary, combined transient NOE experiments and MMC simulations provide a simple approach to analyse glycosidic linkage flexibility.