ASSESSING GLYCOSIDIC LINKAGE FLEXIBILITY - CONFORMATIONAL-ANALYSIS OFTHE REPEATING TRISACCHARIDE UNIT OF AEROMONAS-SALMONICIDA

A detailed conformational analysis was performed for the synthetic bra nched 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 c urves from 1D transient NOE experiments, employing Gaussian-shaped inv ersion pulses at 600 MHz. Eight of the NOE curves were interglycosidic and thus useful for an analysis of glycosidic linkage orientations. M etropolis Monte Carlo (MMC) simulations and minimum-energy calculation s with the program GEGOP were used to obtain theoretical NOE curves wh ich were compared to the experimental ones. MMC simulations with diffe rent temperature parameters of 310, 600, 900 and 2000 K allowed identi fication of NOEs which are sensitive towards different conformation di stributions - not only different conformations - at both glycosidic li nkages in 1. A comparison of trisaccharide 1 with the constituent disa ccharides 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 qua ntitative evaluation was facilitated by the introduction of entropy-re lated flexibility parameters. Our study indicates a notable restrictio n of flexibility, especially at the (1->3) linkage in 1. Although over all flexibility in 1 is reduced as compared to the constituent disacch arides 2 and 3, it cannot be neglected altogether. In summary, combine d transient NOE experiments and MMC simulations provide a simple appro ach to analyse glycosidic linkage flexibility.