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Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed to investigate the bound conformation of the trisaccharide repeating unit of the Streptococcus Group A cell-wall polysaccharide. Thus, the conformations of propyl 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyran osyl)- alpha-L-rhamnopyranoside [C(A')B] (1) as a free ligand and when complexed to the monoclonal antibody Strep 9 were examined. Improved insights about the conformational preferences of the glycosidic linkages of the trisaccharide ligand showed that the free ligand populates various conformations in aqueous solution, thus displaying relatively flexible behavior. The NOE HNAc-H2A', which was not detected in previous work, accounts for a conformation at the beta-(1-->3) linkage with a phi angle of approximately 180 degrees. Observed TRNOEs for the complex are weak, and their analysis was further complicated by spin diffusion. With the use of transferred rotating-frame Overhauser enhancement (TRROE) experiments, the amount of spin diffusion was assessed experimentally, proving that all of the observed long-range TRNOEs arose through spin diffusion. Four interglycosidic distances, derived from the remaining TRNOEs and TRROEs, together with repulsive constraints, derived from the absence of TRROE effects, were used as input parameters in simulated annealing and molecular mechanics calculations to determine the bound conformation of the trisaccharide. Complexation by the antibody results in the selection of one defined conformation of the carbohydrate hapten. This bound conformation, which is a local energy minimum on the energy maps calculated for the trisaccharide ligand, shows only a change from a +gauche to a -gauche orientation at the psi angle of the alpha-(1-->2) linkage when compared to the global minimum conformation. The results infer that the bound conformation of the Streptococcus Group A cell-wall polysaccharide is different from its previously proposed solution structure (Kreis et al., 1995).

C(CH,),], 1.64 (s, 6, CH,); IR (CC14) 2956 (s), 2919 (s), 1477 (m), 1444 (m), 1394 (m), 1363 (m), 1192 (m), 1141 (m), 1073 (m); MS m l e 168 (M'), 111, 97, 84, 69, 57, 41 (base).(E)-2,2,3,4,5,5-Hexamethyl-3-hexene from above showed spectral properties comparable to those already r e p~r t e d .~ Pyrolysis. Samples of a solution containing 5 pL of 1,5 pL of tridecane, and 0.5 mL of tetradecane (prefiltered through a small column of basic alumina) were sealed in washed (1% ammonia water) and dried melting point capillaries. Samples were heated in a stirred, constant-temperature bath filled with silicone oil, heated with a quartz heater, and controlled (cycled, i 0.4 "C, about an accurately (i0.02 "C) measured temperature) with an Omega 2001 regulator. After pyrolysis, each sample was removed from the bath, immediately quenched in ice water, and analyzed by GLC. Peak areas were determined by triangulation. Integration against tridecane showed the total amount of l and 2 remained essentially constant throughout the pyrolysis period. Correction was made for the small impurity by subtracting a constant amount from each area for 1. In order to minimize the significance of this 4.7% correction, data points beyond 90% conversion to 2 were excluded from the analysis.

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