Chem. Pharm. Bull. 55(7) 992—995 (2007)

exposed structural components of the outer membrane of Gram-negative bacteria. LPSs consist of lipid A, a core region, and an O-antigen region in many bacteria. LPSs play an important role in the pathogenicity of Gram-negative infections. In experimental animals, purified LPSs induce numerous pathophysiological activities that can lead to shock and death, a condition called “Sepsis”. Most of these biological activities, including the induction of inflammatory cytokines, are expressed by the lipid A moiety, a ubiquitous component of LPSs that is structurally similar and serologically cross-reacting among Enterobacteriaceae, via toll-like receptor 4 (TLR4). However, numerous recent studies have revealed that LPSs possessing the mannose homopolymer (MHP) as the O-antigen region, such as the LPS from Escherichia coli (E. coli) O9 (O9 LPS), exhibit several characteristic immunological activities differ from those of the well-known endotoxic shock. In addition, we previously described that the mannan moieties of Candida albicans water-soluble fraction, CAWS, from fungi shows similar immunological properties to Gramnegative bacterial O9 LPS. These characteristic activities should be exhibited by recognizing mannose residues via lectins, such as mannose-binding lectin (MBL). Therefore, analyzing the structure of O-antigens, especially mannoserich regions, is important for understanding some of the biological activities of LPSs. The O-antigen structure of O9 LPS has been studied using methylation analysis, smith degradation, optical rotation and enzymatic methods, however, a precise assignment by NMR spectroscopy has not been conducted. In general, focusing on the anomeric proton as the starting point for assignment, TOCSY and HSQC-TOCSY experiments are useful for the analysis of individual sugar components in polysaccharides. However, in some cases, the assignment can be empirically based due to signals that heavily overlap and small J coupling constants. For the reasons mentioned above, we attempted to apply recently developed pulse sequences, the heteronuclear 2-bond correlation (H2BC) experiments in order to achieve an unambiguous assignment of the high mannose type polysaccharide, the O-antigen from O9 LPS. In the present study, we report on the structure of the Oantigen of O9 LPS, and completely assign all protons and carbons using 1D-, 2D-NMR spectroscopy including the DQF-COSY, TOCSY, NOESY, HSQC, H2BC, HSQCTOCSY, and HMBC methods.