I. Introduction and Scope 4589 II. NMR Methods for Structural Determination 4591 A. Carbohydrate Structural Elements and Classical NMR Methods 4591 1. Number of Sugar Residues 4592 2. Constituent Monosaccharides 4592 3. Anomeric Configuration 4593 4. Linkage and Sequence 4594 5. Position of Appended Groups 4594 6. Classical NMR Methods 4594 B. New NMR Methods 4596 C. Computer-Assisted Structural Analysis 4598 D. NMR Spectroscopy of Mixtures 4599 III. Large and Unusual Oligosaccharides 4600 IV. Polysaccharides 4603 V. NMR Analysis of Small Sample Amounts 4604 A. NMR Probes and Sample Tubes 4605 B. Examples of Structural Analysis with Small Sample Amounts 4606 VI. Conclusions 4610 VII. Acknowledgments 4610 VIII. References 4610 † This paper is dedicated to the late Professor R. U. Lemieux in memory of an always exiting and stimulating collaboration on structural studies of oligosaccharides.Jens Ø. Duus received his M.Sc. degree in Chemical Engineering from the Technical University of Denmark in 1989. Following this, he continued his graduate study with Professor Klaus Bock at the Carlsberg Laboratory and was granted his Ph.D. degree from the Technical University of Denmark in 1993. During his graduate study he was twice a Visiting Scientist at Dupont Experimental Station in Wilmington, DE working with Dr. S. Sabesan. From 1993 to 1994 he was a Research Fellow in Chemistry at the Beckman Institute at the California Institute of Technology working with Professor Harry B. Gray. After returning to the Carlsberg Laboratory, he was appointed Senior Scientist. His main fields of research are structure and dynamics of biomolecules with emphasis on carbohydrates using NMR and fluroescence.

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A novel two-dimensional NMR pulse sequence, H2BC, for long-range correlation of 1H and 13C nuclei is presented. The experiment has several attractive features compared to the widely used HMBC experiment, for example, (a) typically strong enhancement of correlations over two bonds while suppressing those over more bonds, that is, resolving ambiguities in standard HMBC spectra and showing two-bond correlations not present in HMBC spectra, (b) independence of long-range 1H-13C coupling constants, (c) full homo- and heteronuclear decoupling in the indirect dimension and heteronuclear decoupling in the acquisition dimension, (d) pure 2D absorption peak shapes, and (e) a pulse sequence duration significantly shorter than that of HMBC. The experiment is quite complementary to HMBC and does not effect correlations to quaternary carbons that must be obtained by HMBC.

Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.Like other members of the family Enterobacteriaceae, the lipopolysaccharides (LPSs) 1 of Klebsiella pneumoniae consist of three structural domains, (i) the hydrophobic lipid A, which is a major component of the outer leaflet of the Gram-negative outer membrane, and (ii) the core oligosaccharide, which is linked to lipid A and provides the attachment site for (iii) the long chain polysaccharide (O antigen; O chain). Typically, structural diversity is greater in the regions of LPS extending from the cell surface (i.e. the O chains). Varying chemical structures in the O chains gives rise to a number of serologically distinct O antigens. In the Klebsiellae there are 11 known O-chain structures, but structural similarities lead to some serological cross-reactivities, so the actual number of unique O serotypes is less (1, 2). Several of the O-antigens are based on a structure designated D-galactan I with a repeat unit compris-2 When present alone, this structure provides the 2a antigen (3), but it can be capped by additional structural domains or modified by side chain acetyl or galactosyl residues to generate additional unique antigens (3-6). For example, in the most clinically prevalent serotype, the O1 antigen, D-galactan I chains are capped by a domain with a different repeat unit structure, [-. Genetic (7) and chemical (8, 9) analyses indicate that D-galactan I chains are linked directly to the lipid A core structure, whereas D-galactan II is confined to the distal end of some of the available D-galactan I chains (10). D-Galactan II provides the epitope(s) that defines the O1 antigen (9), and its presence is required for the resistance of the bacteria to complement-mediated killing in the host; K. pneumoniae mutants that only produce D-galactan I are therefore serum-sensitive (11, 12). However, not all Klebsiella O serotypes are based on D-galactan I. The prevalent O3 and O5 serotypes comprise mannan O chains with structures identical to the Escherichia coli O9 and O8 antigens, respectively (13,14). The remaining Klebsiella O-chain structures are heteropolymers.The biosynthesis of the polysaccharide O chain has been investigated in some serotypes of K. pneumoniae by biochemical and genetic experimental approaches. Much of the data for the O3 and O5 serotypes is derived from the related equivalent systems in E. coli. However, the genetic loci required for synthesis of the corresponding O chain structures in E. coli (O8 ec and O9 ec ) and K. pneumoniae (O5 kp and O3 kp , respectively) are essentially identical and reflect lateral gene trans...

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