Efficacy, safety and tolerability of aliskiren, a direct renin inhibitor, in women with hypertension: a pooled analysis of eight studies.

Hypertension is a major risk factor for cardiovascular disease, which is the leading cause of mortality in women in developed countries. This pooled analysis assessed the antihypertensive efficacy, safety and tolerability of monotherapy with the direct renin inhibitor aliskiren (150 mg and 300 mg) over 8-12 weeks in women with mild-to-moderate hypertension (mean sitting diastolic blood pressure (msDBP) ≥95 and <110 mm Hg) across eight randomized and double-blind trials. Safety and tolerability were assessed in the five placebo-controlled trials in the analysis. In the 1527 women enrolled in these studies, aliskiren 150 mg and 300 mg produced significantly greater blood pressure (BP) reductions (14.1/11.0 and 16.1/12.3 mm Hg, respectively) compared with placebo (7.2/7.6 mm Hg; P<0.0001). BP reductions with aliskiren monotherapy in women were similar to those observed in men, and consistent across subgroups of age, metabolic syndrome and obesity. The overall incidence of adverse events in women was similar with aliskiren treatment (150 mg, 42.3%; 300 mg, 46.0%) and placebo (39.0%); adverse events with aliskiren were more frequent in women than in men, consistent with previous studies of gender differences in drug tolerability. In conclusion, aliskiren monotherapy at 150 mg and 300 mg doses provided effective, dose-dependent BP-lowering in women with mild-to-moderate hypertension, and it was well tolerated.

The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region.

BACKGROUND: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. RESULTS: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. CONCLUSION: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations.

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants.

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (1D(CH)) and long-range H-H residual dipolar couplings (nD(HH)) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the nD(HH) in natural abundance based on a 13C resolved 1H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the 1D(CH) and the nD(HH) residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 1-->6 glycosidic linkages.

Comparison of the conformation and dynamics of a polysaccharide and of its isolated heptasaccharide repeating unit on the basis of nuclear Overhauser effect, long-range C-C and C-H coupling constants, and NMR relaxation data.

A comparison of the conformation and dynamics of the cell wall polysaccharide of S. mitis J22 and the heptasaccharide repeating unit made from this polysaccharide was performed on the basis on nmr data. We have previously reported a model for this highly flexible polysaccharide in which four residues of the antigenic epitope adopt a defined conformation as do the two residues of the lectin-binding epitope. These domains are connected by a 6-substituted galactofuranoside residue that acts as a flexible hinge and the repeating subunits are joined by phosphodiester linkages that provide further flexibility. Homonuclear nuclear Overhauser effect (NOE) and long-range C-C and C-H scalar coupling constants measured in uniform (13)C-labeled samples of the polysaccharide and heptasaccharide were very similar, indicating a similar conformational average in solution. Significant differences in the solution dynamics were found from the heteronuclear relaxation data, T(1), T(1 rho), and NOE, which reflect the faster molecular tumbling of the heptasaccharide. Internal motions occurring on a picosecond time scale are relatively uniform along the polymer while dynamics on the time scale longer than a few nanoseconds is characteristic of hinge motion.

Conformational studies of human milk oligosaccharides using (1)H-(13)C one-bond NMR residual dipolar couplings.

1H-(13)C one-bond dipolar coupling values were measured for natural abundance samples of the human milk oligosaccharides "lacto-N-fucopentaose" (LNF-1 LNF-2, and LNF-3), "lacto-N-difucohexaose" (LND-1), "lacto-N-tetraose" (LNT), and "lacto-N-neo-tetraose" (LNnT), four of which have Lewis blood group epitopes. Each oligosaccharide was dissolved in a 7.5% solution of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelle liquid crystals oriented in the NMR magnetic field. The dipolar coupling data and NOE were fitted to conformational models with calculations of an optimum orientation tensor which best represents the dipolar coupling values for a fragment hypothesized to adopt a single conformation. In the case of LNF-1, LNF-2, LNF-3, and LND-1, the models confirm previous conformational models for the Lewis epitopes based on NOE and molecular dynamics simulations. Extensions of the model provided new structural data for the remaining residues. In all cases, upper limits for the errors in the glycosidic angles of the models were estimated. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.

The use of NMR residual dipolar couplings in aqueous dilute liquid crystalline medium for conformational studies of complex oligosaccharides.

C-H dipolar coupling values were measured for a natural-abundance sample of the pentasaccharide beta-D-Galp-(1-->3)-[alpha-L-Fucp-(1-->4)]-beta-D-GlcNAcp-(1 -->3)-beta-D- Galp-(1-->4)-beta-D-Glcp ('lacto-N-fucopentaose 2') (LNF-2), in a 7.5% solution of dimyristoyl phosphatidylcholine-dihexanoyl phosphatidylcholine bicelle liquid crystals oriented in the NMR magnetic field. Interpretation of the dipolar coupling data and NOE confirms the conformational model for the Lewis(a) trisaccharide epitope based on NOE, molecular dynamics simulations, and scalar coupling data and provided new structural information for the remaining residues of the pentasaccharide. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.

Conformation of a rigid tetrasaccharide epitope in the capsular polysaccharide of Vibrio cholerae O139.

A newly reported strain of Vibrio cholerae, known as strain O139 Bengal, is the first instance of an encapsulated strain that has caused epidemic cholera. The O-antigenic capsule is the critical antigen for protective immunity. Since mapping of the antigenic epitopes will assist in the development of a protein conjugate vaccine based on the capsular polysaccharide, we have undertaken a study of the three-dimensional conformation of the polysaccharide. It contains six residues in the repeating subunit with the unusual feature of a 4,6 cyclic phosphate on a beta-galactopyranoside. A structural epitope composed of four of the residues is somewhat similar to the Lewis(b) blood group tetrasaccharide. Polysaccharide samples enriched in (13)C have been prepared by growth of the bacteria in (13)C-enriched medium. Multidimensional heteronuclear NMR and molecular modeling studies are reported, which show that the O139 tetrasaccharide adopts a compact and tightly folded conformation that is relatively rigid and similar to the Le(b) conformation. The cyclic phosphate on the beta-galactopyranoside residue is in contact with the colitose residue linked to the beta-GlcNAc.

Structure and conformation of complex carbohydrates of glycoproteins, glycolipids, and bacterial polysaccharides.

For nuclear magnetic resonance determinations of the conformation of oligosaccharides in solution, simple molecular mechanics calculations and nuclear Overhauser enhancement measurements are adequate for small oligosaccharides that adopt single, relatively rigid conformations. Polysaccharides and larger or more flexible oligosaccharides generally require additional types of data, such as scalar and dipolar coupling constants, which are most conveniently measured in 13C-enriched samples. Nuclear magnetic resonance relaxation data provide information on the dynamics of oligosaccharides, which involves several different types of internal motion. Oligosaccharides complexed with lectins and antibodies have been successfully studied both by X-ray crystallography and by nuclear magnetic resonance spectroscopy. The complexes have been shown to be stabilized by a combination of polar hydrogen bonding interactions and van der Waals attractions. Although theoretical calculations of the conformation and stability of free oligosaccharides and of complexes with proteins can be carried out by molecular mechanics methods, the role of solvent water for these highly polar molecules continues to present computational problems.

New strategy for the conformational analysis of carbohydrates based on NOE and 13C NMR coupling constants. Application to the flexible polysaccharide of Streptococcus mitis J22.

For complex oligosaccharides, which are relatively rigid with modest excursions from a single minimum energy conformation, it is straightforward to build conformational models from NOE data. Other oligosaccharides are more flexible with transitions between distinct minima separated by substantial energy barriers. We show that modeling based on scalar coupling data is superior to NOE-based modeling for the latter case. Long range 13C-13C and 13C-1H coupling constants measured for the heptasaccharide repeating subunit of the cell wall polysaccharide from Streptococcus mitis J22 are correlated with individual glycosidic dihedral angles, effectively uncoupling the degrees of freedom of the oligosaccharide and allowing a search for combinations of dihedral angles which are energetically reasonable, i.e., with no bad van der Waals contacts, and which can be combined to satisfy all the measured J values. Allowed values of the individual angles can then be combined to search for overall oligosaccharide conformations which contribute to the ensemble. We show that while the polysaccharide from S. mitis J22 is flexible, requiring multiple conformations, most of the flexibility is localized to a few bonds and only a rather small number of conformations is required to reproduce the experimental NOE and scalar coupling data.

Structure determination of the capsular polysaccharide from Vibrio vulnificus strain 6353.

Vibrio vulnificus is a pathogenic gram-negative bacterium, endemic to brackish waters, which is often isolated from sediments, from the water column or from shellfish. It is associated with wound infections and septicemia in humans and the virulence of V. vulnificus has been strongly associated with encapsulation. The capsular polysaccharide purified from a virulent strain of V. vulnificus 6353 did not show cross reactivity with antibodies to the capsular polysaccharide of a related pathogenic strain of V. vulnificus (MO6-24) the structure of which was recently reported. NMR spectroscopic analysis of the purified polysaccharide from strain 6353 showed that the polymer is composed of four sugar residues per repeating subunit including 2,6-dideoxy-2-N-acetylamino-alpha-D-glucose (QuiNAc), 2-deoxy-2-N-acetylamino-alpha-D-galactose (alpha-D-GalNAc), 2-deoxy-2-N-acetylamino-alpha-D-galcturonic acid (alpha-D-GalNAcA) and 2-N-acetylamino-alpha-D-glucuronamide (alpha-D-GlcNAcANH2). The 1H- and 13C-NMR spectra were completely assigned by homonuclear and heteronuclear NMR spectroscopy. Sugar types and anomeric configurations were determined from proton homonuclear coupling constants and glycosidic linkages were determined from 1H-13C heteronuclear multiple bond correlation spectra. Sugar identities were confirmed by high performance anion-exchange chromatography and absolute configurations were determined by gas chromatography in combination with molecular modeling and NMR spectroscopy. The structure of the polysaccharide repeating unit is: [-->4)-alpha-D-GalpNAc-(1-->3)-alpha-D-GalpNAcA-(1-->3)-alpha-D-++ +QuipNAc-(1-->]n alpha-D-GlcpNAcANH2 (1-->4)- -->. While there are some common features shared among the structures of the capsular polysaccharides of pathogenic strains of V. vulnificus, there are distinct differences in the detailed structures.

Structure of a muramic acid containing capsular polysaccharide from the pathogenic strain of Vibrio vulnificus ATCC 27562.

Vibrio vulnificus strains isolated from septicemia cases and from the environment show a wide variety of capsular types. In an attempt to find common structural features which can be correlated with pathogenicity and toxicity, we have determined structures of the capsular polysaccharides (CPS) from several pathogenic strains. We report the complete structure of the polysaccharide from the pathogenic V. vulnificus strain ATCC 27562 using a combination of homonuclear and heteronuclear one-dimensional and two dimensional NMR experiments. The 13C and 1H NMR spectra, including the exchangeable amide proton resonances, have been completely assigned. The amide linkage between Ser and C6 of GalA has been unambiguously determined by water-suppressed 2D NOESY. To verify the structure established by NMR, we have fragmented the polymer employing the Smith degradation procedure. The Smith product identified by NMR and matrix-assisted laser desorption mass spectrometry is consistent with the proposed structure for the CPS, which is composed of D-GlcNAc, MurNAc, D-GalA, L-Rha and is serine-linked as shown: [formula: see text]

Measurement of long-range carbon-carbon coupling constants in a uniformly enriched complex polysaccharide.

A quantitative coherence transfer scheme for 1H-detected measurement of long-range carbon-carbon coupling constants in NMR spectra of complex carbohydrates is described. It is applied to a uniformly highly 13C-enriched monosaccharide and to a complex cell wall polysaccharide from Streptococcus mitis J22 having seven distinct sugars in the repeating subunit. Coupling values within the ring were compared to published values for monosaccharides to demonstrate the validity of the method. An attempt was made to relate coupling constants between carbon atoms across the glycosidic linkage to the dihedral angles of a recently published flexible model for the polysaccharide which is based on 3JCH data. The experimental coupling constants do not agree with any single conformation demonstrating that the repeating subunit of the polysaccharide must be flexible. This conclusion is in accord with results of molecular modeling nuclear Overhauser effect and 3JCH data.

Hemorrhagic ocular complications associated with the use of systemic thrombolytic agents.

OBJECTIVE: This study aimed to report three patients with hemorrhagic ocular and orbital complications associated with the use of systemic thrombolytic agents. DESIGN: The study design was a retrospective small case series. PARTICIPANTS: Three eyes of three patients were studied. INTERVENTION: Surgical procedures to reduce intraocular pressure or relieve optic nerve compression were performed. MAIN OUTCOME MEASURES: Visual acuity and intraocular pressure were measured. RESULTS: Three patients received an intravenous thrombolytic agent on diagnosis of an acute myocardial infarction. One patient had a spontaneous suprachoroidal hemorrhage develop with secondary acute angle closure glaucoma shortly after receiving tissue plasminogen activator. Another patient had an orbital hemorrhage develop on receiving tissue plasminogen activator 4 days after an uncomplicated cataract extraction. The third patient experienced an orbital hemorrhage while receiving streptokinase 1 day after undergoing an external levator resection. Two patients suffered significant visual loss due to glaucoma or compressive optic neuropathy. CONCLUSIONS: The onset of eye pain or visual loss after the administration of a systemic thrombolytic agent should alert the physician to the possibility of an ocular or adnexal hemorrhage. Prompt diagnosis and treatment can improve the likelihood of a favorable visual outcome.

Structural and antigenic types of cell wall polysaccharides from viridans group streptococci with receptors for oral actinomyces and streptococcal lectins.

Lectin-mediated interactions between oral viridans group streptococci and actinomyces may play an important role in microbial colonization of the tooth surface. The presence of two host-like motifs, either GalNAc beta1-->3Gal (Gn) or Gal beta1-->3GalNAc (G), in the cell wall polysaccharides of five streptococcal strains accounts for the lactose-sensitive coaggregations of these bacteria with Actinomyces naeslundii. Three streptococcal strains which have Gn-containing polysaccharides also participate in GalNAc-sensitive coaggregations with strains of Streptococcus gordonii and S. sanguis. Each Gn- or G-containing polysaccharide is composed of a distinct phosphodiester-linked hexa- or heptasaccharide repeating unit. The occurrence of these polysaccharides on 19 additional viridans group streptococcal strains that participate in lactose-sensitive coaggregations with actinomyces was examined. Negatively charged polysaccharides that reacted with Bauhinia purpurea agglutinin, a Gal and GalNAc binding plant lectin, were isolated from 17 strains by anion exchange column chromatography of mutanolysin-cell wall digests. Results from nuclear magnetic resonance and immunodiffusion identified each of 16 polysaccharides as a known Gn- or G-containing structural type and one polysaccharide as a new but closely related Gn-containing type. Unlike the reactions of lectins, the cross-reactions of most rabbit antisera with these polysaccharides were correlated with structural features other than the host-like motifs. Gn-containing polysaccharides occurred primarily on the strains of S. sanguis and S. oralis while G-containing polysaccharides were more common among the strains of S. gordonii and S. mitis examined. The findings strongly support the hypothesis that lectin-mediated recognition of these streptococci by other oral bacteria depends on a family of antigenically diverse Gn- and G-containing cell wall polysaccharides, the occurrence of which may differ between streptococcal species.

Classification of Vibrio vulnificus strains by the carbohydrate composition of their capsular polysaccharides.

Pathogenic bacteria are often classified on the basis of the complex polysaccharides found on the surface, usually capsular polysaccharides or lipopolysaccharides. It is common in clinical practice to use reactivity with antisera specific to the various cell surface carbohydrates for this purpose. In this work, we describe a chemotyping method for bacterial capsular polysaccharides which is based on a carbohydrate analysis of an acid hydrolysate of the capsule. High-performance anion-exchange chromatography at high pH (HPAE) with electrochemical detection, which is used for analysis of the hydrolysate, shows preferential sensitivity for sugars. A single acid hydrolysis condition is chosen for screening a large collection of bacterial isolates and a computerized autosampler is used to make possible a large number of rapid analyses. This procedure does not yield a quantitative carbohydrate analysis for the sample but produces a fingerprint which can be used to discriminate among isolates which have different capsular polysaccharide structures. The procedure has been applied to a collection of 120 isolates of Vibrio vulnificus, a water-born species common in shellfish which causes septicemia in immunocompromised individuals, most often from eating of raw oysters. The collection of bacterial isolates includes strains from both clinical cases of septicemia and from such environmental sources such as sea water, sediments, and shellfish. Our results show that a number of unusual sugars including many amino sugars are found in these polysaccharides and that a wide variety of capsular carbotypes in V. vulnificus may be readily distinguished by the HPAE fingerprint.

Dynamics of uniformly 13C-enriched cell wall polysaccharide of Streptococcus mitis J22 studied by 13C relaxation rates.

We have studied the dynamics of the motion of a complex polysaccharide having seven sugar residues in the repeating subunit and which is a receptor for lectin interaction in the coaggregation of oral bacteria. Measurements of the longitudinal and the rotating frame relaxation rates and the heteronuclear nuclear Overhauser effects were carried out on a uniformly 13C-enriched sample using pulse sequences chosen to minimize the effects 13C-13C coupling and cross relaxation. T1 and T1 rho measurements both showed single exponential decay for the anomeric carbon atom resonances of the polysaccharide. The results show the polymer to be highly flexible with a hinge at the (1-->6)-linked galactofuranoside residue. Since there is no generally accepted scheme for interpreting polysaccharide dynamics, several different methods of data analysis were used including a reduced spectral density function method as well as several different methods in which a series of isotropically decaying rotational correlation functions are assumed. The different analyses all show that there are differing amounts of internal motion in the different residues of the polysaccharide. One possible interpretation of the data, which uses an extended version of the model-free treatment, indicates that picosecond motion is exhibited to a similar degree by all the residues in addition to a slower motion on the nanosecond time scale whose amplitude is greatest in the hinge region around the (1-->6)-linked galactofuranoside residue in the polysaccharide.

Molecular modeling of the flexible cell wall polysaccharide of Streptococcus mitis J22 on the basis of heteronuclear NMR coupling constants.

A method for constructing conformational models of flexible complex polysaccharides on the basis of NMR data and molecular modeling is described and is applied to a polysaccharide which is a lectin-binding receptor important in coaggregation of oral bacteria. The method involves uniform biosynthetic enrichment of the polysaccharide with 13C which allows accurate measurements of heteronuclear coupling constants from a three-dimensional coupled HMQC-NOESY spectrum. The improved resolution of the 3D spectrum also provides a large number of accurate values of NOE cross peak volumes in a decoupled HMQC-NOESY spectrum. While it was not possible to construct a model for the flexible polysaccharide directly from the NOE data, a model was successfully built from the coupling constant data. Possible values of glycosidic dihedral angles were extracted from the 3JCH data to build models which were evaluated by molecular modeling calculations. A simple average over a linear combination of low-energy conformations was selected which matched the experimental 3JCH data within experimental error. Simulation of the NOE data for this same combination of conformers gave excellent agreement with experimental NOESY data. Molecular dynamics trajectories both with and without coupling constant constraints do not represent the experimental NOE and 3JCH data as well as the linear combination model. While the polysaccharide has some flexibility in the antigenic site, the lectin-binding site, which contains a furanoside with (1-->6)-linkages, provides a more flexible hinge in the polysaccharide.

Comparison of NMR and molecular modeling results for a rigid and a flexible oligosaccharide.

Three-bond heteronuclear coupling constants (3JCH) are extremely useful in describing flexible models for oligosaccharides. We show that antiphase methods for measuring 3JCH in oligosaccharides have limited reliability but that the coupling constants can be reliably measured in natural abundance by quantitative J-correlation methods. Interpretation of 3JCH data for a pentasaccharide (lacto-N-fuco-pentaose 2) from human milk are consistent with a rigid model for the Lewis(a) trisaccharide epitope but for an antigenic tetrasaccharide fragment from the cell wall polysaccharide of viridans streptococci, 3JCH data imply a considerably more flexible model. Nuclear Overhauser effect (NOE) data are reported for a heptasaccharide repeating unit isolated from the cell wall polysaccharide of Streptococcus gordonii 38. The results for a tetrasaccharide fragment are similar to data reported for the same fragment in the cell wall polysaccharide from S.mitis J22. This result implies a similar conformation for the tetrasaccharide fragment in the polysaccharide and in the heptasaccharide and also implies that anisotropy of motion is not significant in the interpretation of the nuclear Overhauser effects in the polysaccharide. Interpretation of the NOE results for the tetrasaccharide fragment, like the 3JCH data, implies a flexible model with three conformations in fast exchange. The results of the two experimental techniques are combined with molecular modeling results including molecular dynamics simulation to provide a clear delineation between flexible and rigid oligosaccharide epitopes. The blood group Lewis(a) trisaccharide antigenic determinant is highly restricted in its motions by steric interactions while the antigenic tetrasaccharide fragment of the S.gordonii 38 heptasaccharide is considerably more mobile. We propose that some branched oligosaccharides are relatively rigid and some are flexible depending on subtle details of the linkages.

Antibodies that react with the capsular polysaccharide of Vibrio vulnificus are detectable in infected patients, and in persons without known exposure to the organism.

In serious infections with Vibrio vulnificus, IgG antibodies to the capsular polysaccharide of the infecting strain were demonstrable in patient serum. It was not possible to show that persons with probable increased exposure to V. vulnificus (shellfish industry workers) had increased levels of antibodies to any one of three capsular types tested when compared with persons who would be expected to have had minimal exposure to the organism (Seventh Day Adventists). Antibodies that reacted with the capsular polysaccharides were demonstrable in persons without a history of V. vulnificus infection, suggesting that cross-reacting antibodies are present in the general population.

A flexible model for the cell wall polysaccharide of Streptococcus mitis J22 determined by three-dimensional 13C edited nuclear overhauser effect spectroscopy and 13C-1H long-range coupling constants combined with molecular modeling.

We report on the conformation of a tetrasaccharide fragment in the repeating subunit of the cell wall polysaccharide of Streptococcus mitis J22, a receptor for the lectin of Actinomyces viscosus T14V in a bacterial coaggregation that is important in the ecological interactions of oral bacteria. Although there is considerable overlap of the 1H-nmr signals, some cross peaks can be extracted from conventional two-dimensional nuclear Overhauser effect spectroscopy (NOESY) data on the polysaccharide. These data cannot be fit to a single conformation of the tetrasaccharide fragment. Therefore we have prepared a polysaccharide sample fully enriched in 13C from which we have determined accurate NOESY cross-peak volumes in a three-dimensional heteronuclear-resolved spectrum that allows accurate determination of many more NOESY cross peaks than does conventional two-dimensional spectroscopy. We have also used the 13C enriched polysaccharide to measure accurate values of long-range 13C-1H coupling constants that can be correlated with glycosidic dihedral angles. Molecular modeling calculations on the polysaccharide fragment, including molecular dynamics simulations, identify multiple low-energy conformations. This result is to be contrasted with previous calculations on blood group oligosaccharides in our laboratory using similar methods that showed relatively rigid conformations with little flexibility of the glycosidic linkages. The present NOESY and 3JCH data can be reconciled with a model for the antigenic tetrasaccharide in which three distinct conformations are in fast exchange. We propose that some carbohydrate epitopes such as those of the blood group oligosaccharides are relatively rigid while others such as the tetrasaccharide fragment in these studies exhibit much greater flexibility.