The core oligosaccharide component from Mannheimia (Pasteurella) haemolytica serotype Al lipopolysaccharide contains L-glycero-D-manno- and D-glycero-D-manno-heptoses: Analysis of the structure and conformation by high-resolution NMR spectroscopy

Abstract

Previous studies from our laboratory have indicated that the lipopolysaccharide (LPS) from Mannheimia haemolytica serotype A1 contains both L-glycero-D-manno-heptose and D-glycero-D-manno-heptose residues. NMR methods making use of 1D 1H selective excitation and 2D (1H, 13C) and (1H, 31P) heteronuclear experiments were used for the structural determination of the major core oligosaccharide components of the deacylated low-molecular-mass LPS obtained following sequential treatment with anhydrous hydrazine and aq KOH. The core oligosaccharide region was found to be composed of a branched octasaccharide linked to the deacylated lipid A moiety via a 3-deoxy-4-phospho-D-manno-oct-2-ulosonate residue having the structure,[Formula: see text]Heterogeneity was found to be present at several linkages. NMR methods were devised to distinguish between the diastereomeric forms of the heptose residues. Synthesized monosaccharides of L-D- and D-D-heptose were used as model compounds for analysis of the 1H and 13C NMR chemical shifts and proton coupling constants. Molecular modeling using a Monte Carlo method for conformational analysis of saccharides was used to determine the conformation of the inner core of the oligosaccharide and to establish the stereochemical relationships between the heptoses.Key words: LPS, NMR, conformation, oligosaccharide, heptose.

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

“…The sample volume was ϳ150 l. The measured pH in D 2 O (pD) uncorrected for D 2 O was 3. Standard two-dimensional homonuclear COSY, TOCSY, and NOESY spectra and selective 1D-1 H TOCSY and NOESY spectra were acquired for the assignment of proton resonances within the glycan and were performed as described previously (19,20). Proton assignments were then used to identify cross-peaks in heteronuclear 1 H- 13 C heteronuclear single quantum coherence and HMBC spectra required for complete structure determination.…”

Targeted Metabolomics Analysis of Campylobacter coli VC167 Reveals Legionaminic Acid Derivatives as Novel Flagellar Glycans

“…One-dimensional TOCSY experiments were then used to assign chemical shifts and to measure coupling constants. These experiments are particularly useful for assigning overlapping resonances originating from sugar ring protons and for resolving mixtures of compounds (25)(26)(27). One-dimensional TOCSY of H3ax (Fig.…”

Molecular Modeling Study of the Carbohydrate Region of the Endotoxin from Burkholderia cenocepacia ET‐12

“…The only exceptions were the C5-C6 bond of l-glycero-d-manno heptose (l,d-Hep) residue, which was always considered to be in a gg conformation, and the C6-C7 bond of 3-deoxyd-manno-oct-2-ulopyranosonic acid (d-Kdo), and d-glycero-d-talooct-2-ulopyranosonic acid (d-Ko), which were instead considered to be in the tg conformation, as reported. [8,9] Each disaccharide was energetically minimized using a dielectric constant of 78.5.…”

“…The only exceptions were for the C5-C6 bond of the lglycero-α-d-manno-heptose (α-l,d-Hep) residue, which was always considered to be in a gg conformation, and for the C6-C7 bond of Kdo and Ko, which were instead considered to be in the tg conformation, as previously reported. [8,9] Thus, considering all the possible values that can be assumed by each couple of…”