The last mannose residue was present at 4.889 ppm and was representative of a 6-substituted mannose, given the downfield shift value of its C-6 resonance.

At higher selleck kinase inhibitor fields (4.52 ppm) another anomeric proton signal was present, which was attributable to the galactopyranose residue present in its β-anomeric configuration (3 J H-1, H-2 = 8.1 Hz). Analysis of the TOCSY spectrum made it possible to determine the H-1 to H-4 resonances. In contrast, the H-5 resonance, as in all galacto-configured systems, was only visible by NOESY owing to its low coupling constant with H-4, which impaired any transfer of magnetization. The chemical shifts of carbon signals of this latter spin system were taken from HSQC, and indicated there was no glycosylation shift, suggesting the presence of an unsubstituted β-galactopyranose residue. On the basis of the above chemical and NMR data, and in accordance with reported data [48], it was likely that the EPS was an α-(1→6)-linked,

highly branched, comb-like mannopyranan polysaccharide structure with mannopyranose units branched at C-2 with 2-substituted mannose residues. In order to confirm this structural hypothesis, we carried out an enzymatic hydrolysis on 10 mg of the EPS using an exo-mannosidase that is able to cleave the branching mannose residues selleck compound starting from the non-reducing ends. As expected, after purification by gel filtration chromatography, two products were identified. enough The lower molecular size fraction was mannose (6 mg). The polysaccharide fraction that eluted in the void LY2228820 concentration volume (3 mg) was analysed by NMR spectroscopy, and although still present as part of a heterogeneous polymer, this fraction consisted of only one major residue. The comparison of proton anomeric signal intensities between the polymer and the mannosidase-degraded product showed a remarkable increase in the signal at δ4.89 (6-substituted

mannose) with respect to all the other signals (Figure 4). However, it was not possible to observe the galactose signal in this polymer, likely because the amount of galactose in the entire EPS was very low and in the presence of the predominant mannose, disappeared due to background noise. The methylation analysis was in good agreement with this observation, and showed a substantially higher content of 6-substitued mannose. Following the exo-mannosidase hydrolyses of the terminal mannose units, it was confirmed that 6-substituted mannose was a constituent of the mannan backbone and that 2- substituted and 3-substituted mannose were present in the oligosaccharide arms. Figure 4 HSQC and the 1 H-NMR spectra of the mannosidase-digested polymer that demonstrates the presence of a single abundant peak at 4.89 ppm, which represents the anomeric proton signal of the 6-substituted mannose. After establishing the nature of the backbone, an acetolysis reaction was used to determine the identity and length of the branches.