NMR characterization of uniformly 13C- and/or 15N-labeled, unsulfated chondroitins with high molecular weights

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric ¹H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with ¹³C and ¹⁵N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly ¹³C- and/or ¹⁵N-labeled chondroitin polysaccharides –4)-β-D-glucuronopyranosyl-(1–3)-2-acetamido-2-deoxy-β-D-galactopyranosyl-(1– with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for ¹³C and ¹⁵N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. ¹H and ¹³C NMR signals were assigned mainly based on HSQC and ¹³C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-β-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the β-D-glucuronic acid residue by the observation of ¹³C peak splitting due to ¹J_CN coupling in ¹³C- and ¹⁵N-labeled chondroitin. The T₂* and T₁ were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.