During the year, a further 17 monosaccharide and oligosaccharide intermediates of Vibrio cholera antigens, for the preparation of a hexasaccharide-protein conjugate have been analyzed by high-resolution one-dimensional (1D) H-1 and 1D C-13 NMR at 500 MHz and 126 MHz, respectively, making a cumulative total of 80. Validation of the chemical structures was based on integration of the 1D H-1 NMR spectra, and on counting of carbon resonances in the 13C spectra, especially in the well-separated spectral regions of the C=O, aromatic C, C-1, C-N, and C-CH3 moieties. The NMR spectra were also used to assess the purity of the synthetic preparations, for example, determination of the extent of completion of hydrogenolytic removal of benzyl protecting groups by measurement of the content of aromatic groups. H-1 NMR spectra at 500 MHz have been measured for the characterization of 12 native polysaccharide fractions isolated from Shigella sonnei. Comparison of the integrals of the polysaccharide H-1 and rhamnose methyl signals facilitated detection and quantitation of core polysaccharide in the samples. The techniques provide complex fingerprint spectra that can be compared with literature data to verify identity. A sample of the E. coli O148 O-specific polysaccharide has been characterized by H-1 NMR. When we came to publish our work on immunochemical studies of Shigella flexneri 2a and 6, etc, a referee of the manuscript was concerned that acetyl groups on the polysaccharide might react with our conjugation-assisting reagent O-(3-thiopropyl)hydroxylamine, under the conditions of the conjugation reaction. We therefore conducted a study in which the O-SP of S. flexneri 6 was treated with an equimolar proportion of the hydroxylamine derivative under these conditions. The solution in D2O was monitored by H-1 NMR spectroscopy at various times over a period of 22 h. No changes in the relative intensities of the OAc and NAc resonances of the polysaccharide or in any other parts of the spectra were observed that would indicate an on-going chemical reaction.
| Kubler-Kielb, Joanna; Vinogradov, Evgeny; Mocca, Christopher et al. (2010) Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates. Carbohydr Res 345:1600-8 |
