NMR Analysis of Synthetic Oligosaccharide Fragments of the OSP of E. coli O-148

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Abstract

The NMR techniques that have been employed include one-dimensional (1D) 1H, 1D 13C, two-dimensional (2D) 1H-1H correlation spectroscopy (COSY), 2D 1H-1H total correlation spectroscopy (TOCSY), 2D 13C-1H heteronuclear single quantum correlation spectroscopy (HSQC), 2D 13C-1H heteronuclear multiple bond correlation (HMBC), 2D 1H-coupled 13C-1H HSQC, and 1D 1H-coupled 13C NMR. The COSY technique gives information about 1H spectral assignments, the TOCSY procedure provides separate 1H sub-spectra for individual sugar residues, the HSQC method provides 13C spectral assignments by correlating the 1H and 13C chemical shifts, and the HMBC pulse sequence delineates the positions of substituents by correlating their 1H or 13C chemical shifts with those of nuclei in the sugar rings. The 1H-coupled 1D 13C and 1H-coupled 2D HSQC techniques are used to measure 13C-1H coupling constants of the oligosaccharides. The latter 2D method is particularly useful when the 1H-coupled, 1D 13C NMR spectra are insufficiently dispersed for a facile analysis. 1JC-1,H-1 values were measured, typically giving information on the configuration (alpha or beta) of all the anomeric linkages in the oligosaccharide. Confirmatory information is often available from the 3JH-1,H-2 values measured from the 1H NMR spectra, with the exception of mannopyranose and rhamnopyranose, for which the values of the alpha and beta anomers are similar.[unreadable] A problem has been encountered with broadness of the 1H spectra of the 8-mer and 12-mer in D2O solutions, which could inhibit the analysis and assignment of the spectra. Thinking that this broadness might be due to viscosity of the solutions of the 8-mer and 12-mer, we acquired 1H and 13C spectra of the 12-mer at elevated temperatures, 50 C and 60 C, but this did not help. We are investigating the possibility that the unexpected broadness of the spectra is due to paramagnetic metal ion contaminants in the 8-mer and 12-mer. Carbohydrates are often excellent metal-complexing agents, and the occasional spectral broadening observed in the presence of scavenged paramagnetic metal ions is a well-known phenomenon. We have therefore treated the 8-mer with Chelex 100 cation exchange resin (sodium form) in an effort to remove any polyvalent, paramagnetic ions. Following this treatment, the 8-mer has shown improved 1H spectral resolution.