NMR Verification of Structures of Bacterial Saccharide Precursors for Vaccines

点击翻译按钮获取中文摘要

About 40 monosaccharide and oligosaccharide intermediates for the preparation of a hexasaccharide-protein conjugate have been analyzed by high-resolution NMR at 500 MHz. The techniques used were: one-dimensional (1D) 1H and 1D 13C NMR. Two-dimensional (2D) methods such as COSY, TOCSY, and HSQC were also used in selected cases, if additional information on spectral assignments was needed. Validation of the chemical structures was based on integration of the 1D 1H NMR spectra, and on counting of carbon resonances in the 13C spectra, especially in well-separated spectral regions, such as those of the C=O, aromatic C, C-1, C-N, and C-CH3 moieties. 1D 13C Distortionless Enhancement by Polarization Transfer (DEPT) was also used to differentiate 13CH2 resonances from those of 13CH and 13CH3. This simple spectral editing technique yields a 13CH2 count in the form of 13C resonances of inverted phase, and was particularly applicable to these intermediates because of the mixed CH, CH2, CH3 resonances of the amide and linker side-chains in these structures. NMR service spectra measured at 500 MHz have been provided for the identification of 20 polysaccharide fragments isolated from Shigella sonnei, including products of nitrous acid deamination that are being used in structural studies, and spectra that facilitate detection of core polysaccharide in the samples. The techniques used include 1D 1H and 13C NMR, but sometimes 2D COSY, TOCSY, and HSQC, too. Acquisition of the spectra was performed at an elevated sample temperature of 50 C to reduce the viscosity of the solutions, thus permitting the measurement of sharper spectra. The techniques provide complex fingerprint spectra that can be compared with literature data to verify identity. Service spectra were also measured for nine fractions isolated from Klebsiella pneumoniae type 2, including a series of three samples of the polysaccharide that had been subjected to sonication for 5, 15, or 30 minutes to test a molecular weight reduction procedure. One sample of a polysaccharide found in S. flexneri type 2a has been identified by 1D 1H and 13C NMR.