NMR Verification of Structures of Bacterial Saccharide Precursors for Vaccines

疫苗用细菌糖前体结构的核磁共振验证

• 批准号: 8149329
• 负责人: Rachel Schneerson
• 金额: $ 0.7万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149329
• 关键词: Structure; Vaccines

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.

本年度，分别在500 MHz和126 MHz高分辨率一维H-1和一维C-13核磁共振分析了霍乱弧菌抗原的17个单糖和寡糖中间体，用于制备六糖-蛋白偶联物，累计总数为80个。化学结构的验证是基于一维H-1核磁共振谱的整合，以及13C光谱中的碳共振计数，特别是在C=O，芳香C， C-1， C- n和C- ch3部分的分离良好的光谱区域。核磁共振光谱还用于评价合成产物的纯度，例如，通过测定芳香族的含量来确定苯保护基团的氢解去除完成程度。