NMR Verification of Structures of Bacterial Saccharide Precursors for Vaccines

在这一年中，又用高分辨率一维（1D）H-1和1D C-13 NMR分别在500 MHz和126 MHz分析了17种霍乱弧菌抗原的单糖和寡糖中间体，用于制备六肽-蛋白质缀合物，累积总数为80。化学结构的验证是基于1D H-1 NMR光谱的积分，以及13 C光谱中碳共振的计数，特别是在C=O、芳香族C、C-1、C-N和C-CH 3部分的良好分离的光谱区域中。NMR谱也用于评估合成制备物的纯度，例如，通过测量芳族基团的含量来确定苄基保护基团的氢解除去的完成程度。 测定了12个宋内志贺菌天然多糖组分的500 MHz ~ 1H NMR谱。比较多糖H-1和鼠李糖甲基信号的积分有助于检测和定量样品中的核心多糖。该技术提供了复杂的指纹图谱，可以与文献数据进行比较，以验证身份。对E. coli O 148 O-特异性多糖的结构用H-1 NMR进行了表征。 当我们发表我们对福氏志贺菌2a和6等的免疫化学研究工作时，稿件的一位审稿人担心多糖上的乙酰基可能会在缀合反应的条件下与我们的缀合辅助试剂O-（3-硫代丙基）羟胺反应。因此，我们进行了一项研究，其中S。在这些条件下用等摩尔比例的羟胺衍生物处理Flexneri 6。在22小时内的不同时间通过H-1 NMR光谱监测D2 O中的溶液。未观察到多糖的0Ac和NAc共振的相对强度或光谱的任何其他部分的变化，这将表明正在进行的化学反应。

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.