NMR Verification of Structures of Bacterial Saccharide Precursors for Vaccines

年内，另有17个霍乱弧菌抗原的单糖和低聚糖中间体分别在500兆赫和126兆赫的高分辨率一维(1D)H-1和一维C-13核磁共振(1D C-13)进行分析，累积得到80个中间体。化学结构的验证是基于1DH-1核磁共振谱的积分，以及13C谱中碳共振的计数，特别是在C=O、芳香C、C-1、C-N和C-CH3部分的谱区。核磁共振谱也被用来评估合成制剂的纯度，例如，通过测量芳香基团的含量来确定苯基保护基氢解脱除的完成程度。 测定了宋内氏志贺氏菌12个天然多糖组分在500 MHz的H-1核磁共振波谱。比较多糖H-1和鼠李糖甲基信号的积分有助于样品中核心多糖的检测和定量。这些技术提供了复杂的指纹图谱，可以与文献数据进行比较来验证身份。用核磁共振氢谱对大肠杆菌O148 O专一性多糖的结构进行了表征。 当我们来发表我们对福氏志贺氏菌2a和6等的免疫化学研究时，一位稿件的推荐人担心，在接合反应的条件下，多糖上的乙酰基可能会与我们的接合辅助剂O-(3-硫丙基)羟胺反应。因此，我们进行了一项研究，在此条件下，用等摩尔比例的羟胺衍生物处理福氏志贺氏菌6的O-SP。用H-1核磁共振波谱在22h的不同时间监测了D2O中的溶液，没有观察到多糖的OAC和NAC共振的相对强度或光谱的任何其他部分的变化，这表明存在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.