NMR Verification of Structures of Bacterial Saccharide Precursors for Vaccines

霍乱弧菌抗原的另外20种单糖和寡糖中间体，用于制备六肽-蛋白质缀合物，已分别在500 MHz和126 MHz下通过高分辨率一维（1D）1H和1D 13 C NMR进行了分析。化学结构的验证是基于1D 1H 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。 在500 MHz下测量的NMR光谱已被提供用于鉴定从福氏志贺菌2a型的脂多糖分离的6个多糖片段，和从该细菌的突变体版本制备的7个多糖级分。比较多糖H-1和鼠李糖甲基信号的积分有助于检测样品中的核心多糖。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。 用1D ~ 1H和~（13）C NMR研究了A型流感嗜血杆菌荚膜多糖的一种新制剂，发现其纯度高，与本实验室以前制剂的谱图非常一致。几年前，通过二维NMR方法分析了这些光谱，得到了多糖的D-葡萄糖残基的1H-1H和C-1，H-1偶合常数以及D-葡萄糖和D-核糖醇组分的13 C-31 P偶合常数。还获得了β-D-吡喃葡萄糖基和D-核糖醇残基的1H和13 C NMR信号的完整归属，这些残基通过D-葡萄糖的O-4和D-核糖醇的O-5之间的磷酸二酯键连接。

A further 20 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) 1H and 1D 13C NMR at 500 MHz and 126 MHz, respectively. 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 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. NMR spectra measured at 500 MHz have been provided for the identification of six polysaccharide fragments isolated from the lipopolysaccharide of Shigella flexneri type 2a, and seven polysaccharide fractions prepared from a mutant version of this bacterium. Comparison of the integrals of the polysaccharide H-1 and rhamnose methyl signals facilitated detection of core polysaccharide in the samples. The techniques provide complex fingerprint spectra that can be compared with literature data to verify identity. A new preparation of the capsular polysaccharide of Haemophilus influenzae type a has been investigated by 1D 1H and 13C NMR and found to have excellent purity, and close agreement with the spectra of previous preparations in this laboratory. These spectra had been analyzed several years ago by two-dimensional NMR methods to give sets of 1H-1H and C-1,H-1 coupling constants for the D-glucose residue and 13C-31P coupling constants for the D-glucose and D-ribitol components of the polysaccharide. Also obtained, were complete assignments of the 1H and 13C NMR signals of the beta-D-glucopyranosyl and D-ribitol residues, which are joined by a phosphoric diester linkage between O-4 of the D-glucose and O-5 of the D-ribitol.