NMR Verification of Structures of Bacterial Saccharide Precursors for Vaccines

用500 MHz的高分辨核磁共振分析了制备六糖-蛋白质偶合物所需的约40个单糖和低聚糖中间体。所使用的技术是：一维(1D)1H和1D 13C核磁共振。如果需要有关光谱分配的额外信息，也可以在选定的情况下使用COSY、TOCSY和HSQC等二维(2D)方法。化学结构的验证是基于1D 1H核磁共振谱的积分，以及13C谱中碳共振的计数，特别是在分离良好的谱区，如C=O、芳香C、C-1、C-N和C-CH3部分。一维13C极化转移无畸变增强(DEPT)也被用来区分13CH2和13CH3的共振。这种简单的光谱编辑技术以反相的13C共振的形式产生13CH2计数，并且特别适用于这些中间体，因为这些结构中的酰胺和连接体侧链的混合CH、CH2、CH3共振。 提供了在500 MHz下测量的核磁共振服务谱，用于鉴定从宋内氏志贺氏菌中分离出的20个多糖片段，包括用于结构研究的亚硝酸脱氨产物，以及有助于检测样品中核心多糖的光谱。使用的技术包括1D、1H和13C核磁共振，但有时也包括2D COSY、TOCSY和HSQC。光谱的采集是在50摄氏度的高温下进行的，以降低溶液的粘度，从而允许测量更清晰的光谱。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。 还测量了从2型肺炎克雷伯氏菌分离出的九个组分的服务光谱，其中包括一系列三个样品的多糖，这些样品经过5、15或30分钟的超声波处理以测试分子量降低过程。 从2a型福氏志贺氏菌中分离得到一种多糖，经1D、1H和13C核磁共振确证。

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.