NMR Analysis of Synthetic Oligosaccharide Fragments of the OSP of E. coli O-148

大肠杆菌 O-148 OSP 合成寡糖片段的 NMR 分析

• 批准号: 7734847
• 负责人: Rachel Schneerson
• 金额: $ 6.78万
• 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/7734847
• 关键词: Carbohydrates; Carrier Proteins; Cation Exchange Resins; Cell Nucleus; Chemicals; Coupling; Escherichia coli; Galactose; Glucose; Glycosides; Individual; Ions; Mannose; Measures; Metals; Methods; O Antigens; Object Attachment; Oligosaccharides; Physiologic pulse; Positioning Attribute; Preparation; Procedures; Proteins; Pulse taking; Resolution; Shigella dysenteriae; Sodium; Solutions; Spectrum Analysis; Techniques; Temperature; Thinking; Vaccines; Viscosity; chelex; improved; metal complex; outer surface lipoprotein; quantum; sugar; two-dimensional

The NMR techniques that have been employed include one-dimensional (1D) 1H, 1D 13C, two-dimensional (2D) 1H-1H correlation spectroscopy (COSY), 2D 1H-1H total correlation spectroscopy (TOCSY), 2D 13C-1H heteronuclear single quantum correlation spectroscopy (HSQC), 2D 13C-1H heteronuclear multiple bond correlation (HMBC), 2D 1H-coupled 13C-1H HSQC, and 1D 1H-coupled 13C NMR. The COSY technique gives information about 1H spectral assignments, the TOCSY procedure provides separate 1H sub-spectra for individual sugar residues, the HSQC method provides 13C spectral assignments by correlating the 1H and 13C chemical shifts, and the HMBC pulse sequence delineates the positions of substituents by correlating their 1H or 13C chemical shifts with those of nuclei in the sugar rings. The 1H-coupled 1D 13C and 1H-coupled 2D HSQC techniques are used to measure 13C-1H coupling constants of the oligosaccharides. The latter 2D method is particularly useful when the 1H-coupled, 1D 13C NMR spectra are insufficiently dispersed for a facile analysis. 1JC-1,H-1 values were measured, typically giving information on the configuration (alpha or beta) of all the anomeric linkages in the oligosaccharide. Confirmatory information is often available from the 3JH-1,H-2 values measured from the 1H NMR spectra, with the exception of mannopyranose and rhamnopyranose, for which the values of the alpha and beta anomers are similar. A problem has been encountered with broadness of the 1H spectra of the 8-mer and 12-mer in D2O solutions, which could inhibit the analysis and assignment of the spectra. Thinking that this broadness might be due to viscosity of the solutions of the 8-mer and 12-mer, we acquired 1H and 13C spectra of the 12-mer at elevated temperatures, 50 C and 60 C, but this did not help. We are investigating the possibility that the unexpected broadness of the spectra is due to paramagnetic metal ion contaminants in the 8-mer and 12-mer. Carbohydrates are often excellent metal-complexing agents, and the occasional spectral broadening observed in the presence of scavenged paramagnetic metal ions is a well-known phenomenon. We have therefore treated the 8-mer with Chelex 100 cation exchange resin (sodium form) in an effort to remove any polyvalent, paramagnetic ions. Following this treatment, the 8-mer has shown improved 1H spectral resolution.

已采用的NMR技术包括一维(1D) 1H、1D 13C、二维(2D) 1H-1H相关光谱(COSY)、2D 1H-1H全相关光谱(TOCSY)、2D 13C-1H异核单量子相关光谱(HSQC)、2D 13C-1H异核多重键 关联 (HMBC)、2D 1H 耦合 13C-1H HSQC 和 1D 1H 耦合 13C NMR。 COSY 技术提供有关 1H 光谱归属的信息，TOCSY 程序为各个糖残基提供单独的 1H 子光谱，HSQC 方法通过关联 1H 和 13C 化学位移提供 13C 光谱归属，HMBC 脉冲序列通过关联 1H 或 13C 化学位移与糖环中的原子核化学位移来描绘取代基的位置。 1H耦合1D 13C和1H耦合2D HSQC技术用于测量寡糖的13C-1H耦合常数。当 1H 耦合、1D 13C NMR 光谱分散度不足以方便分析时，后一种 2D 方法特别有用。测量 1JC-1,H-1 值，通常给出寡糖中所有异头键的构型（α 或 β）信息。验证性信息通常可以从 1H NMR 谱测量的 3JH-1,H-2 值中获得，但吡喃甘露糖和吡喃鼠李糖除外，它们的 α 和 β 端基异构体值相似。 D2O 溶液中 8 聚体和 12 聚体的 1H 光谱存在宽度问题，这可能会抑制光谱的分析和分配。我们认为这种宽度可能是由于 8 聚体和 12 聚体溶液的粘度造成的，因此我们在高温（50°C 和 60°C）下获得了 12-聚体的 1H 和 13C 光谱，但这并没有帮助。我们正在研究光谱意外宽广是由于 8 聚体和 12 聚体中的顺磁性金属离子污染物造成的可能性。碳水化合物通常是优异的金属络合剂，并且在清除的顺磁金属离子存在下偶尔观察到的光谱展宽是众所周知的现象。因此，我们用 Chelex 100 阳离子交换树脂（钠型）处理 8 聚体，以去除任何多价顺磁性离子。经过这种处理后，8 聚体的 1H 光谱分辨率得到了改善。