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

四糖(4-聚体)、八糖(8-聚体)和十二糖(12-聚体)的核磁共振研究已经完成。以前在D2O溶液中遇到的8-聚体和12-聚体的1H谱宽的问题已经通过在溶液中添加丙酮-d6来解决，这使得可以更准确地在光谱仪中进行磁场匀场。找到的最好的程序是在丙酮-d6的氘信号上进行填补，然后将光谱仪的场频比锁定在d2O信号上。其基本原理是，基于D2O信号的锁定电平对垫片不敏感。 除了少数例外，4-聚体、8-聚体和12-聚体的5-甲氧羰基戊基糖苷的~1H和~(13)C核磁共振谱已用2D核磁共振方法完全归属。此外，还测定了这三种寡糖糖苷的几乎完整的1H-1H偶联常数。根据Karplus考虑对这些偶联常数的解释证实了存在的糖残基的类型，并证实了吡喃糖环的椅子构象。用1H偶联的2D HSQC核磁共振观察到很大的1J(C-1，H-1)值(168.9-175.8赫兹)，表明合成设计的三个寡糖糖苷中的所有键都是α-异构型。 通过观察一个或两个13C1H对的HMBC连接性，证实了这三个寡糖糖苷中的所有连接位置。核磁共振的结构证明允许制备低聚糖的蛋白质结合物，这些蛋白质结合物将用于针对大肠杆菌O148和1型痢疾沙门氏菌的潜在疫苗成分的免疫原性测试。 我们以前发现伯氏杆菌天然抗原物的两个主要成分是我们合成的1-O-palmitoyl-2-O-oleoyl-3-O-alpha-D-galactopyranosyl-sn-glycerol和1-O-oleoyl-2-O-palmitoyl-3-O-alpha-D-galactopyranosyl-sn-glycerol，，现在我们用高分辨核磁共振对其进行了完全表征。这些结构异构体的~1H核磁共振谱非常相似，由于我们需要一种可靠地区分这些异构体的方法，因此我们研究了13C核磁共振的应用。通过对这两种双甘油酯的~(13)C核磁共振谱中的羰基~(13)C区的分辨率增强，揭示了它们在~(13)C化学位移上的特征差异，可用于区分异构体和评价其化学纯度。通过对核磁共振谱的详细分析，可以完成~1H和~(13)C信号的归属，还可以得到以下概括：(A)主要的~(13)C=O化学位移是由于甘油部分上的取代基位置，(B)微小的~(13)C=O位移是由于脂肪酸的类型，(C)1-取代基C=Os在较低的场下共振，2-取代基C=Os，(D)棕榈基C=Os处于比油酰基C=Os更低的场。

NMR studies of the tetrasaccharide (4-mer), octasaccharide (8-mer), and dodecasaccharide (12-mer) glycosides have been completed. The problem encountered previously with broadness of the 1H spectra of the 8-mer and 12-mer in D2O solutions has been solved by addition of acetone-d6 to the solutions, which permitted more accurate shimming of the magnetic field in the spectrometer. The best procedure found was to shim on the deuterium signal of the acetone-d6, and then lock the field-frequency ratio of the spectrometer on the D2O signal. The rationale is that the lock level based on the D2O signal is insensitive to shimming. With few exceptions, complete assignments of the 1H and 13C NMR spectra of the 5-methoxycarbonylpentyl glycosides of the 4-mer, 8-mer, and 12-mer have been obtained by 2D NMR methods. Also, almost complete sets of 1H-1H coupling constants have been measured for the three oligosaccharide glycosides. Interpretation of these coupling constants according to Karplus considerations confirmed the types of sugar residues present, and confirmed the chair conformations of the pyranose rings. The observation of exclusively large 1J(C-1,H-1) values (168.9-175.8 Hz) by 1H-coupled 2D HSQC NMR indicates the alpha anomeric configuration for all linkages in the three oligosaccharide glycosides, as synthetically designed. All of the linkage positions in the three oligosaccharide glycosides have been confirmed by the observation of specific HMBC connectivities for one or two 13C1H pairs per linkage. Proof of structure by NMR allows for preparation of protein conjugates of the oligosaccharides, which will be used for immunogenic testing of potential vaccine components against E. coli O148 and S. dysenteriae type 1. We found previously that two major components of the native antigenic material from B. burgdorferi are 1-O-palmitoyl-2-O-oleoyl-3-O-alpha-D-galactopyranosyl-sn-glycerol and 1-O-oleoyl-2-O-palmitoyl-3-O-alpha-D-galactopyranosyl-sn-glycerol, which we have synthesized and now characterized completely by high-resolution NMR. The 1H NMR spectra of these structural isomers are extremely similar, and since we required a method that would reliably distinguish these isomers, we investigated the application of 13C NMR. Resolution enhancement of the carbonyl 13C region of the 13C NMR spectra of these two diglycerides revealed characteristic differences in 13C chemical shift that can be used to distinguish the isomers and assess their chemical purity. Detailed analysis of the NMR spectra allowed complete 1H and 13C signal assignments to be made, and also the following generalizations: (a) The major 13C=O chemical shift is due to the substituent position on the glycerol moiety, (b) the minor 13C=O shift is due to the type of fatty acid, (c) the 1-sustituent C=Os resonate at lower field that the 2-substituent C=Os, (d) the palmitic C=Os are at lower field than the oleoyl C=Os.