Vinogradov E V, Stuike-Prill R, Bock K, Holst O, Brade H
Division of Biochemical Microbiology, Forschungsinstitut Borstel, Institut für Experimentelle Biologie und Medizin, Germany.
Eur J Biochem. 1993 Dec 1;218(2):543-54. doi: 10.1111/j.1432-1033.1993.tb18408.x.
Lipopolysaccharide from Vibrio cholerae strain H11 (non-O1) was de-O-acylated, dephosphorylated, reduced, de-N-acylated, N-acetylated, and the products were separated by high-performance anion-exchange chromatography (HPAE). A decasaccharide, 1, was isolated as the major product, representing the core oligosaccharide attached to the reduced GlcN-disaccharide lipid A backbone. Its structure was established by compositional and methylation analyses, and extensive NMR investigations including 1H,1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser enhancement spectroscopy (NOESY), as well as heteronuclear 13C,1H COSY. In another reaction sequence the lipopolysaccharide was hydrolysed with dilute acetic acid and reduced with NaBH4. The resulting core fractions were separated by HPAE giving seven individual octasaccharides differing at the reducing 3-deoxy-D-manno-octulosonic acid (Kdo) residue. A major product, 2, was isolated and investigated by the same methods as described for the decasaccharide 1. The following structures are proposed for compounds 1 and 2: alpha-D-GlcNAcp-(1-7)-[beta-D-Galp-(1-3)-]-alpha-Hepp-(1-2)- alpha-Hepp- (1-3)-[beta-D-Glcp-(1-4)-]- [alpha-D-Glcp-(1-6)-]-alpha-Hepp-(1-5)-R, where R is alpha-Kdop-(2-6)-beta-D-GlcNAcp-(1-6)-D-GlcNAcol in 1 and 4,8-anhydro-Kdool in 2, and Hep is L-glycero-D-manno-heptose. In lipopolysaccharide, the terminal residue of alpha-D-glucosamine possessed a free amino group, as proved by deamination with nitrous acid and the 1H-NMR spectrum of de-O-acylated lipopolysaccharide. The conformational preferences of the terminal core heptasaccharide was assessed by Monte Carlo simulations combined with restrained calculations of side chains based on experimentally determined proton-coupling constants. These calculations, confirmed by NOE data, displayed several long-range interactions, which resulted in a well-defined three-dimensional structure of the core oligosaccharide.
对霍乱弧菌H11株（非O1）的脂多糖进行脱O - 酰化、脱磷酸化、还原、脱N - 酰化、N - 乙酰化处理，产物通过高效阴离子交换色谱（HPAE）分离。分离出一种十糖1作为主要产物，它代表连接在还原型葡糖胺 - 二糖脂质A主链上的核心寡糖。通过组成分析和甲基化分析，以及包括1H,1H相关光谱（COSY）、全相关光谱（TOCSY）、核Overhauser增强光谱（NOESY）以及异核13C,1H COSY在内的广泛核磁共振研究确定了其结构。在另一个反应序列中，脂多糖用稀乙酸水解并用NaBH4还原。所得的核心级分通过HPAE分离，得到七个在还原型3 - 脱氧 - D - 甘露 - 辛酮糖酸（Kdo）残基上不同的单个八糖。分离出一种主要产物2，并采用与十糖1相同的方法进行研究。为化合物1和2提出了以下结构：α - D - 葡糖胺 - 1 - 磷酸 - (1→7)-[β - D - 半乳糖 - (1→3)-]-α - 庚糖 - (1→2)-α - 庚糖 - (1→3)-[β - D - 葡萄糖 - (1→4)-]-[α - D - 葡萄糖 - (1→6)-]-α - 庚糖 - (1→5)-R，其中在1中R为α - Kdo - (2→6)-β - D - 葡糖胺 - 1 - 磷酸 - (1→6)-D - 葡糖胺醇，在2中为4,8 - 脱水 - Kdo醇，Hep为L - 甘油 - D - 甘露 - 庚糖。在脂多糖中，α - D - 葡糖胺的末端残基具有游离氨基，这通过亚硝酸脱氨和脱O - 酰化脂多糖的1H - NMR光谱得到证实。通过蒙特卡罗模拟结合基于实验测定的质子耦合常数对侧链进行受限计算来评估末端核心七糖的构象偏好。这些计算结果得到NOE数据的证实，显示出几种长程相互作用，从而形成了核心寡糖明确的三维结构。
