Isolation and NMR elucidation of 3 saponins from Blighia sapida

QS-21是一种复杂的皂素，已被用作疫苗佐剂，这一特性激发了人们对这类化合物的兴趣。在这项合作中，我们从大头翁中分离得到了三个复杂的皂苷：大头翁皂苷A、B和C，并用高分辨核磁共振确定了它们的结构。用质谱仪测定了其相对分子质量。 我们的结果表明，Blighoside A(分子量为1086道尔顿)含有一个未支化的四糖，含有阿拉伯糖、鼠李糖、葡萄糖和第二个阿拉伯糖，通过其中一个阿拉伯糖部分连接到一个30碳三萜苷元的C-3上。鼠李糖连接到第一个阿拉伯糖的C-2，葡萄糖连接到鼠李糖的C-3，第二个阿拉伯糖连接到葡萄糖的C-4，葡萄糖在O-3被乙酰化。 Blighoside B(Mw 1070 Dalton)含有与Blighoside A相同的四糖部分，但与另一种略有不同的三萜齐墩果酸相连，其中Hederagenin的C-23现在是甲基而不是羟甲基。 Blighoside C(分子量1504道尔顿)含有六个与齐墩果酸相连的单糖单元。这种三萜的C-3上连接着一种线性六糖，它由两个木糖、鼠李糖、葡萄糖、第二个鼠李糖和另一个葡萄糖组成。第二木糖连接到第一木糖的C-3，第一鼠李糖连接到第二木糖的C-2，第一葡萄糖连接到前一鼠李糖的C-3，第二鼠李糖连接到前一葡萄糖的C-4，以及最终葡萄糖连接到前一鼠李糖的C-3。此外，第一个葡萄糖在O-3和O-6处乙酰化，末端葡萄糖在O-4和O-6处乙酰化。 用于确定结构的核磁共振方法包括使用2D TOCSY数据集的1D切片来生成单个单糖残基的单独的子谱，这使得可以通过测量邻近的1H1H偶合常数来识别糖的类型。还使用了2D HMBC实验，给出了1H/13C通过键的连接性，定义了结构中的核邻居，以及糖键和乙酰基取代的位置。2D ROESY实验表明，1H/1H通过空间相互作用，特别是对邻近糖残基中的近端质子。 通过2D1H耦合的HSQC谱获得了类似糖残基的核磁共振信号的额外分辨率，这也允许识别糖中的赤道质子或轴向质子，以及通过测量每个糖的1JC-1，H-1耦合常数来确定每个糖的异常构型。

QS-21 is a complex saponin that has been used as a vaccine adjuvant, a property that has stimulated interest in this general class of compounds. In this collaboration, three complex saponins, Blighosides A, B, and C have been isolated from the tree Blighia sapida and their structures determined by high-resolution NMR. The molecular weights of the blighosides were obtained by mass spectrometry. Our results show that Blighoside A (MW 1086 Daltons) has an unbranched tetrasaccharide containing arabinose, rhamnose, glucose, and a second arabinose linked through one of the arabinose moieties to C-3 of a 30-carbon triterpene aglycone, hederagenin. The rhamnose is linked to C-2 of the first arabinose, the glucose to C-3 of the rhamnose, and the second arabinose to C-4 of the glucose, which is acetylated at O-3. Blighoside B (MW 1070 Daltons) contains the same tetrasaccharide moiety as Blighoside A, but linked to a slightly different triterpene, oleanolic acid, in which C-23 of hederagenin is now a methyl group instead of a hydroxymethyl group. Blighoside C (MW 1504 Daltons) has six monosaccharide units linked to oleanolic acid. Attached to C-3 of this triterpene is a linear hexasaccharide comprised of two xyloses, rhamnose, glucose, a second rhamnose, and another glucose. The second xylose is linked to C-3 of the first xylose, the first rhamnose to C-2 of the second xylose, the first glucose to C-3 of the preceding rhamnose, the second rhamnose to C-4 of the preceding glucose, and the final glucose to C-3 of the preceding rhamnose. Additionally, the first glucose is acetylated at O-3 and O-6, and the terminal glucose at O-4 and O-6. The NMR methods used for determination of structure included the use of 1D slices of 2D TOCSY data sets to generate separate sub-spectra for the individual monosaccharide residues, which allowed identification of sugar type by measurement of vicinal 1H1H coupling constants. Also used were 2D HMBC experiments that gave 1H/13C through-bond connectivities that defined nuclear neighbors in the structures, and also the sugar linkages and positions of acetyl substitution. 2D ROESY experiments indicated 1H/1H through-space interactions, especially for proximal protons in adjacent sugar residues. Additional resolution of the NMR signals of similar sugar residues was achieved by 2D 1H-coupled HSQC spectra, which also allowed identification of equatorial or axial protons in the sugar, as well as determination of the anomeric configuration of each sugar by measurement of their 1JC-1,H-1 coupling constants.