Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States.
Anal Chem. 2020 Oct 6;92(19):13221-13228. doi: 10.1021/acs.analchem.0c02379. Epub 2020 Sep 2.
Plant cell wall polysaccharide analysis encompasses the utilization of a variety of analytical tools, including gas and liquid chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. These methods provide complementary data, which enable confident structural proposals of the many complex polysaccharide structures that exist in the complex matrices of plant cell walls. However, cell walls contain fractions of varying solubilities, and a few techniques are available that can analyze all fractions simultaneously. We have discovered that permethylation affords the complete dissolution of both soluble and insoluble polysaccharide fractions of plant cell walls in organic solvents such as chloroform or acetonitrile, which can then be analyzed by a number of analytical techniques including MS and NMR. In this work, NMR structure analysis of 10 permethylated polysaccharide standards was undertaken to generate chemical shift data providing insights into spectral changes that result from permethylation of polysaccharide residues. This information is of especial relevance to the structure analysis of insoluble polysaccharide materials that otherwise are not easily investigated by solution-state NMR methodologies. The preassigned NMR chemical shift data is shown to be vital for NMR structure analysis of minor polysaccharide components of plant cell walls that are particularly difficult to assign by NMR correlation data alone. With the assigned chemical shift data, we analyzed the permethylated samples of destarched, alcohol-insoluble residues of switchgrass and poplar by two-dimensional NMR spectral profiling. Thus, we identified, in addition to the major polysaccharide components, two minor polysaccharides, namely, <5% 3-linked arabinoxylan (switchgrass) and <2% glucomannan (poplar). In particular, the position of the arabinose residue in the arabinoxylan of the switchgrass sample was confidently assigned based on chemical shift values, which are highly sensitive to local chemical environments. Furthermore, the high resolution afforded by the H NMR spectra of the permethylated switchgrass and poplar samples allowed facile relative quantitative analysis of their polysaccharide composition, utilizing only a few milligrams of the cell wall material. The concepts herein developed will thus facilitate NMR structure analysis of insoluble plant cell wall polysaccharides, more so of minor cell wall components that are especially challenging to analyze with current methods.
植物细胞壁多糖分析包括利用多种分析工具，包括气相和液相色谱、质谱 (MS) 和核磁共振 (NMR) 光谱。这些方法提供互补的数据，使我们能够对存在于植物细胞壁复杂基质中的许多复杂多糖结构提出有信心的结构建议。然而，细胞壁包含不同溶解度的部分，并且只有少数技术可以同时分析所有部分。我们发现，全甲基化可以使植物细胞壁的可溶性和不溶性多糖部分完全溶解在有机溶剂中，如氯仿或乙腈中，然后可以通过多种分析技术进行分析，包括 MS 和 NMR。在这项工作中，对 10 种全甲基化多糖标准品进行了 NMR 结构分析，以生成化学位移数据，这些数据提供了有关多糖残基全甲基化导致的光谱变化的见解。这一信息对于分析不溶性多糖材料的结构特别重要，否则这些材料很难通过溶液状态 NMR 方法进行研究。所分配的 NMR 化学位移数据对于通过 NMR 相关数据单独分配特别困难的植物细胞壁次要多糖成分的 NMR 结构分析至关重要。有了分配的化学位移数据，我们通过二维 NMR 光谱分析对无定形糖、醇不溶残渣的柳枝稷和杨树进行了全甲基化样品分析。因此，除了主要多糖成分外，我们还鉴定了两种次要多糖，即 <5% 的 3 连接阿拉伯木聚糖（柳枝稷）和 <2% 的葡甘露聚糖（杨树）。特别是，基于对局部化学环境高度敏感的化学位移值，能够自信地分配柳枝稷样品中阿拉伯木聚糖中阿拉伯糖残基的位置。此外，全甲基化柳枝稷和杨树样品的 H NMR 谱提供的高分辨率允许仅使用几毫克细胞壁材料对其多糖组成进行简单的相对定量分析。因此，本文提出的概念将有助于不溶性植物细胞壁多糖的 NMR 结构分析，特别是对于目前方法分析特别具有挑战性的次要细胞壁成分。
