REDUCTIVE CLEAVAGE METHOD FOR GLYCAN STRUCTURAL ANALYSIS

聚糖（聚合碳水化合物）结构的测定是一种 在许多生物医学研究领域， 它们参与细胞识别过程， 肿瘤、细菌、真菌和病毒中的抗原决定簇，及其 作为识别元件参与细菌和病毒感染。 由于其结构的复杂性，没有单一的方法来建立 其结构是可用的。虽然许多化学和光谱学 已经开发了用于成分和结构分析的方法 它们是非常费力的。有些程序需要相对 大量的样品，和其他的，虽然更敏感， 通常适用于所有种类的单糖或所有 可能遇到的结构特征的组合。 针对标准方法的缺点，我们开始了一项计划， 开发了一种新的聚糖结构分析方法。事实上，过去的研究 已经表明，它是敏感的，并适用于所有类别的 单糖和主要类别的共价连接，非- 糖取代基。该方法被称为“还原法”， 裂解法”，因为它的突出特点是区域特异性 完全甲基化聚糖中所有糖苷键的还原裂解。 该方法与标准甲基化分析有很大不同， 然而，由于（1）聚糖中的所有键合位置和环形式 同时建立，和（2）完全甲基化的 适合分离和鉴定的聚糖衍生物， 在没有中间转移的一锅反应中完成。 根据以往工作的结果，我们认为， 方法有可能作为聚糖的通用方法 结构表征，包括确定 组成单体、它们的环形式、它们的连接位置、它们的 异头构型和它们的序列。为了让这种潜力 并且为了使该方法可用于日常使用， 在其他实验室，分析数据（气液色谱 保留指数和质谱）的完整的真实的 标准必须随时可用。此外，为了进行测序 潜力的方法来实现，新的程序，选择性 必须开发糖苷键的还原裂解。 因此我们 建议提供这些真实的标准，并开发新的选择性 还原裂解程序。指导这一总体发展 方法将应用于糖蛋白的结构分析， 衍生的寡糖和细菌脂多糖 牙周病原体

The determination of structure of glycans (polymeric carbohydrates) is an important problem in many areas of biomedical investigation because of their involvement in cellular recognition processes, their presence as antigenic determinants in tumors, bacteria, fungi, and viruses, and their involvement as recognition elements in bacterial and viral infections. Because of their structural complexity, no single method for establishing their structure is available. Although many chemical and spectroscopic methods have been developed for the compositional and structural analysis of glycans, they are very laborious. Some procedures require relatively large amounts of sample, and others, although more sensitive, are not generally applicable to all classes of monosaccharides or to all combinations of structural features that can be encountered. In response to the shortcomings of standard methods, we began a program to develop a new method for glycan structural analysis. Indeed, past studies have shown that it is sensitive, and applicable to all classes of monosaccharides and to the major classes of covalently-attached, non- carbohydrate substituents. The method is referred to as the "Reductive Cleavage Method" because its salient feature is the regiospecific reductive cleavage of all glycosidic bonds in a fully methylated glycan. This method is substantially different from standard methylation analysis, however, because (1) all linkage positions and ring forms in the glycan are established simultaneously, and (2) conversion of the fully methylated glycan to derivatives suitable for separation and identification is accomplished in a one-pot reaction without intermediate transfers. Based upon the results of past work, we feel that the Reductive Cleavage Method has the potential to serve as a general method for glycan structural characterization, to include establishing the identities of constituent monomers, their ring form, their position(s) of linkage, their anomeric configuration and their sequence. In order for this potential to be realized and in order for the method to be accessible for routine use in other laboratories, analytical data (gas-liquid chromatographic retention indices and mass spectra) for a complete set of authentic standards must be readily available. Moreover, in order for the sequencing potential of the method to be realized, new procedures for the selective reductive cleavage of glycosidic bonds must be developed. We therefore propose to provide these authentic standards and to develop new selective reductive cleavage procedures. Guiding the overall development of this method will be its application to the structural analysis of glycoprotein- derived oligosaccharides and bacterial lipopolysaccharides derived from periodontal pathogens.