REDUCTIVE CLEAVAGE METHOD FOR GLYCAN STRUCTURAL ANALYSIS

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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.