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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL TECHNOLOGIES FOR PRODUCING RENEWABLE CHEMICALS AND POLYMERS FROM CARBOHYDRATES DERIVED FROM AGRICULTURAL FEEDSTOCKS

Location: Renewable Product Technology Research Unit

Title: Nickel-catalyzed proton-deuterium exchange (HDX) for linkage analysis of complex carbohydrates

Authors
item Price, Neil
item Hartman, Trina
item Vermillion, Karl

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 13, 2014
Publication Date: N/A

Technical Abstract: The structural assignment of complex carbohydrates typically requires the analysis of at least three parameters: 1. composition; 2. linkage; and 3. substituents. These are often assigned on a small scale by gas chromatography/mass spectrometry (GC/MS). Linkage positions are determined by permethylation analysis, during which non-linked hydroxyls on the parent carbohydrate are converted to acid-stable methyl ethers. Permethylation analysis is effective for many carbohydrates, but requires a non-aqueous polar solvent to dissolve both the carbohydrate sample and the generally more polar alkylating agent. This often leads to problems of poor solubility and miscibility, even with solvents such as DMSO or DMF, and this is compounded by the need for a strong inorganic base to catalyze the permethylation reaction. Under-methylation can occur due to both residual water or because of the inherent insolubility of the carbohydrate under study. This is a major drawback because an under-methylated position on the sugar can be interpreted as the location of a substituent or of a linkage position. Other problems are the loss and/or mobility of alkaline-labile groups, such as commonly occurring O-acetyl or O-acyl moieties, oxidation when using NaOH/MeI, and the toxicity of the solvents amd methylating reagents. We now report a novel alternative approach to linkage analysis that makes use of the non-reversible exchange of C-H protons on the carbohydrate backbone. The exchange reaction is conducted in deuterated water catalyzed by Raney nickel, and results in the selective exchange of C-H protons adjacent to free hydroxyl groups. Hence, the position of the residual C-H protons is indicative of the position of glycosidic linkages or other substituents, and can be readily assessed by HSQC-NMR or, following suitable derivatization, by GC/MS analysis. Moreover, because the only changes to the parent sugar are proton/deuterium exchanges, the composition and linkage analysis can be assessed by GC/MS in a single step, significantly lowering the time required for analysis. Kinetic parameters such as temperature, catalyst, water/D2O ratios, and the use of internal exchange standards have been assessed. We have verified this new technique by linkage analysis of several complex oligosaccharides and polysaccharides, including fructans that because of heat-lability are often difficult to assign.

Last Modified: 10/25/2014
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