Nickel-catalyzed proton-deuterium exchange (HDX) procedures for glycosidic linkage analysis of complex carbohydrates

Authors: Price, Neil; Hartman, Trina; Vermillion, Karl

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2015
Publication Date: 6/15/2015
Citation: Price, N.P.J., Hartman, T.M., Vermillion, K.E. 2015. Nickel-catalyzed proton-deuterium exchange (HDX) procedures for glycosidic linkage analysis of complex carbohydrates. Analytical Chemistry. 87(14):7282-7290. doi: 10.1021/acs.analchem.5b01505.

Interpretive Summary: Complex carbohydrates such as polysaccharides are most often composed of long chains made up of various monosaccharide building blocks. The identity of the component monosaccharides may vary, as may the way in which they are linked together. Hence, branched and unbranched polysaccharides are common, such as cellulose, starch, or hemicelluloses. Determining the position at which these branches occur is important for understanding the structure and properties of carbohydrates, and this is generally referred to as linkage analysis. The most common approach to linkage analysis for carbohydrates is permethylation, which requires the chemical methylation of all of the non-linked positions on the building blocks. Unfortunately, this is not suitable for all polysaccharides, and it also requires toxic reagents and solvents. We have developed a new method for linkage analysis based on the replacement of the protons attached to the carbohydrate backbone with deuterium atoms. The deuterium comes from deuterated water, which is also used as the solvent for the exchange reaction. We have found that the positions on the carbohydrate that are linked do not exchange with deuterium, and we have devised two methods to determine these positions using laboratory instrumental techniques. This has several advantages: 1. It is water-based, and uses less toxic reagents; 2. The composition of the monosaccharide building blocks and their linkages can be determined in one step; and 3. It can be used for less stable carbohydrates that are degraded by the current permethylation chemistry. The new method may be applicable to most carbohydrate types, and will be of potential use to the U.S. sugar and biofuels industries.

Technical Abstract: The structural analysis of complex carbohydrates typically requires the assignment of three parameters: monosaccharide composition, the position of glycosidic linkages between monosaccharides, and the position and nature of non-carbohydrate substituents. The glycosidic linkage positions are often determined by permethylation analysis, but this can be complicated by high viscosity or poor solubility, resulting in under-methylation. This is a drawback because an under-methylated position may be misinterpreted as the erroneous site of a linkage or substituent. Here we describe an alternative approach to linkage analysis that makes use of a non-reversible deuterium 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 assigned 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 determined in a single step.