IMPROVEMENTS IN PROCEDURES FOR PER-O-METHYLATION OF CARBOHYDRATES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Improved procedures for per-O-methylation of carbohydrates are being developed, consistent with our results from detailed investigation of the possible oxidative degradation mechanisms occurring during the derivatization in dimethyl sulfoxide with methyl iodide in the presence of base. We demonstrated that degradation occurs only under anhydrous conditions when there is a long reaction time between the carbohydrate dissolved in dimethyl sulfoxide and methyl iodide, followed by reaction with the base. We found that it can be totally avoided by treating the carbohydrate with powdered sodium hydroxide before introduction of methyl iodide under non-anhydrous conditions, or by adding a trace of water in dimethyl sulfoxide before methyl iodide, or by using N, N-dimethyl acetamide as the solvent (Ciucanu and Costello, J. Am. Chem. Soc. , 2003, 125, 16213-16219). Later results reported by others (Novotnoy et al., ASMS 2005) that took advantage of this chemical insight have demonstrated that microscale reactions are feasible using capillaries packed with solid NaOH. We have evaluated this approach, and other steps that may help to improve efficiency and minimize contamination for low amounts of sample. We also participated in a round-robin analysis of a set of O-linked glycopeptide samples that were analyzed by several laboratories within a project sponsored by the Human Glycoproteomics Initiative, based in Osaka, Japan. A manuscript describing this was recently published in Molecular and Cellular Proteomics.