IMPROVEMENTS IN PROCEDURES FOR PER-O-METHYLATION OF CARBOHYDRATES

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Improved procedures for per-O-methylation of carbohydrates are being developed, based on investigation of the possible oxidative degradation mechanisms occurring during the derivatization in dimethyl sulfoxide with methyl iodide in the presence of base. We have 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. Evidence was obtained that the 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 are now evaluating this approach, as well as other steps that may help to improve efficiency for low amounts of sample. We are also participating in a round-robin analysis of a set of O-linked glycopeptide samples that are being analyzed by several laboratories within a project sponsored by the Human Glycoproteomics Initiative, based in Osaka, Japan. A manuscript describing the first step in this comparison among laboratories, analysis of N-linked glycoprotein glycans, was recently published in Glycobiology and the paper on O-linked glycans is almost ready for submission.