IMPROVEMENTS IN PROCEDURES FOR PER-O-METHYLATION OF CARBOHYDRATES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 正在开发改进的碳水化合物的Per-O-甲基化程序，其基础是在碱存在下二甲基亚砜与甲基碘衍生化过程中可能发生的氧化降解机理的研究。我们已经证明，只有在无水条件下，溶解在二甲基亚砜中的碳水化合物与甲基碘反应时间较长，然后与碱反应，才会发生降解。有证据表明，只有在无水条件下，溶解在二甲基亚砜中的碳水化合物与甲基碘反应时间较长，然后与碱反应，才能发生降解。我们发现，在无水条件下，在引入甲基碘之前用氢氧化钠粉末处理碳水化合物，或者在甲基碘之前在二甲基亚砜中加入微量水，或者使用N，N-二甲基乙酰胺作为溶剂(Ciucanu和Costello，J.Am)，可以完全避免这种情况化学。SoC。，2003年，125125,16213-16219)。后来由其他人(Novotnoy等人，ASMS 2005)报道的利用这种化学洞察力的结果表明，使用填充固体NaOH的毛细管进行微规模反应是可行的。我们现在正在评估这一方法，以及其他可能有助于提高低样本量效率的步骤。我们还参与了一组O连接糖肽样本的循环分析，这些样本正在由设在日本大阪的人类糖蛋白组学倡议赞助的一个项目中的几个实验室进行分析。最近在《糖生物学》上发表了一篇手稿，描述了实验室间比较的第一步--N-连接糖蛋白多糖的分析，而关于O-连接多糖的论文几乎准备好提交。

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.