PURIFICATION, LINKAGE AND NMR OF SACCHARIDE PREPARATION

糖制备的纯化、连接和核磁共振

基本信息

批准号：
7721588
负责人：
Parastoo Azadi
金额：
$ 0.14万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-01 至 2009-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7721588
关键词：
Acetates Aliquot Avicel Biological Assay Blood capillaries Butanols Carbohydrates Chemicals Chromatography Color Computer Retrieval of Information on Scientific Projects Database Deuterium Dimethyl Sulfoxide Electrons Ethanol Freeze Drying Funding Grant Hydrolysis Incubated Institution Isotopes Laboratories Mass Fragmentography Measures Methanol Methods Methylation NMR Spectroscopy Object Attachment Phase Phenols Polymers Preparation Protons Relative (related person)Research Research Personnel Resources Running Sampling Sep-Pak C18 Signal Transduction Silicon Dioxide Sodium Chloride Sodium Hydroxide Solvents Source Staging Sugar Alcohols Sulfuric Acids TOCSY Temperature Trifluoroacetic Acid Tube United States National Institutes of Health Water acetic anhydride capillary detector genetic linkage analysis ionization methyl iodide microcrystalline cellulose reversed phase chromatography seal

项目摘要

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. Purification Normal Phase Chromatography Sample was dissolved in butanol:ethanol:water 4:1:1 and loaded onto a 2ml column of Avicel microcrystalline cellulose then eluted with 7ml of the above solvent. Sample was eluted with 6ml ethanol:water 1:1. Most of the color and carbohydrate eluted in this stage as judged by phenol sulfuric acid assay while most of the salt eluted in the load. Reverse Phase Chromatography Material from the 50% ethanol wash of the normal phase column was further purified by loading onto a Waters C18 Sep Pak in 5% methanol. 1ml of 5% methanol was then run through the column and 4ml 50% methanol was used for elution. All color and most carbohydrate eluted in this stage. Glycosyl linkage NaOH method: For glycosyl linkage analysis, the sample was permethylated, depolymerized, reduced, and acetylated; and the resultant partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Initially, an aliquot of sample was permethylated by the method of Ciucanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). This involved incubating the sample in 1M NaOH in DMSO for 10 minutes at room temperature then adding 400ul of methyl iodide and incubating 10 more minutes. 80ul of 2.0M NaOH in DMSO were then added and after 10 more minutes 80ul of methyl iodide were added. 40 minutes after this, 80 ul of NaOH were added and allowed to incubate 40 minutes whereupon 2ml of water were added and the methyl iodide evaporated by bubbling. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121¿C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode); separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary columnInitially, an aliquot of sample was permethylated by the method of Ciucanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The permethylation was repeated twice in order to aid complete methylation of the polymer. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode); separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. NMR Spectroscopy The sample was deuterium-exchanged by lyophilization from D2O (99.9 % D, Aldrich) and dissolved in 40 uL D2O (99.996 % D, Cambridge Isotope Laboratories). 1-D Proton and 2-D gradient HSQC, and TOCSY NMR spectra were acquired in a capillary probe on a Varian Inova-600 MHz spectrometer at 298 K (25 ¿C). Proton chemical shifts were measured relative to the HDO signal (S=4.60 ppm at 40 ¿C).

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。对于中心来说，它不一定是研究者的机构。纯化正相色谱法将样品溶解在丁醇:乙醇:水4:1:1中并加载到2ml Avicel微晶纤维素柱上，然后用7ml上述溶剂洗脱样品，并用6ml乙醇:水1:1洗脱。根据苯酚硫酸测定判断，碳水化合物在此阶段洗脱，而大部分盐在负载中洗脱。反相色谱法将来自正相柱的50%乙醇洗液的材料通过加载到在5%甲醇中的Waters C18 Sep Pak上来进一步纯化，然后使1ml 5%甲醇流过该柱并使用4ml 50%甲醇进行洗脱。颜色和大部分碳水化合物在此阶段洗脱。糖基连接氢氧化钠法：对于糖基键分析，将样品全甲基化、解聚、还原和乙酰化；并按照 York 等人 (1985)Methods Enzymol 的描述，通过气相色谱-质谱 (GC-MS) 分析所得部分甲基化糖醇乙酸酯 (PMAA)。 118:3-40。最初，通过 Ciucanu 和 Kerek (1984) CarboHydr. 131:209-217 的方法对等分样品进行全甲基化（用干燥 DMSO 中的氢氧化钠和碘甲烷处理），这涉及将样品在 DMSO 中孵育。室温下孵育10分钟，然后加入400ul碘甲烷并再孵育10分钟。然后加入80ul 2.0M NaOH的DMSO溶液，再过10分钟后，加入80ul碘甲烷，40分钟后，加入80ul NaOH并温育40分钟，随后加入2ml水并蒸发碘甲烷。样品处理后，使用 2 M 三氟乙酸（2）水解全甲基化材料。 h 在密封管中于 121°C 下），用 NaBD4 还原，并使用乙酸酐/三氟乙酸乙酰化所得 PMAA 在连接至 5970 MSD（质量选择检测器，电子轰击电离模式）的 Hewlett Packard 5890 GC 上进行分析；在 30 m Supelco 2330 键合相熔融石英毛细管上进行分离首先，通过Ciucanu和Kerek (1984) CarboHydr. 131:209-217的方法对等分样品进行全甲基化(用氢氧化钠和碘甲烷在干燥DMSO中处理)，重复两次以帮助完成。样品处理后，使用 2 M 三氟乙酸水解全甲基化材料（在密封中水解 2 小时）。在 30 m Supelco 2330 键合相熔融石英毛细管柱上进行分离。核磁共振波谱通过冻干将样品从 D2O（99.9% D，Aldrich）中进行氘交换，并溶解在 40 uL D2O（99.996% D，剑桥同位素实验室）中，获得 1-D 质子和 2-D 梯度 HSQC，并获得 TOCSY NMR 谱。在 Varian Inova-600 MHz 光谱仪上的毛细管探头中，温度为 298 K (25 °C) 相对于 HDO 信号进行测量（S=4.60 ppm，40 °C）。