Expanding the Glycomics toolbox: Chemo-enzymatic synthesis of well-defined N-glycan standards

项目摘要/摘要： 意义：N-连接蛋白糖基化是一类重要的生物分子 注意它们在疾病诊断和治疗开发中的应用。一个主要的障碍是快速和 简便易行的N-葡聚糖结构鉴定源于缺乏可用于 生物分析标准物质。无法有力地解释蛋白质N-糖基化阻碍了发育 发现生物标志物的新技术并阻碍重组糖蛋白的进步 工程学，如抗体。目前商业上可用的参考材料要么提供了有限的 不能代表生物组成或缺乏确切信息的N-糖链数量 结构装配。我们的图书馆将包括最大的商业上可用的定义明确的N- 这将是对当前可用标准的重大进步。战略：利用优势 在糖基转移酶和水解酶的严格底物要求中，酶促方法将是 用来生产高纯度结构的集合。具体目标：该项目描述了一种健壮的半 合成酶法制备人血清ALL中含量最丰富的20种N-糖链 丰度大于1%的，合计占血清总N-糖蛋白的50%以上。 每种化合物将具有99%的纯度，并将包含可自由还原的异构体末端，从而提供灵活性 用于多种分析技术。任务1：从商品中提取唾液酸化糖肽 提供蛋黄粉和制备高级中间体，这是所有需要的共同 目标。任务2：对称和非对称结构的双天线目标的合成。任务3： 合成一系列三天线的唾液酸化区域异构体。任务4：开发第二条生产线 其中每个N-糖链包含一个可用作内部参照物的统一标记的13C6 GlcNAc 用于量化。初步数据：已经设计了一种可伸缩的提取策略来访问GRAM- 粗提法从蛋黄粉中提取所需唾液酸化糖肽起始原料的量 技术将在任务1-4中采用的合成方法已通过验证，无需 进行进一步的优化。可交付成果：完成后，所描述的库将代表 全面收集结构上定义的N-葡聚糖标准，并将提供迫切需要的工具 科学界。

Project Summary / Abstract: Significance: N-linked protein glycosylation represents an important class of biomolecules receiving significant attention for their application in disease diagnosis and therapeutic development. A major hurdle with rapid and facile N-glycan structural identification stems from the lack of well-defined reagents that can be used as bioanalytical reference material. The inability to robustly interpret protein N-glycosylation prevents development of new technologies for uncovering biomarkers and hinders the advancement of recombinant glycoprotein engineering, such as antibodies. Current commercially available reference material either provides a limited number of N-glycans which are not representative of biological composition or lack details regarding exact structural assembly. Our library will comprise the largest commercially available collection of well-defined N- glycans and will represent a significant advancement to current available standards. Strategy: Taking advantage of the strict substrate requirements of glycosyltransferases and hydrolases, an enzymatic approach will be utilized to produce a collection of high purity structures. Specific Aim: This project describes a robust semi- synthetic, enzymatic methodology for producing the 20 most abundant N-glycans found in human serum all having an abundance greater than 1%, and together, comprise greater than 50% of the total serum N-glycome. Each compound will have a purity of >99% and will contain a free-reducing anomeric terminus providing flexiblity for use in a multitude of analytical techniques. Task 1: Extraction of a sialylated glycopeptide from commercially available egg yolk powder and the preparation of an advanced intermediate which is common to all desired targets. Task 2: Synthesis of bi-antennary targets with symmetric and asymmetric architectures. Task 3: Synthesize a collection of tri-antennary, sialylated regio-isomers. Task 4: Development of a second product line where each N-glycan contains one uniformly labeled 13C6 GlcNAc which can be used as an internal reference for quantification. Preliminary Data: A scalable extraction strategy has been devised for accessing gram- quantities of the desired sialylated glycopeptide starting material from egg yolk powder using robust extraction technology. The synthetic methodologies to be employed in tasks 1 – 4 have been validated removing the need for further optimization. Deliverables: When complete, the described library will represent the most comprehensive collection of structurally defined N-glycan standards and will deliver an urgently needed tool to the scientific community.