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

项目概要/摘要： 意义：N-连接蛋白糖基化代表一类重要的生物分子， 关注其在疾病诊断和治疗开发中的应用。一个主要的障碍， 简单的N-聚糖结构鉴定源于缺乏可用作 生物分析参考物质。不能有力地解释蛋白质N-糖基化阻碍了发展 发现生物标志物的新技术，阻碍了重组糖蛋白的发展 工程，如抗体。目前市售的参考材料提供了有限的 N-聚糖的数量不代表生物组成或缺乏关于确切的 结构装配。我们的图书馆将包括最大的商业上可获得的定义明确的N- 聚糖，并将代表对当前可用标准的重大进步。策略：利用 由于糖基转移酶和水解酶对底物的严格要求， 用于产生高纯度结构的集合。具体目标：该项目描述了一个强大的半- 用于生产人血清中发现的20种最丰富的N-聚糖的合成酶促方法， 具有大于1%的丰度，并且一起构成大于50%的总血清N-糖组。 每种化合物将具有>99%的纯度，并且将含有提供柔性的自由还原异头末端 用于多种分析技术。任务1：从市售产品中提取唾液酸化糖肽 蛋黄粉和制备一种高级中间体， 目标的任务2：合成具有对称和非对称架构的双天线目标。任务三： 合成一系列三触角唾液酸化区域异构体。任务4：开发第二条产品线 其中每个N-聚糖含有一个均匀标记的13 C6 GlcNAc，其可用作内部参比 用于量化。初步数据：一个可扩展的提取策略已被设计用于访问克- 使用稳健提取法从蛋黄粉中获得所需量的唾液酸化糖肽起始物料 技术.任务1 - 4中采用的合成方法已经过验证， 进一步优化。可扩展性：完成后，所描述的库将代表 全面收集结构确定的N-聚糖标准品，并将提供急需的工具， 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.