Chemical and Enzymatic Synthesis of N-Glycan Libraries and Glycoarrays

N-聚糖文库和糖阵列的化学和酶法合成

• 批准号: 191313488
• 负责人: Professor Dr. Carlo Unverzagt
• 金额: --
• 依托单位: Arbeitsgruppe Bioorganische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/191313488?language=en
• 关键词: Chemical; Enzymatic; Synthesis; Glycan; Libraries

The importance of carbohydrates in biological systems has stimulated the recent development of new screening technologies. With the advent of glycomics, the characterization of entire glycomes of individual cell types, organs and organisms can be attempted. The variety of natural oligosaccharides is typically determined by mass spectrometry but leaves bioscientists with many unresolved questions. Glycan structures can be determined from minute quantities, however elucidation of the biological properties requires much larger quantities of the compounds. In order to detect the biological recognition of glycans by lectins or antibodies miniaturized methods were developed. Robust nanoscale chemical immobilization of glycans on surfaces by micro contact printing (glycan arrays) allows the analysis of the specificity of carbohydrate binding proteins in a high throughput manner. These arrays are based on the availability of many defined carbohydrate structures, which need to immobilized after chemical derivatization. Despite worldwide efforts the number of glycans printed on current arrays is still low and represents glycomes only very incompletely. The main reason is the difficult isolation of pure oligosaccharides from natural sources and thus the required structures need to provided by synthesis. Access to oligosaccharides is quite demanding and requires specialized labs for each class of compounds. Thus current arrays are focusing on a limited number of structures whereas the systematic variation of the most important structures shows many gaps. Despite several shortcomings the carbohydrate binding specificity of an unknown protein can be determined using glycan arrays in most of the cases. This project aims at:1) developing further the methods for modular synthesis of complex N-glycans and their selective deprotection to universally applicable N-glycan azides.2) the enzymatic elongation of all mammalian N-glycan core structures by glycosyltransferases in solution.3) generating stable conjugates of N-glycans for arrays and NMR-measurements.4) use of N-glycanarrays for the systematic elucidation of specificity profiles of lectins followed by detailed structure determination using NMR and crystallography.5) establishing a rapid semisynthesis of all mammalian N-glycan core structures of the complex type using recombinant glycosyltransferases and an N-glycan isolated from egg yolk.

碳水化合物在生物系统中的重要性刺激了新的筛选技术的发展。随着糖组学的出现，可以尝试表征单个细胞类型、器官和生物体的整个糖组。天然低聚糖的种类通常由质谱法测定，但给生物科学家留下了许多未解决的问题。聚糖结构可以从微量确定，但是生物学特性的阐明需要大量的化合物。为了检测凝集素或抗体对聚糖的生物识别，开发了小型化方法。通过微接触印刷（聚糖阵列）将聚糖稳固地纳米级化学固定在表面上允许以高通量方式分析碳水化合物结合蛋白的特异性。这些阵列基于许多确定的碳水化合物结构的可用性，这些结构需要在化学衍生化后固定化。尽管全世界都在努力，但在当前阵列上打印的聚糖的数量仍然很低，并且仅非常不完全地代表糖组。主要原因是从天然来源中分离纯寡糖很困难，因此需要通过合成提供所需的结构。获取低聚糖的要求很高，需要专门的实验室来处理每一类化合物。因此，目前的阵列集中在有限数量的结构，而最重要的结构的系统变化显示出许多差距。尽管存在一些缺点，但在大多数情况下，可以使用聚糖阵列来确定未知蛋白质的碳水化合物结合特异性。本项目旨在：1）进一步开发复杂N-聚糖的模块化合成方法及其选择性脱保护以获得普遍适用的N-聚糖叠氮。2）通过溶液中的糖基转移酶酶促延伸所有哺乳动物N-聚糖的核心结构。3）生成用于阵列和NMR测量的N-聚糖的稳定缀合物。4）使用N-聚糖阵列系统阐明凝集素的特异性特征，然后使用NMR和晶体学进行详细结构测定。5）使用重组糖基转移酶和从蛋黄中分离的N-聚糖建立所有哺乳动物N-聚糖复杂型核心结构的快速半合成。