Chemical and Enzymatic Synthesis of N-Glycan Libraries and Glycoarrays

碳水化合物在生物系统中的重要性刺激了近年来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。天然寡糖的种类通常由质谱测定，但留给生物科学家许多未解决的问题。聚糖的结构可以从微小的量确定，但是生物学特性的阐明需要大量的化合物。为了通过凝集素或抗体检测聚糖的生物识别，小型化方法得到了发展。通过微接触印刷（聚糖阵列）在表面上稳定的纳米级化学固定聚糖允许以高通量的方式分析碳水化合物结合蛋白的特异性。这些阵列是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。尽管全世界都在努力，但目前印刷在阵列上的聚糖的数量仍然很低，而且只代表了非常不完整的聚糖。主要原因是很难从天然来源中分离出纯低聚糖，因此需要通过合成来提供所需的结构。获得低聚糖是相当苛刻的，每一类化合物都需要专门的实验室。因此，目前的阵列集中在有限数量的结构上，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，未知蛋白质的碳水化合物结合特异性可以使用聚糖阵列来确定。本项目旨在：1)进一步发展复合n -聚糖的模块化合成方法及其对普遍适用的n -聚糖叠氮化物的选择性脱保护。2)通过糖基转移酶在溶液中对所有哺乳动物n -聚糖核心结构的酶促延伸。3)生成用于阵列和核磁共振测量的n -聚糖的稳定共轭物。4)利用n -聚糖阵列系统阐明凝集素的特异性谱，然后利用核磁共振和晶体学进行详细的结构测定。5)利用重组糖基转移酶和从蛋黄中分离的n -聚糖建立了所有哺乳动物n -聚糖核心结构的快速半合成。

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.