Chemoenzymatic synthesis of unsymmetric N-glycans for high resolution glycome analyses and glycan arrays

存在于各种糖缀合物中的聚糖是细胞的基本组分。生物分子的聚糖部分影响它们的性质，从而影响它们所参与的生物过程。然而，碳水化合物在生物系统中的作用仍然知之甚少。这主要归因于从天然来源获得纯聚糖和糖缀合物的途径有限。此外，天然聚糖的结构解析由于存在大量异构体结构以及样品量有限而受到阻碍。在大多数情况下，这些缺点只能通过合成来克服。对于基于聚糖的相互作用研究，需要满足额外的要求。理想情况下，糖基化的生物分子应使用天然实体进行研究，以建立结构-活性关系。然而，在大多数情况下，这是不可行的。由于额外的合成努力和宏观分析中珍贵材料的损失，限制出现了。作为替代方案，开发了聚糖微阵列。该技术最大限度地减少了聚糖的消耗，并允许并行进行许多实验。目前使用的许多N-聚糖阵列的组成不是系统的，而且打印的化合物的纯度经常没有公开。缺乏不对称取代的N-聚糖，其包含大部分天然存在的N-聚糖，但很难获得。我们已经开发了一种化学酶的方法来多样化选择性苄基化的N-聚糖，从而提供访问完整的图书馆的不对称半乳糖基化的多触角N-聚糖。这些N-聚糖容易在其叠氮基处衍生化，并且将用于补充具有对称N-聚糖的现有微阵列。通过提供完整的迄今为止不可用的参比化合物，我们打算改进糖组学中N-聚糖分析的方法，以实现精确的结构归属。该项目旨在：1）开发复杂N-聚糖酶促多样化的方法; 2）合成具有基于苄基化N-聚糖叠氮的完整取代模式的非对称N-聚糖的半乳糖基化参考库; 3）建立扩展的N-聚糖阵列，以详细阐明凝集素特异性; 4）合成用于糖组学和糖蛋白分析的非对称N-聚糖标记参考库; 5）开发具有核心修饰的非对称N-聚糖参考库的合成方法。

Glycans occurring in various glycoconjuates are essential components of cells. The glycan part of biomolecules influences their properties and thus the biological processes they are involved in. However, the effects of carbohydrates within biological systems are still poorly understood. This is mainly attributed to limited access to pure glycans and glycoconjugates from natural sources. Additionally, the structure elucidation of natural glycans is hampered by the presence of numerous isomeric structures in combination with limited amounts of sample. In most cases these shortcomings can only be overcome by synthesis.For glycan-based interaction studies additional requirements need to be met. Ideally, glycosylated biomolecules should be investigated using the natural entity for establishing structure-activity relationships. However, in most cases this is not feasible. Limitations arise through the additional synthetic effort and the loss of precious material in macroscopic assays. As an alternative, glycan microarrays were developed. This technology minimizes the consumption of glycans and allows to conduct many experiments in parallel. The composition of many of the currently used N-glycan arrays is not systematic and furthermore the purity of the printed compounds is frequently not disclosed. There is a lack of unsymmetrically substituted N-glycans, which comprise the majority of naturally occurring N-glycans but are quite difficult to obtain. We have developed a chemoenzymatic method to diversify selectively benzylated N-glycans thus providing access to complete libraries of unsymmetrically galactosylated multiantennary N-glycans. These N-glycans are readily derivatized at their azido group and will serve to complement an existing microarray with symmetrical N-glycans. By providing complete sets of hitherto unavailable reference compounds we intend to improve the methods for N-glycan analysis in glycomics allowing for precise structure assignment. The project aims at:1) developing methods for the enzymatic diversification of complex N-glycans.2) the synthesis of galactosylated reference libraries of unsymmetric N-glycans with complete substitution patterns based on benzylated N-glycan azides.3) establishing an extended N-glycan array for the detailed elucidation of lectin specificities.4) the synthesis of labelled reference libraries of unsymmetric N-glycans for use in glycomics and glycoprotein profiling.5) developing methods for the synthesis of reference libraries of unsymmetric N-glycans bearing core modifications.