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

糖链存在于各种糖结合物中，是细胞的基本成分。生物分子中的葡聚糖部分会影响它们的性质，从而影响它们所参与的生物过程。然而，人们对碳水化合物在生物系统中的影响仍然知之甚少。这主要归因于从天然来源获得纯多糖和糖共轭化合物的机会有限。此外，由于存在大量的异构体结构，结合有限的样本量，天然多聚糖的结构解释受到了阻碍。在大多数情况下，这些缺点只能通过合成来克服。对于基于多糖的相互作用研究，需要满足额外的要求。理想情况下，糖基化生物分子应该使用天然实体来研究，以建立结构-活性关系。然而，在大多数情况下，这是不可行的。限制是由于额外的合成努力和宏观分析中贵重物质的损失而产生的。作为另一种选择，开发了葡聚糖微阵列。这项技术最大限度地减少了多糖的消耗，并允许并行进行许多实验。目前使用的许多N-葡聚糖阵列的组成不是系统的，而且印刷化合物的纯度通常不被披露。缺乏不对称取代的N-葡聚糖，它构成了大多数自然产生的N-葡聚糖，但很难获得。我们开发了一种化学酶方法来使选择性的苄基化N-糖链多样化，从而提供了不对称半乳糖化多天线N-糖链的完整文库。这些N-葡聚糖很容易在其叠氮基团上衍生，并将用对称的N-葡聚糖来补充现有的微阵列。通过提供一整套迄今无法获得的参考化合物，我们打算改进糖组学中N-糖链分析的方法，从而实现精确的结构分配。该项目的目标是：1)开发复杂N-糖链的酶多样化方法；2)合成具有基于苄基化N-糖叠氮的完全取代模式的半乳糖化不对称N-糖基参考库；3)建立一个扩展的N-糖链阵列，用于详细阐明凝集素的特异性；4)合成用于糖组学和糖蛋白图谱的标记N-糖链参考库；5)开发具有核心修饰的不对称N-糖链参考库的合成方法。

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.