Facile Chemical Methods for Reverse Synthesis and Large Scale Production of Biomedically Relevant Glycan Libraries

 描述（由申请人提供）：功能性糖组学的最新发现，对聚糖结构和功能的系统研究，证明了聚糖在许多生物医学过程中的重要性，包括蛋白质折叠、细胞-细胞粘附、信号传导和发病机制。在许多这些事件中，聚糖功能通过其与聚糖结合蛋白（GBP）的相互作用来实现。这些可以广义地定义为结合聚糖决定簇的任何蛋白质，例如凝集素、糖胺聚糖结合蛋白和抗体。在没有自动化聚糖测序和合成能力的情况下，功能性糖组学的主要障碍是具有确定结构的聚糖的普遍不可用。最全面的聚糖文库由NIH资助的功能性糖组学联盟（Consortium for Functional Glycomics）生产，并且主要以约600种聚糖的聚糖微阵列形式提供，其占估计包含在人类糖组中的总聚糖决定簇的不到20%。因此，很明显，功能糖组学进化的下一个主要发展必须扩大可用聚糖结构的数量和多样性。我们的指导性假设是，人/动物/植物糖组中天然存在的聚糖被大量GBP识别，每种GBP在聚糖结合和结合模式中显示出特异性。尽管合成化学家做出了巨大的努力和进步，但目前化学-酶法合成聚糖的速度远远落后于糖组学的爆炸性兴趣。目前用于分离天然聚糖的技术受到昂贵的酶和苛刻的化学条件的限制。因此，它们主要集中在需要相对小的微克量的结构分析上，并且对于大规模生产不实用。 聚糖文库和功能研究。此外，糖苷配基部分通常被去除，引起了关于聚糖功能完整性的问题。为了直接解决这些问题，我们开发了新的化学方法，以实施创新的“反向合成”方法，从丰富的天然来源中的糖缀合物中大量生产大量聚糖。基于我们在初步研究中的成功，我们能够通过简单的一步或两步程序从糖蛋白中制备毫克量的标记N-和O-聚糖，以及从鞘糖脂中制备聚糖。我们将改进温和的化学反应以操纵糖苷配基部分;即，鞘糖脂的脂质部分和糖蛋白的肽链。新的化学方法允许使用大量（克至千克）的起始材料（组织/器官），并将提供足够的聚糖用于详细的结构分析和功能研究。该项目的成功将产生 含有数百毫克数量的直接与生物医学相关的复杂天然聚糖的文库。这些库将推动聚糖相互作用的研究， 正常细胞功能和宿主-病原体生物学。

 DESCRIPTION (provided by applicant): Recent discoveries in Functional Glycomics, the systematic study of structure and function of glycans, demonstrate the importance of glycans in many biomedical processes, including protein folding, cell-cell adhesion, signaling, and pathogenesis. Glycan functions in many of these events are fulfilled by their interactions with glycan-binding proteins (GBPs). These may be broadly defined as any protein, e.g. lectins, glycosaminoglycan-binding proteins, and antibodies, which binds to glycan determinants. Without automated glycan sequencing and synthesis capabilities, the major impediment to Functional Glycomics is the general unavailability of glycans with defined structures. The most comprehensive glycan library has been produced by the NIH-funded Consortium for Functional Glycomics, and is largely available as a glycan microarray of ~600 glycans, which represents less than 20% of the total glycan determinants estimated to be contained within the human glycome. Thus, it is obvious that the next major developments in the evolution of Functional Glycomics must expand the number and diversity of available glycan structures. Our guiding hypothesis is that naturally-occurring glycans in human/animal/plant glycomes are recognized by a large repertoire of GBPs, each of which displays specificity in glycan binding and mode of binding. Despite great efforts and progress from synthetic chemists, the current pace of chemo-enzymatic syntheses of glycans lags far behind the explosive interest in glycomics. Current techniques for the isolation and separation of natural glycans are limited by expensive enzymes and harsh chemical conditions. Therefore they are mainly focused on structural analysis requiring relatively small microgram quantities and are not practical for large-scale production of glycan libraries and functional studies. In addition, the aglycon moieties are typically removed, raising questions about functional integrity of the glycans. To directly address these questions, we have developed novel chemistry to implement an innovative "reverse synthesis" approach to produce large numbers of glycans in significant quantities from glycoconjugates in abundant natural sources. Building upon our success in preliminary studies, we are able to prepare milligram quantities of tagged N- and O-glycans from glycoproteins, and glycans from glycosphingolipids, through simple one- or two-step procedures. We will refine our mild chemical reactions to manipulate the aglycon moieties; i.e., lipid moiety of glycosphingolipids and the peptide chain of glycoproteins. The new chemical methods allow use of large-scale amounts (gram to kilogram) of starting material (tissue/organ) and will provide enough glycans for both detailed structural analyses and functional studies. Success in this project will generate libraries containing many hundreds of complex natural glycans in milligram quantities that are directly biomedically relevant. These libraries will drive studies of glycan interactions important in normal cellular functions and host-pathogen biology.