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

 描述(申请人提供)：功能糖组学中的最新发现，即对多糖结构和功能的系统研究，证明了多糖在许多生物医学过程中的重要性，包括蛋白质折叠、细胞-细胞黏附、信号传递和发病机制。其中许多事件中的糖链功能是通过它们与糖链结合蛋白(GBP)的相互作用来实现的。它们可以被广泛地定义为任何蛋白质，例如凝集素、糖胺聚糖结合蛋白和抗体，它与糖决定簇结合。如果没有自动化的糖链测序和合成能力，功能性糖链的主要障碍是通常无法获得具有特定结构的糖链。最全面的糖链文库是由美国国立卫生研究院资助的功能糖链联盟建立的，主要以约600个糖链的糖链微阵列形式提供，仅占人类糖链所含总糖链决定因素的不到20%。因此，很明显，功能性糖链进化的下一个主要发展必须扩大可用的糖链结构的数量和多样性。我们的指导性假设是，人/动物/植物糖中的自然产生的多糖可以被大量的Gbps识别，每个Gbps在糖结合和结合模式上都表现出特异性。尽管合成化学家做出了巨大努力并取得了进展，但目前化学-酶法合成多糖的速度远远落后于人们对糖组学的爆炸性兴趣。目前分离和分离天然多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较小微克量的结构分析上，而不适用于大规模生产 糖链文库和功能研究。此外，糖苷元部分通常会被去除，这引发了对多糖功能完整性的质疑。为了直接解决这些问题，我们开发了一种新的化学方法来实施创新的“反向合成”方法，从丰富的自然来源中提取大量的糖共轭化合物来生产大量的多糖。在前期研究取得成功的基础上，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-葡聚糖，以及从神经鞘糖脂中制备毫克量的标记N-和O-葡聚糖。我们将改进我们的温和化学反应，以操纵糖苷元部分，即糖鞘脂脂的脂类部分和糖蛋白的肽链。新的化学方法允许使用大规模(克到公斤)的起始材料(组织/器官)，并将为详细的结构分析和功能研究提供足够的葡聚糖。这个项目的成功将产生 包含数百个毫克量的复杂天然多糖的文库，这些多糖与生物医学直接相关。这些文库将推动对多糖相互作用的重要研究 在正常的细胞功能和宿主病原体生物学中。

 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.