SYNTHESES APPLICATIONS OF ALPHA GALACTOSYL EPITOPES

描述：（申请人摘要）主要的异种活性抗原负责 超急性排斥反应最近被确定为碳水化合物 含有末端α Gal 1 -3Gal序列（α-Gal表位）的结构。 最常见的α-Gal表位是二糖α Gal 1 - 3Gal 1（1）， 三乙酰α Gal 1 - 3 β Gal 1 - 4 β GlcNAc（2）和 α Gal 1 - 3 β Gal 1 - 4 β Glc（3）和五糖 α Gal 1 - 3 β Gal 1 - 4 β GlcNAc 1 - 3 β Gal 1 - 4 β Glc（4）。α-Gal表位是 在大多数哺乳动物的细胞上大量表达，除了 人类猿类和旧大陆猴子相反，天然抗体与 对α-Gal表位（抗-Gal）的特异性仅存在于人类和其他老年人中。 世界灵长类动物。α-Gal和抗-Gal相互作用的发现， 导致了通过以下方法来克服超急性排斥反应的实验尝试 通过含有α-Gal的亲和力耗尽接受者的抗-Gal 柱（抗Gal免疫吸附方法）或通过 注入可溶性合成α-Gal寡糖（抗Gal中和 方法）。此外，α-Gal-抗原缀合物显示出增强抗原结合。 天然人抗Gal抗体和α-Gal免疫毒素的呈递 糖缀合物被用于靶向产生抗Gal的B细胞 抗体的所有这些生物医学应用都需要获得大量的 α-Gal寡糖以及合成衍生的α-Gal的量 对抗Gal抗体具有高亲和力的类似物和模拟物。因此， 研究计划旨在开发合成技术， 大规模生产α-半乳糖低聚糖和寻找 有效的α-Gal模拟物。 1)α-Gal抗原表位及其多价体合成方法的改进 衍生物.第三必需糖基转移酶β 1，3 GlcNAc 转移酶，将被克隆和过表达。然后一锅酶系统 将开发合成4与原位再生糖 核苷酸UDP-Gal和UDP-GlcNAc通过多个酶循环。进一步 探索结构上定义的α-Gal表位的多价效应 将合成α-Gal低聚物和α-Gal树枝状聚合物。的结合 这些α-Gal衍生物与人抗Gal的比率将通过以下方法测量： 竞争ELISA、流式细胞术和免疫组织化学测定。 2)α-Gal/抗-Gal相互作用的结构研究。统一的C-13 标记的α-Gal三糖α Gal 1 - 3 β Gal 1 - 4 β Glc，5个 构象受限的α-Gal类似物和七种单脱氧α-Gal 二糖衍生物将被合成并用于NMR实验， 确定与单克隆抗半乳糖苷结合的α-半乳糖苷的活性构象 抗体，并研究α-Gal的柔性和可控刚性 表位 3)α-Gal模拟文库和高通量筛选。一项新技术 平台（从拆分合成到流式细胞术筛选和MS结构分析） 分析），以产生和筛选大量（更多） 10的6次方）的糖肽，以鉴定前导α-Gal模拟物 序列的天然和非天然氨基酸将用于构建两个 用于筛选抗-Gal的不同表型的组合文库 共有结合结构。 总之，这项研究计划的成功将使α-Gal 低聚糖容易获得，将提供关于α-Gal的基本数据 /抗-Gal相互作用，并将发现潜在的α-Gal模拟物 可用作免疫诊断剂的结构、碳水化合物 治疗剂或疫苗。

DESCRIPTION: (Applicant's Abstract) The major xenoactive antigens responsible for hyperacute rejection have recently been identified as carbohydrate structures containing terminal alpha Gal1-3Gal sequence (alpha-Gal epitopes). The most common alpha-Gal epitopes are disaccharide alpha Gal1-3Gal1 (1), trisaccharides alphaGal1-3betaGal1-4betaGlcNAc (2) and alphaGal1-3betaGal1-4betaGlc (3), and pentasaccharide alphaGal1-3betaGal1-4betaGlcNAc1-3betaGal1-4betaGlc (4). Alpha-Gal epitopes are abundantly expressed on the cells of most mammals, with the exception of humans, apes and Old World monkeys. Conversely, the natural antibody with specificity to alpha-Gal epitope (anti-Gal) exists only in humans and other Old World primates. The discovery of the interaction of alpha-Gal and anti-Gal has led to experimental attempts to overcome hyperacute rejection by either depleting the recipient's anti-Gal through an alpha-Gal containing affinity column (anti-Gal immunoadsorption approach) or antagonizing anti-Gal by infusing soluble synthetic alpha-Gal oligosaccharides (anti-Gal neutralization approach). Moreover, alpha-Gal-antigen conjugates were shown to enhance antigen presentation by the natural human anti-Gal antibody, and alpha-Gal-immunotoxin glycoconjugates are being used to target B cells which generate anti-Gal antibody. All these biomedical applications require access to a substantial amount of alpha-Gal oligosaccharides as well as synthetically derived alpha-Gal analogs and mimetics with high-affinity to anti-Gal antibodies. Thus, this research program is aimed at development of synthetic technology for large-scale production of alpha-Gal oligosaccharides and at searching for potent alpha-Gal mimetics. 1) Improvement on the synthesis of alpha-Gal epitopes and its multivalent derivatives. The third necessary glycosyltranferase, beta1,3 GlcNAc transferase, will be cloned and overexpressed. Then a one-pot enzymatic system will be developed to synthesize 4 with in situ regeneration of sugar nucleotides UDP-Gal and UDP-GlcNAc through multiple enzyme cycles. To further explore the polyvalent effect of alpha-Gal epitope, structurally defined alpha-Gal oligomers and alpha-Gal dendrimers will be synthesized. The binding of these alpha-Gal derivatives to human anti-Gal will be measured by competition ELISA, flow cytometry, and immunohischemistry assays. 2) Structural studies on alpha-Gal / anti-Gal interaction. Uniformly C-13 labeled alpha-Gal trisaccharide alphaGal1-3betaGal1-4betaGlc, five conformationally constrained alpha-Gal analogs and seven monodeoxy alpha-Gal disaccharide derivatives will be synthesized and used in NMR experiments to determine the active conformation of alpha-Gal bound to monoclonal anti-Gal antibody, and to study the flexibility and controlled rigidity of alpha-Gal epitopes. 3) Alpha-Gal mimetic library and high-throughput screening. A new technology platform (from split synthesis to flow cytometry screening and to MS structural analysis) will be established to generate and screen a large number (greater than ten to the six power) of glycopeptides to identify lead alpha-Gal mimetic sequences. Both natural and unnatural amino acids will be used to construct two combinatorial libraries for screening against different phenotype of anti-Gal for consensus binding structures. In summary, success of this research program will make alpha-Gal oligosaccharides easily accessible, will provide fundamental data on alpha-Gal / anti-Gal interaction and will discover lead potent alpha-Gal mimetic structures that are useful as immunodiagnostic agents, carbohydrate therapeutics, or vaccines.