Chemical and Biological Studies of the Guadinomines

描述（由申请人提供）：该奖学金有三个目的。第一个目标是通过合成和生物合成相结合的方法来创造胍二胺的类似物。假设驱动的类似物将被合成，每个都解决一个问题，即哪种结构特征是III型分泌系统（TTSS）抑制所必需的。类似物也将被制成，作为前体定向生物合成的底物，这将允许快速形成胍二胺类似物。第二个目的是设计一种改进的胍二胺分离方案。天然胍二胺A和B在培养基中的含量为~ 1mg /L，需要繁琐的纯化程序。由于胍二胺A和B都含有邻二胺，这是一种罕见的结构亚基，我们将利用其反应性来提高其分离性。我们已经证明，在室温下，二胺与含氟醛干净地反应，无需添加催化剂即可生成动物（May， Khosla -未发表的结果）。这将允许从培养基中的剩余物质中提取固相氟。第三个目的是确定TTSS对胍二胺的抑制范围，并确定它们如何影响其靶标。我们已经证明植物病原体黄单胞菌受到瓜苷胺A的抑制（May， Mudgett -未发表的结果），并将继续研究天然瓜苷胺及其类似物的植物病原体。我们将使用肠道耶尔森菌试验来确定哪些效应蛋白、结构蛋白和伴侣蛋白受到胍二胺的影响。这一知识将使第二代设计新的和更有效的类似物。

DESCRIPTION (provided by applicant): This fellowship has three Aims. The first Aim is to use the create analogs of the guadinomines through a combined synthetic and biosynthetic approach. Hypothesis-driven analogs will be synthesized, each addressing a question about which structural features are necessary for Type III Secretion System (TTSS) inhibition. Analogs will also be made that should serve as substrates for precursor-directed biosynthesis, which should allow the rapid formation of guadinomine analogs. The second Aim is to devise an improved isolation protocol for guadinomines. Natural guadinomines A and B are present at levels of ~1 mg/L of culture media, and require tedious purification procedures. Since guadinomines A and B both contain a vicinal diamine, an uncommon structural subunit, we will exploit its reactivity to enhance its isolation. We have shown that diamines react cleanly with fluorine-containing aldehydes to make aminals at room temperature without added catalysts (May, Khosla - unpublished results). This will allow for solid phase fluorous extraction from the remaining material in the culture media. The third Aim is to determine the scope of TTSS inhibition of the guadinomines, and determine how they affect their targets. We have shown that the plant pathogen Xanthomonas is inhibited by guadinomine A (May, Mudgett - unpublished results) and will continue to study plant pathogens with natural guadinomines and their analogs. We will use a Yersinia enterolitica assay to determine which effector proteins, structural proteins, and chaperones are affected by guadinomines. This knowledge will enable a second-generation design of new and more potent analogs. PUBLIC HEALTH RELEVANCE: Pathogenic Gram-negative bacteria use a Type III Secretion System (TTSS) to infect hosts. The goals of the proposal are to study how the guadinomines inhibit TTSS, and to make new and more potent analogs.