New Methods for Simultaneous Arming and SAR Studies of Natural Products

描述(申请人提供)：天然产品具有巨大的结构多样性，并与不同生物体中的各种蛋白质相互作用，由于结构上的同源性，在人类中也是如此。这项提议寻求开发一个试剂和反应工具箱，使天然产品(NP)的同时武装和结构-活性研究(SAR)成为可能。特别行政区的研究可以确定NP衍生物潜在的新活性，并确保最初的生物活性不会丧失。虽然考虑到这项RFA的范围，这笔赠款并不是一个具体的目标，但令人武装的方面导致了NP衍生物，它们可以随后通过夏普莱斯-惠斯根环加成反应连接到各种标签上，从而能够进行生物学研究，如作用机制(MOA)研究(包括非靶标研究)，并为基础细胞生物学提供有用的探针。长期目标是开发一种更系统化的方法来挖掘NPs作为促进化学遗传学的细胞途径的激活剂/失活剂的丰富潜力。该团队包括一名合成有机化学家(D.Romo)、一名分析化学家(G.Vigh)、一名生物有机/生物合成化学家(C.Watanabe)和一名生物化学家/分子生物学家(J.Liu，Johns Hopkins)。我们建议进一步开发一系列使用双功能试剂的化学和位置选择性反应，以获得也是有用的细胞探针的NP衍生物。这些研究将有助于从根本上理解复杂NPs背景下各种反应的化学选择性和位置选择性。我们已经与合成、生物合成和分离化学家建立了合作关系，他们已经为拟议的研究提供或同意提供~25个生物活性纳米颗粒。要研究的主要反应是Rh(II)促进的O-H插入反应，C-H胺化反应和烯烃氮杂化反应，以及温和的芳基卤化反应。在最初的非选择性步骤之后，我们将使用健壮/通用的NP衍生物纯化方法，重新分析得到的纯衍生物(SAR研究)，并确定最适合标记的位置，即找到保持最大生物活性的衍生物。我们还将在各种细胞分析中筛选NP衍生物的潜在新生物活性，并向分子库小分子储存库提供大量资料。初始反应的化学和位置选择性版本的后续发展将依赖于筛选各种手性金属络合物，以及用于反应监测(LC-MSN)和半制备高效液相色谱纯化的可靠方法，以通过一种类型的“双重不对称合成”来调节位置选择性。 与公共卫生相关：该项目的长期目标是开发一种更系统化的方法，以挖掘天然产品作为细胞途径激活剂和失活剂的丰富潜力。天然产物作为确定人类疾病新治疗靶点的工具和药物开发的先导结构，有着丰富的历史。这些研究的结果将与包括糖尿病、炎症和癌症在内的人类疾病直接相关，因为提出了与这些疾病相关的天然产物的衍生新方法(这项提议)。后续的搜救研究(这项建议)将确定潜在的新生物活性和合适的探针附着地点，以便随后进行与这些疾病相关的行动模式研究(不在本建议中)。

DESCRIPTION (provided by applicant): Natural products have immense structural diversity and interact with a wide range of proteins in diverse organisms and, due to structural homology, also in humans. This proposal seeks to develop a toolbox of reagents and reactions that will enable simultaneous arming and structure- activity studies (SAR) of natural products (NPs). The SAR studies may identify potential new activities of the NP derivatives and will also ensure that initial biological activity has not been lost. While not a Specific Aim of this grant given the scope of this RFA, the arming aspect leads to NP derivatives that are equipped for subsequent conjugation to various tags via Sharpless-Huisgen cycloadditions, enabling biological studies such as mechanism of action (MOA) studies (including off-targets) and provide useful probes for basic cell biology. The long-term objective is to develop a more systemized approach to mine the rich potential of NPs as activators/inactivators of cellular pathways contributing to chemical genetics. The team includes a synthetic organic chemist (D. Romo), an analytical chemist (G. Vigh), a bioorganic/biosynthetic chemist (C. Watanabe), and a biochemist/molecular biologist (J. Liu, Johns Hopkins). We propose to further develop a set of chemo- and site selective reactions employing bifunctional reagents to obtain NP derivatives that are also useful cellular probes. These studies will contribute to a fundamental understanding of the chemo- and site selectivity of various reactions in the context of complex NPs. We have established collaborations with synthetic, biosynthetic, and isolation chemists and they have provided or agreed to provide ~25 bioactive NPs for the proposed studies. Primary reactions to be studied are Rh(II)-promoted O-H insertion reactions, C-H aminations and alkene aziridinations with metal nitrenoids, and mild aryl halogenations. After an initial site non-selective step, we will employ robust/versatile NP derivative purification methods, re- assay the resulting pure derivatives (SAR studies), and determine the most appropriate site for tag attachment, i.e. find the derivatives that retain the greatest bioactivity. We will also screen for potential novel bioactivities of the NP derivatives in various cellular assays and make quantities available to the Molecular Libraries Small Molecule Repository. The subsequent development of both chemo and site-selective versions of the initial reactions will rely on screening various chiral metal complexes, and robust methods for reaction monitoring (LC-MSn) and semi-preparative HPLC purification, to modulate site-selectivity via a type of "double asymmetric synthesis." PUBLIC HEALTH RELEVANCE: The long-term objective of this project is the development of a more systematized approach to mine the rich potential of natural products as activators and inactivators of cellular pathways. Natural products have a rich history as tools for identification of novel therapeutic targets for human disease and as lead structures for drug development. Results from these studies will be directly relevant to human disease including diabetes, inflammation, and cancer since novel methods are proposed for derivatization of natural products (this proposal) pertinent to these disease. Subsequent SAR studies (this proposal) will be pursued to identify potential new bioactivities and suitable sites for probe attachment enabling subsequent mode of action studies (not in this proposal) relevant to these diseases.