New Methods for Simultaneous Arming and SAR Studies of Natural Products

描述（由申请人提供）：天然产物具有巨大的结构多样性，并与各种生物体中的各种蛋白质相互作用，由于结构同源性，也与人类相互作用。该提案旨在开发一个试剂和反应工具箱，以实现天然产物（NP）的同时武装和结构活性研究（SAR）。SAR研究可以确定NP衍生物的潜在新活性，并且还将确保初始生物活性没有丧失。虽然考虑到该RFA的范围，这不是该授权的具体目标，但武装方面导致NP衍生物，其配备用于随后通过Sharpless-Huisgen环加成与各种标签缀合，从而实现生物学研究，例如作用机制（MOA）研究（包括脱靶），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NP作为促进化学遗传学的细胞途径的激活剂/灭活剂的丰富潜力。该团队包括一位合成有机化学家（D。Romo），分析化学家（G。Vigh），生物有机/生物合成化学家（C。Watanabe）和生物化学家/分子生物学家（J. Liu，Johns霍普金斯）。我们建议进一步开发一套化学和位点选择性反应，采用双功能试剂，以获得NP衍生物，也是有用的细胞探针。这些研究将有助于从根本上理解复杂纳米粒子背景下各种反应的化学和位点选择性。我们已经与合成、生物合成和分离化学家建立了合作关系，他们已经提供或同意提供约25种生物活性纳米颗粒用于拟议的研究。主要研究的反应是Rh（II）促进的O-H插入反应，C-H胺化和烯烃氮杂环丙烷与金属氮杂环戊烯的反应，以及温和的芳基卤代反应。在初始位点非选择性步骤之后，我们将采用稳健/通用的NP衍生物纯化方法，重新测定所得纯衍生物（SAR研究），并确定用于标签附着的最合适位点，即找到保留最大生物活性的衍生物。我们还将在各种细胞试验中筛选NP衍生物的潜在新生物活性，并将数量提供给分子库小分子库。初始反应的化学和位点选择性版本的后续开发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）和半制备型HPLC纯化的稳健方法，以通过一种类型的“双重不对称合成”来调节位点选择性。" 公共卫生相关性：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灭活剂的丰富潜力。天然产物作为鉴定人类疾病的新治疗靶点的工具和作为药物开发的先导结构具有丰富的历史。这些研究的结果将与人类疾病（包括糖尿病、炎症和癌症）直接相关，因为提出了与这些疾病相关的天然产物衍生化的新方法（本提案）。将进行后续SAR研究（本提案），以确定潜在的新生物活性和探针连接的合适位点，从而能够进行与这些疾病相关的后续作用模式研究（不在本提案中）。

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.