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，并与多种生物中的多种蛋白质相互作用，并且由于结构同源性，也适用于人类。本提案旨在开发一个试剂和反应工具箱，使天然产物（NPs）的同时武装和结构活性研究（SAR）成为可能。SAR研究可以确定NP衍生物的潜在新活性，也将确保初始生物活性不会丧失。考虑到该RFA的范围，虽然不是该资助的特定目标，但武装方面导致NP衍生物通过Sharpless-Huisgen环添加，可用于随后与各种标签结合，从而实现诸如作用机制（MOA）研究（包括脱靶）等生物学研究，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/灭活剂的丰富潜力。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套使用双功能试剂的化学和位点选择反应，以获得同样有用的细胞探针的NP衍生物。这些研究将有助于对复杂NPs背景下各种反应的化学和位点选择性的基本理解。我们已经与合成、生物合成和分离化学家建立了合作关系，他们已经或同意为拟议的研究提供约25个生物活性NPs。研究的主要反应是Rh（II）促进的O-H插入反应，C-H胺化和烯烃与金属类氮酰胺的叠氮化反应，以及轻度芳基卤化反应。在初始的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，重新测定得到的纯衍生物（SAR研究），并确定最合适的标签附着位点，即找到保留最大生物活性的衍生物。我们还将在各种细胞分析中筛选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发展将依赖于筛选各种手性金属配合物，以及反应监测（LC-MSn）和半制备型HPLC纯化的稳健方法，通过一种“双不对称合成”来调节位点选择性。

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.