Reagents for Chemical Oligophosphorylation, Synthesis of Oligophosphate-Organic Molecule Conjugates, and Biochemical Studies

用于化学低磷酸化、低磷酸盐-有机分子缀合物的合成以及生化研究的试剂

基本信息

批准号：
10551903
负责人：
CHRISTOPHER C CUMMINS
金额：
$ 29.17万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-01-31
项目状态：
未结题

项目摘要

Phosphorylated biomolecules play essential roles in human physiology, health, and medicine. Biological tar- gets for phosphorylation include nucleosides, lipids, amino acids, peptides, and proteins. It has been discovered recently that polyphosphorylation of proteins is an important post-translational modification, spurring researchers to synthesize chemical probes containing oligophosphate chains of specific lengths as tools to explore what has been termed the human polyP-ome. This development exposes the need for well-defined chemical reagents to enable phosphate chains of a desired length to be conjugated to an organic molecule of interest. Recently we reported the first well defined, crystalline reagent for the triphosphorylation of C, N, and O nucleophiles. This was obtained by activation of trimetaphosphate using a modern peptide coupling reagent, and now we propose to extend the methodology to afford new reagents for tetra-, penta-, and hexa-phosphorylation of C, N, and O nucle- ophiles; this is what we term our class I family of reagents for oligophosphorylation of organic molecules. We also propose to develop a class II family of reagents that is derived from the class I family by oligophosphorylation of the classic Wittig reagent, H2CPPh3. The class II family of reagents opens up the possibility to make the con- nection between an oligophosphate and a desired aldehyde-containing organic molecule via the Wittig reaction; in this case the constructs so obtained will contain a non-hydrolyzable P–C bond next to the new olefinic junction between the oligophosphate and the organic substrate. When using either the class I or II reagents to make the connection between an organic molecule and an oligophosphate chain, the initially formed product will contain an intact cyclophosphate residue. We propose to isolate and characterize such intermediates. Some of these will be stable under physiological conditions and will be targeted for further study. We will study the ring-opening of the cyclic intermediates by a variety of nucleophiles; use of hydroxide will give simply a terminal phosphate group at the end of the oligophosphate chain, while other nucleophiles are expected to result in target constructs that may contain linkages to two different organic residues at either end of the linear oligophosphate chain. In collabora- tion with the Raines group (MIT Chemistry) we propose to undertake collaborative biochemical studies of bovine pancreatic ribonuclease A (RNase A) inhibition by oligophosphates and their organic-molecule conjugates. Small molecule ribonuclease inhibitors are valuable biochemical tools for studies of RNA for which success often relies on shutting down all ribonucleolytic activity. Also in collaboration with Raines we propose to map out via protein crystallography the binding mode for oligophosphates and their organic conjugates in the RNase A active site, to aid in the iterative design of improved inhibitors.

磷酸化的生物分子在人类生理，健康和医学中起重要作用。生物焦油获得磷酸化包括核苷，脂质，氨基酸，胡椒粉和蛋白质。它已经被发现最近，蛋白质的多磷酸化是一种重要的翻译后修饰，刺激了研究人员综合包含特定长度的寡磷酸盐链的化学问题，以探索什么工具被称为人类息肉。这种发展暴露了对明确定义的化学试剂的需求使所需长度的磷酸盐链与有机分子共轭。最近我们报道了第一个良好定义的Crystalline试剂，用于C，N和O核粉状的三磷酸化。是通过使用现代肽偶联试剂激活三磷酸来获得的，现在我们建议扩展了用于提供新试剂的方法，用于C，N和O核的四，五体和Hexa-磷酸化。 Ophiles;这就是我们认为有机分子寡磷酸化试剂的I类。我们也是提议开发II类试剂家族，该家族是通过寡磷酸化从I级家族衍生而来的经典的Wittig试剂H2CPH3。 II级试剂家族开辟了使该概念的可能性通过Wittig反应，寡磷酸盐和所需的含有醛的有机分子之间的缝合；在这种情况下，因此获得的构建体将在新的烯烃结合在寡磷酸盐和有机底物之间。当使用I类或II级试剂时有机分子与寡磷酸链之间的连接，最初形成的产品将包含一个完整的环磷酸住宅。我们建议分离并表征此类中间体。其中一些将是在生理条件下稳定，将被针对进一步研究。我们将研究开放的环各种核磷酸盐的循环中间体；使用氢氧化物将仅提供一个末端磷酸组寡磷酸酯链的末端，而其他核磷酸盐预计将导致目标结构在线性寡磷酸盐链的任一端都包含与两个不同的有机残基的联系。在合作中 - 我们建议与Raines Group（MIT化学）进行牛协作生化研究寡磷酸盐及其有机分子结合物抑制胰腺核糖核酸酶A（RNase A）。小的分子核糖核酸酶抑制剂是有价值的生化工具，用于研究成功的RNA 关闭所有核糖核解活性。另外，我们建议通过蛋白质进行绘制晶体学在RNase中的寡磷酸盐及其有机偶联物的结合模式在活性位点到有助于改善抑制剂的迭代设计。