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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、健康和医学中起着重要作用。生物焦油用于磷酸化的底物包括核苷、脂质、氨基酸、肽和蛋白质。已经发现最近，蛋白质的多磷酸化是一种重要的翻译后修饰，这促使研究人员合成含有特定长度寡磷酸链的化学探针，作为探索被称为人类polyP-ome。这一发展暴露了对定义明确的化学试剂的需求，使所需长度的磷酸盐链能够与感兴趣的有机分子共轭。最近我们报道了第一个定义明确的结晶试剂，用于C、N和O亲核试剂的三磷酸化。这通过使用现代肽偶联试剂活化三偏磷酸盐获得，现在我们提出扩展了该方法，以提供用于C、N和O核的四-、五-和六-磷酸化的新试剂，这是我们称之为用于有机分子的寡磷酸化的I类试剂家族。我们也建议开发一种II类试剂家族，其通过寡磷酸化衍生自I类家族经典的维蒂希试剂H2 CPPh 3 II类试剂家族开辟了制造这种试剂的可能性，通过Wittig反应使低聚磷酸酯与所需的含二氢呋喃的有机分子连接; 在这种情况下，如此获得的构建体将在新的烯键连接处附近含有不可水解的P-C键在低聚磷酸盐和有机基质之间。当使用I类或II类试剂制备当有机分子与低聚磷酸盐链之间存在连接时，最初形成的产物将含有完整的环磷酸盐残留物我们建议分离和表征这样的中间体。其中一些将在生理条件下稳定，并将作为进一步研究的目标。我们将研究环开放的通过各种亲核试剂形成环状中间体;使用氢氧化物将在寡磷酸链的末端，而其它亲核试剂预期产生靶构建体，在线性低聚磷酸酯链的任一末端含有与两个不同有机残基的连接。在合作中- 与Raines小组（麻省理工学院化学）合作，我们建议进行牛的生物化学研究。胰腺核糖核酸酶A（RNase A）抑制寡磷酸盐及其有机分子共轭物。小分子核糖核酸酶抑制剂是研究RNA的有价值的生物化学工具，RNA的成功通常依赖于停止所有的核糖核酸溶解活性与Raines合作，我们还提出通过蛋白质晶体学研究了寡磷酸盐及其有机缀合物在RNase A活性位点的结合模式，有助于改进的抑制剂的迭代设计。