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

磷酸化生物分子在人体生理、健康和医学中发挥着重要的作用。生物焦油- 磷酸化的GET包括核苷、脂类、氨基酸、多肽和蛋白质。它已被发现 最近，蛋白质的多聚磷酸化是一种重要的翻译后修饰，这促使了研究人员 合成含有特定长度的寡磷酸链的化学探针，作为探索 被称为人类息肉。这一发展暴露了对定义明确的化学试剂的需求 使所需长度的磷酸链能够连接到感兴趣的有机分子上。最近我们 报道了第一个定义良好的结晶试剂，用于C，N和O的三磷化亲核试剂。这 是通过使用现代多肽偶联剂活化三偏磷酸盐而获得的，现在我们建议 扩展方法学以提供用于C，N和O核的四、五和六-磷酸化的新试剂-- 这是我们称之为I类试剂家族的有机分子寡磷酸化试剂。我们也 建议开发由I类家族通过寡磷酸化衍生的II类试剂家族 经典的Wittig试剂，H_2CPPh3。II类试剂系列为使该试剂成为可能提供了条件。 低聚磷与所需含醛有机分子之间通过Wittig反应连接； 在这种情况下，这样得到的构造物将在新的烯烃连接附近包含一个不可水解的P-C键 在低聚磷和有机底物之间。当使用I类或II类试剂制作 在有机分子和低聚磷酸链之间的连接，最初形成的产物将包含 完整的环磷酸盐残留物。我们建议对这些中间体进行分离和表征。其中一些将是 在生理条件下稳定，将作为进一步研究的目标。我们要研究的是开环问题。 由各种亲核试剂形成的环状中间体；使用氢氧化物将只得到末端的磷酸基团 寡磷酸链的末端，而其他亲核试剂预计会产生可能 在线状低聚磷酸链的两端都含有与两个不同有机残基的连接。在协作中- 与Raines小组(麻省理工学院化学)合作，我们建议对牛进行合作生化研究 寡磷酸盐及其有机分子结合物对胰腺核糖核酸酶A的抑制作用。小的 分子核糖核酸酶抑制剂是RNA研究的宝贵生化工具，而RNA研究的成功往往依赖于此 关于关闭所有的核糖核溶解活动。此外，在与Raines的合作中，我们建议通过蛋白质绘制 核糖核酸酶A活性部位低聚磷酸盐及其有机结合物的结合方式 协助改进的抑制剂的迭代设计。

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.