Simulations and Novel Catalytic Schemes for Sensitivity Enhancement in Biomedical Applications of Magnetic Resonance by Para-Hydrogen Induced Nuclear Polarization

对氢激发极化(PhIP)是一种非常有效的超极化技术，可用于核磁共振波谱和核磁共振断层扫描的信号增强。在之前的资助期间，我们开发了一种高效的多肽PhIP标记策略，基于带有不饱和烷基侧链的非天然氨基酸。将它们加入到寡肽中，如向日葵胰蛋白酶抑制剂SFTI-1，它们在保持其生物活性的同时，给出了约1000个数量级的非常强的信号增强。我们第二个项目阶段的主要目标是开发更多的PhIP标记，用于多肽和更大的抑制剂，如结蛋白或粘附体的1H-或13C-核磁共振应用。为此，还将合成其他适合固相合成肽的含烷基侧链的非天然氨基酸。由于超极化的寿命由其弛豫时间决定，对于许多应用来说是至关重要的，我们将根据需要使用选择性的13C标记和烷基的选择性氢化来加宽PhIP的动力学窗口。对小模型多肽的测试实验将确定该标记物的PhIP效率。然后，有效的标记被加入到更大的多肽中，最后，最好的多肽被加入上述抑制剂中。最终目的是找出PhIP增强的核磁共振是否可以确定这些抑制剂及其相关酶的结合常数和动力学。为此，将对现有的实验装置进行修改，以允许在通过PhIP超极化抑制剂后注入酶。同时，我们希望继续寻找有效的RNA适配子的PhIP标记，在小模型上对它们进行评估，并将它们应用于黄素单核苷酸(FMN)作为一个有趣的靶向系统。

Parahydrogen Induced Polarization (PHIP) is a very efficient hyperpolarization technique for the signal-enhancement of NMR spectroscopy and MRI tomography. In the previous funding period, we developed an efficient PHIP labeling strategy for peptides, based on non-natural amino acids with unsaturated alkyl side chains. Incorporated into oligo-peptides, such as the sun-flower trypsin inhibitor SFTI-1, they gave very strong signal enhancements on the order of 1000, while retaining their bioactivity. Our main goal for the second project period is the development of additional PHIP-labels for 1H- or 13C-NMR applications to peptides and larger inhibitors such as knottins or affibodies. To this end, other non-natural amino acids with alkyl-bearing side chains suitable for solid-phase peptide synthesis will be synthesized. Since the lifetime of hyperpolarization, determined by its relaxation times, is crucial for many applications, we will employ as necessary selective 13C-labeling and selective deuteration of the alkyl groups to widen the kinetic window of PHIP. Test experiments on small model peptides will determine the marker's PHIP efficiency. The efficient markers are then incorporated into larger peptides and finally, the best ones, into the above-mentioned inhibitors. The ultimate goal is to find out, whether PHIP-enhanced NMR allows determination of the binding constants and kinetics of these inhibitors and their associated enzymes. For this purpose, the existing experimental setup will be modified so that it allows the injection of the enzyme after hyperpolarization of the inhibitors by PHIP. In parallel, we would like to continue the search for efficient PHIP labels for RNA aptamers, evaluate them on small models and apply them to the flavin mononucleotide (FMN) as an interesting targeting system.