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

仲氢诱导极化（PHIP）是一种非常有效的超极化技术，用于核磁共振波谱和核磁共振成像的信号增强。在上一个资助期，我们开发了一种基于具有不饱和烷基侧链的非天然氨基酸的肽的高效PHIP标记策略。将它们并入寡肽中，例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非常强的信号增强，大约为1000。我们在第二个项目期间的主要目标是开发额外的PHIP标签，用于肽和较大的抑制剂（如knottins或bodies）的1H或13 C-NMR应用。为此，将合成适合于固相肽合成的具有含烷基侧链的其它非天然氨基酸。 由于超极化的寿命，由其弛豫时间，是至关重要的许多应用，我们将采用必要的选择性13 C-标记和选择性氘代的烷基基团，以扩大动力学窗口的PHIP。对小模型肽的测试实验将确定标记物的PHIP效率。然后将有效的标记物掺入较大的肽中，最后将最好的标记物掺入上述抑制剂中。最终的目标是找出，PHIP增强NMR是否允许确定这些抑制剂及其相关酶的结合常数和动力学。为此目的，将修改现有的实验设置，使其允许在通过PHIP超极化抑制剂后注射酶。与此同时，我们希望继续寻找RNA适体的有效PHIP标签，在小模型上对其进行评估，并将其应用于黄素单核苷酸（FMN）作为一种有趣的靶向系统。

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.