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

Subject Area Analytical Chemistry
Term from 2014 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 247467975
 
Final Report Year 2024

Final Report Abstract

The objective of the project was the development and implementation of an efficient Parahydrogen Induced Polarization (PHIP) labeling strategy for bioactive peptides, based on nonnatural amino acids with unsaturated alkyl side chains. In the first period of the project, we had shown that non-natural amino-acids, such as propargyl-tyrosine, incorporated into oligo-peptides, yield very strong signal enhancements on the order of 1000, while retaining their bioactivity. Our main goal for the second project period was the development of additional PHIP-labels for 1H- or 13 C-NMR applications and to find out whether PHIP-enhanced NMR allows the investigation of such large inhibitors while preserving their bioactivity and thus permit the determination of the binding constants and kinetics of these inhibitors and their associated enzymes. In order to develop a universal PHIP labelling strategy for disulfide bridge containing peptides which preserves their bioactivity and tertiary structures, we studied the PHIP labeling of the antiplatelet aggregation inhibitor eptifibatide (Integrilin), a cyclic heptapeptide derived from a protein found in the venom of rattlesnakes. By labelling the disulfide bridge, we synthesized an eptifibatide derivate which yielded 1H-PHIP-NMR enhancements over 1000. Next, we studied the PHIP labelling of octreotide, a somatostatin analogue employed in the diagnostics and treatment of various types of cancer. Introduction of L-propargyl tyrosine as a PHIP marker at different positions of octreotide by manual Solid-Phase Peptide Synthesis (SPPS) led to up to 2000-fold proton signal enhancement (SE). Cell binding studies confirmed that all octreotide variants retained their bioactivity. The application of PHIP to larger biomolecules or polymers is still a mainly unexplored area. A particular challenge is the presence of folding-essential disulfide bridges. To study the feasibility of PHIP on these larger systems, we introduced the L-propargyl tyrosine label at different positions in three synthetic open-chain variants of the miniprotein MCoTI-II. All three variants expressed high reactivity upon the PHIP experiments in the organic solvent MeOD-d4 yielding SEs of up to 964 for the reduced variant MP-5ox and 498.6 for the folded miniprotein. To the best of our knowledge, we were the first to apply hydrogenative PHIP to bioactive peptidic molecules of the size of 29 amino acids. The second major challenge in larger systems is the size of the molecule itself, which reduces the rotational correlation times and causes line broadening and potential cancellation of the typical anti-phase signals of PHIP-NMR. To explore this, we studied PHIP hyperpolarization of the synthetic 236 kDa biopolymer poly-γ- (4-propargyloxy)-benzyl-L-glutamate) (PPOBLG). The hydrogenation with parahydrogen caused signal enhancements of 800 and more for the vinyl protons of the side chain at low substrate concentration. In addition to the main project, we worked on PHIP of fumarate as possible tumor marker for in-vivo MRI applications, SABRE hyperpolarization of an oligopeptide, and finished our work on heterogeneous catalysts for PHIP experiments.

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