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Applications of Para-Hydrogen Polarization to Biological NMR
Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a popular analytic technique in chemistry despite its intrinsic, low sensitivity. Nuclear spin hyperpolarization methods can improve NMR signals by several orders of magnitude and alleviate this disadvantage. Para-hydrogen induced polarization (PHIP) methods can be implemented with simple, economic equipment to create a large spin polarization in seconds.
Signal amplification by reversible exchange (SABRE) produces renewable signal enhancements without the structural modification of substances. SABRE requires polarization transfer catalysts (PTC) that can reversibly bind to ligands and parahydrogen simultaneously, and SABRE efficiencies strongly depend on ligand exchange rates. Biological compounds such as dihydrofolate reductase inhibitors cannot bind to typical PTC well. A series of iridium catalysts are designed with bidentate nitrogen-heterocyclic carbene ligands that can be tuned for both steric hindrance and electron donation properties. These tunable designs are used to optimize ligand exchange and achieve higher SABRE efficiency.
The SABRE technique achieves high efficiency in organic solvents that limit biological applications. Reverse micelles (RM) consist of hydrocarbon bulks and nano-size water droplets embedded in surfactants. RM systems can incorporate hydrophobic compounds including catalysts, ligands, and parahydrogen in their organic phases and encapsulate proteins in their aqueous phases. A nonhydrogenative-PHIP technique is proposed to determine protein-ligand interactions in the micromolar range, and a thiaminase-II-ligand dissociation constant (KD) is accurately determined.
A liquid-injection setup is implemented to deliver and mix the solutions of proteins and ligands for biological applications. It enables ligand hyperpolarization in optimal SABRE conditions before injection, solvent-composition regulation, and concentration adjustment. Low-field NMR spectroscopy, in the millitesla range, has been used for many applications including material characterization, flow measurement, and imaging. Here, it demonstrates the selectivity of hyperpolarized signals and limits other NMR signal interferences. A ^19F signal of a trypsin ligand is enhanced 400-fold, which enables ligand detection in the low field with similar concentrations for the high field. The R2 relaxation rates of this ligand are measured in different mixtures with proteins and competitors and analyzed to accurately determine the KD of competitors. These R2 measurements also benefit from the absence of exchange broadening that is caused by PTC-bound ligands.
Subject
NMRPHIP
SABRE
Para-hydrogen
Protein-Ligand Interaction
Organometallic Catalysts
Low-field NMR.
Citation
Pham, Pierce Phuc (2023). Applications of Para-Hydrogen Polarization to Biological NMR. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /198855.