Nuclear Magnetic Resonance of Hyperpolarized Fluorine for Characterization of Protein–Ligand Interactions

Abstract

Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the (19)F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.

Optimizing dissolution dynamic nuclear polarization

“…However, several examples where d-DNP was used for high-gamma nuclear spins can be found in the literature. Fluorinated molecules are widely used in drug discovery for the detection of protein interactions, and 19 F d-DNP provides a way of dramatically improving the sensitivity [15]. With an enhancement in the order of 1000, the detection of sub-micromolar concentrations of fluorinated binders was demonstrated.…”

“…Although most peer-reviewed papers utilizing d-DNP are concerned with localized spectroscopy or imaging of tumours following the infusion of hyperpolarized 1-13 C pyruvate, the technology of d-DNP can in principle be used in combination with a variety of other NMR or MRI experiments, using an extensive diversity of molecules, and addressing many other nuclear spins. One can cite amongst many other publications in vitro or in-cell studies [13,14], drug screening [15], and monitoring of chemical reactions including the detection of intermediates [16]. Further d-DNP NMR applications can be found in reference [17].…”

Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization

“…However, for quantitative and rapid quenching, ascorbate must be used in excess, and the remaining ascorbate in solution may affect the analyte or sensitive components present in the NMR or MRI system, such as enzymes (5,27,28). Furthermore, the presence of potentially noninnocent additional products arising from the paramagnetic PAs is obviously undesirable for in vivo MRI experiments.…”

2′-O-[(4-CF3-triazol-1-yl)methyl] Uridine – A Sensitive 19F NMR Sensor for the Detection of RNA Secondary Structures

“…[22][23][24][25][26] It may also be worth noting that remarkable breakthroughs have been made in NMR of hyperpolarized fluorine (HPF NMR, several thousand fold enhanced sensitivity) in studies of protein-ligand interactions. 27 There is a likelihood that HPF NMR may soon find applications in oligonucleotides as well. 28 We and other research groups have focused on developing novel fluorine-labeled nucleoside derivatives, aimed at increasing the sensitivity and straightforwardness of 19 F NMR-detection of oligonucleotides.…”