Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins

Abstract

Chemical exchange phenomena are ubiquitous in macromolecules, which undergo conformational change or ligand complexation. NMR relaxation dispersion (RD) spectroscopy based on a Carr-Purcell-Meiboom-Gill pulse sequence is widely applied to identify the exchange and measure the life time of intermediate states on the millisecond time scale. Advances in hyperpolarization methods improve the applicability of NMR spectroscopy when rapid acquisitions or low concentrations are required, through an increase in signal strength by several orders of magnitude. Here, we demonstrate the measurement of chemical exchange from a single aliquot of a ligand hyperpolarized by dissolution dynamic nuclear polarization (D-DNP). Transverse relaxation rates are measured simultaneously at different pulsing delays by dual-channel 19F NMR spectroscopy. This two-point measurement is shown to allow the determination of the exchange term in the relaxation rate expression. For the ligand 4-(trifluoromethyl)benzene-1-carboximidamide binding to the protein trypsin, the exchange term is found to be equal within error limits in neutral and acidic environments from D-DNP NMR spectroscopy, corresponding to a pre-equilibrium of trypsin deprotonation. This finding illustrates the capability for determination of binding mechanisms using D-DNP RD. Taking advantage of hyperpolarization, the ligand concentration in the exchange measurements can reach on the order of tens of μM and protein concentration can be below 1 μM, i.e. conditions typically accessible in drug discovery.

Applications of dissolution dynamic nuclear polarization in chemistry and biochemistry

“…Relaxation dispersion permits the measurement of on and off rates of ligands, as well as the identification of binding mechanisms. Thereby, this technique can enhance the available information compared with basic screening experiments …”

Binding site exchange kinetics revealed through efficient spin–spin dephasing of hyperpolarized129Xe

“…Hyperpolarization ( via DNP) has been implemented for RD measurements in a previous study. 16 However, it mentioned the challenges of such single shot techniques: the non-self-renewing magnetization has to be used with care when multiple data points are required to isolate R 2,ex . Whereas a significant sensitivity enhancement could be achieved, the system was still in fast exchange and showed only moderate amplitudes of R 2,ex .…”

“…One of the systems studied herein even serves as efficient MRI contrast agent that actually surpasses the most efficiently known paramagnetic T 2,ex agents such as EuDOTA chelates 14 and natural d -glucose 15 by 2–3 orders of magnitude. Moreover, the accelerated spatial encoding techniques developed herein are also beneficial for other hyperpolarized agents generated via dynamic nuclear polarization (DNP) or parahydrogen induced polarization (PHIP) that could complement the minimalistic RD analysis with hp nuclei by Liu et al 16 by replacing multiple RF channels with multiple samples. It also paves the way for ultra-sensitive MRI with such tracers from various HP techniques.…”

Determination of binding affinities using hyperpolarized NMR with simultaneous 4-channel detection

“…Examples include NMR based titrations or relaxation dispersion measurements that require multiple pulsing delays. 28 Another approach to high-throughput D-DNP NMR has previously been described, which enables polarization of multiple different sample aliquots simultaneously and dissolving each sample independently. 13–16 The multiplexing method described here could also be combined with this approach to further increase the efficiency of D-DNP NMR experiments.…”