Measuring Protein-Ligand Binding by Hyperpolarized Ultrafast NMR.

Protein-ligand interactions can be detected by observing changes in the transverse relaxation rates of the ligand upon binding. The ultrafast NMR technique, which correlates the chemical shift with the transverse relaxation rate, allows for the simultaneous acquisition of R2 for carbon spins at different positions. In combination with dissolution dynamic nuclear polarization (D-DNP), where the signal intensity is enhanced by thousands of times, the R2 values of several carbon signals from unlabeled benzylamine are observable within a single scan. The hyperpolarized ultrafast chemical shift-R2 correlated experiment separates chemical shift encoding from the readout phase in the NMR pulse sequence, which allows it to beat the fundamental limit on the spectral resolution otherwise imposed by the sampling theorem. Applications enabled by the ability to measure multiple relaxation rates in a single scan include the study of structural properties of protein-ligand interactions.

R2 Relaxometry of SABRE-Hyperpolarized Substrates at a Low Magnetic Field.

Nuclear magnetic resonance (NMR) relaxometry at a low magnetic field, in the milli-Tesla range or less, is enabled by signal enhancements through hyperpolarization. The parahydrogen-based method of signal amplification by reversible exchange (SABRE) provides large signals in a dilute liquid for the measurement of R2 relaxation using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment. A comparison of relaxation rates obtained at high and low fields indicates that an otherwise dominant contribution from chemical exchange is excluded in this low-field range. The SABRE process itself is based on exchange between the free and polarization transfer catalyst-bound forms of the substrate. At a high magnetic field of 9.4 T, typical conditions for producing hyperpolarization including 5 mM 5-fluoropyridine-3-carboximidamide as a substrate and 0.5 mM chloro(1,5-cyclooctadiene)[4,5-dimethyl-1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]iridium(I) as a polarization transfer catalyst precursor resulted in an R2 relaxation rate as high as 3.38 s-1. This relaxation was reduced to 1.19 s-1 at 0.85 mT. A quantitative analysis of relaxation rates and line shapes indicates that milli-Tesla or lower magnetic fields are required to eliminate the exchange contribution. At this magnetic field strength, R2 relaxation rates are indicative primarily of molecular properties. R2 relaxometry may be used for investigating molecular interactions and dynamics. The SABRE hyperpolarization, which provides signal enhancements without requiring a high magnetic field or large instrumentation, is ideally suited to enable these applications.

Biomolecular interactions studied by low-field NMR using SABRE hyperpolarization.

We demonstrate that low-field nuclear magnetic resonance provides a means for measuring biomacromolecular interactions without requiring a superconducting, or even a permanent magnet. A small molecule, 5-fluoropyridine-3-carboximidamide, is designed to be a specific ligand for the trypsin protein, while containing a fluorine atom as a nuclear spin hyperpolarizable label. With hyperpolarization by the parahydrogen based signal amplification by the reversible exchange method, fluorine NMR signals are detectable in the measurement field of 0.85 mT of an electromagnet, at a concentration of less than 100 µM. As a weak ligand for the protein, the hyperpolarized molecule can serve as a reporter for measuring the binding of other ligands of interest, illustrated by the determination of the dissociation constant KD of benzamidine from changes in the observed R2 relaxation rates. A signal enhancement of more than 106 compared to Boltzmann polarization at the measurement field indicates that this experiment is not feasible without prepolarization. The extended magnetic field range for the measurement of biomolecular interactions under near physiological conditions, with a protein concentration on the order of 10 µM or less, provides a new option for screening of ligand binding, measurement of protein-protein interactions, and measurement of molecular dynamics.

Analysis of Large Data Sets in a Physical Chemistry Laboratory NMR Experiment Using Python.

We describe an update to an experiment demonstrating low-field NMR spectroscopy in the undergraduate physical chemistry laboratory. A Python-based data processing and analysis protocol is developed for this experiment. The Python language is used in fillable worksheets in the notebook software JupyterLab, providing an interactive means for students to work with the measured data step by step. The protocol teaches methods for the analysis of large data sets in science or engineering, a topic that is absent from traditional chemistry curricula. Python is among the most widely used modern tools for data analysis. In addition, its open-source nature reduces the barriers for adoption in an educational laboratory.

Screening of Protein-Ligand Binding Using a SABRE Hyperpolarized Reporter.

Hyperpolarization through signal amplification by reversible exchange (SABRE) provides a facile means to enhance nuclear magnetic resonance (NMR) signals of small molecules containing an N-heterocycle or other binding site for a polarization transfer catalyst. A purpose-designed reporter ligand, which is capable of binding both to a target protein and to the catalyst, makes the sensitivity enhancement by this technique compatible with the measurement of a range of biomolecular interactions. The 1H polarization of the reporter ligand 4-amidinopyridine, which is targeting trypsin, is used to screen ligands that are not themselves hyperpolarizable by SABRE. The respective protein-ligand dissociation constants (KD) are determined by an observed change in the R2 relaxation rate of the reporter. A calculation of expected signal changes indicates that the accessible ligand KD values extend over several orders of magnitude, while the concentrations of target proteins and ligands can be reduced considering the sensitivity gains from hyperpolarization. In general, the design of a single, weakly binding ligand for a target protein enables the use of SABRE hyperpolarization for ligand screening or other biophysical studies involving macromolecular interactions.

Hyperpolarized water as universal sensitivity booster in biomolecular NMR.

NMR spectroscopy is the only method to access the structural dynamics of biomolecules at high (atomistic) resolution in their native solution state. However, this method's low sensitivity has two important consequences: (i) typically experiments have to be performed at high concentrations that increase sensitivity but are not physiological, and (ii) signals have to be accumulated over long periods, complicating the determination of interaction kinetics on the order of seconds and impeding studies of unstable systems. Both limitations are of equal, fundamental relevance: non-native conditions are of limited pharmacological relevance, and the function of proteins, enzymes and nucleic acids often relies on their interaction kinetics. To overcome these limitations, we have developed applications that involve 'hyperpolarized water' to boost signal intensities in NMR of proteins and nucleic acids. The technique includes four stages: (i) preparation of the biomolecule in partially deuterated buffers, (ii) preparation of 'hyperpolarized' water featuring enhanced 1H NMR signals via cryogenic dynamic nuclear polarization, (iii) sudden melting of the cryogenic pellet and dissolution of the protein or nucleic acid in the hyperpolarized water (enabling spontaneous exchanges of protons between water and target) and (iv) recording signal-amplified NMR spectra targeting either labile 1H or neighboring 15N/13C nuclei in the biomolecule. Water in the ensuing experiments is used as a universal 'hyperpolarization' agent, rendering the approach versatile and applicable to any biomolecule possessing labile hydrogens. Thus, questions can be addressed, ranging from protein and RNA folding problems to resolving structure-function relationships of intrinsically disordered proteins to investigating membrane interactions.

Interfacing Liquid State Hyperpolarization Methods with NMR Instrumentation.

Advances in liquid state hyperpolarization methods have enabled new applications of high-resolution NMR spectroscopy. Utilizing strong signal enhancements from hyperpolarization allows performing NMR spectroscopy at low concentration, or with high time resolution. Making use of the high, but rapidly decaying hyperpolarization in the liquid state requires new techniques to interface hyperpolarization equipment with liquid state NMR spectrometers. This article highlights rapid injection, high resolution NMR spectroscopy with hyperpolarization produced by the techniques of dissolution dynamic nuclear polarization (D-DNP) and para-hydrogen induced polarization (PHIP). These are popular, albeit not the only methods to produce high polarization levels for liquid samples. Gas and liquid driven sample injection techniques are compatible with both of these hyperpolarization methods. The rapid sample injection techniques are combined with adapted NMR experiments working in a single, or small number of scans. They expand the application of liquid state hyperpolarization to spins with comparably short relaxation times, provide enhanced control over sample conditions, and allow for mixing experiments to study reactions in real time.

Detection of Protein-Ligand Interactions by 19F Nuclear Magnetic Resonance Using Hyperpolarized Water.

The transfer of nuclear spin hyperpolarization from water to ligand 19F spins results in a transient signal change that is indicative of protein-ligand interaction. The 19F nucleus allows for background-free detection of these signals, which are modulated by polarization transfer via pathways similar to those in a hyperpolarized 1H water LOGSY experiment. Quantification of the apparent heteronuclear cross-relaxation rates is facilitated by a simultaneous dual-channel detection of 1H and 19F signals. Calculated cross-relaxation rates for the 1H-19F transfer step indicate that these rates are sensitive to binding to medium- and large-sized proteins. The heteronuclear observation of hyperpolarization transfer from water may be used to screen protein-ligand interactions in drug discovery and other applications.

Time- and site-resolved kinetic NMR for real-time monitoring of off-equilibrium reactions by 2D spectrotemporal correlations.

Nuclear magnetic resonance (NMR) spectroscopy provides detailed information about dynamic processes through line-shape changes, which are traditionally limited to equilibrium conditions. However, a wealth of information is available by studying chemical reactions under off-equilibrium conditions-e.g., in states that arise upon mixing reactants that subsequently undergo chemical changes-and in monitoring the reactants and products in real time. Herein, we propose and demonstrate a time-resolved kinetic NMR experiment that combines rapid mixing techniques, continuous flow, and single-scan spectroscopic imaging methods, leading in unison to a 2D spectrotemporal NMR correlation that provides high-quality kinetic information of off-equilibrium chemical reactions. These kinetic 2D NMR spectra possess a high-resolution spectral dimension revealing the individual chemical sites, correlated with a time-independent, steady-state spatial axis that delivers information concerning temporal changes along the reaction coordinate. A comprehensive description of the kinetic, spectroscopic, and experimental features associated with these spectrotemporal NMR analyses is presented. Experimental demonstrations are carried out using an enzymatically catalyzed reaction leading to site- and time-resolved kinetic NMR data, that are in excellent agreement with control experiments and literature values.

Characterization of protein-ligand interactions by SABRE.

Nuclear spin hyperpolarization through signal amplification by reversible exchange (SABRE), the non-hydrogenative version of para-hydrogen induced polarization, is demonstrated to enhance sensitivity for the detection of biomacromolecular interactions. A target ligand for the enzyme trypsin includes the binding motif for the protein, and at a distant location a heterocyclic nitrogen atom for interacting with a SABRE polarization transfer catalyst. This molecule, 4-amidinopyridine, is hyperpolarized with 50% para-hydrogen to yield enhancement values ranging from -87 and -34 in the ortho and meta positions of the heterocyclic nitrogen, to -230 and -110, for different solution conditions. Ligand binding is identified by flow-NMR, in a two-step process that separately optimizes the polarization transfer in methanol while detecting the interaction in a predominantly aqueous medium. A single scan Carr-Purcell-Meiboom-Gill (CPMG) experiment identifies binding by the change in R 2 relaxation rate. The SABRE hyperpolarization technique provides a cost effective means to enhance NMR of biological systems, for the identification of protein-ligand interactions and other applications.

Application of Relaxation Dispersion of Hyperpolarized 13 C Spins to Protein-Ligand Binding.

Nuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) provided a 13 C signal enhancement between 3000-6000 for the ligand 4-(trifluoromethyl) benzene-1-carboximidamide binding to trypsin. The measurement of 13 C R2 relaxation dispersion was enabled without isotope enrichment, using a series of single-scan Carr-Purcell-Meiboom-Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes.

Polyolefin catalysis of propene, 1-butene and isobutene monitored using hyperpolarized NMR.

Polymerization reactions of the dissolved gases propene, 1-butene, and isobutene catalyzed by [Zr(Cp)2Me][B(C6F5)4] were characterized using in situ NMR. Hyperpolarization of 13C spins by the dissolution dynamic nuclear polarization (DNP) technique provided a signal enhancement of up to 5000-fold for these monomers. For DNP hyperpolarization, liquid aliquots containing monomers were prepared at a temperature between the freezing point of the solvent toluene and the boiling point of the monomer, mixed with the polarizing agent α,γ-bis-diphenylene-ß-phenylallyl free radical, and subsequently frozen. The hyperpolarized signals after dissolution enabled the observation of reaction kinetics, as well as polymer products and side products within a time of 30 s from the start of the reaction. The observed kinetic rate constants for polymerization followed a decreasing trend for propene, 1-butene, and isobutene, with the lowest rate constant for the latter explained by steric bulk. For all reactions, partial deactivation was further observed during the measurement time. The line shape and the chemical shift of the monomer signals with respect to a toluene signal were both dependent on catalyst concentration and reaction time, with the strongest dependence observed for isobutene. These changes are consistent with the characteristics of a rapid binding and unbinding process of the monomer to the catalyst occurring during the reaction.

2D NMR spectroscopy of refolding RNase Sa using polarization transfer from hyperpolarized water.

Polarization transfer from hyperpolarized water through proton exchange is used to enhance the NMR signals of amide protons of the Ribonuclease Sa protein. Spectra of the refolding protein are measured within 6 s after dilution of the denaturant urea, at urea-dependent folding rates adjusted in the range of 0.3-0.8 s-1. Peak patterns including a mixture of folded and unfolded protein at different ratios are observed. The changes in the observed signals indicate that each spectrum accesses a different point in the partial completion of the folding. A comparison to simulated 2D NMR spectra suggests a lower polarization transfer efficiency from water when the protein folds slowly, which may result from the molecular motions in the unfolded protein and the absence of long-range contacts. The ability to acquire 2D NMR spectra under different refolding conditions may open a new avenue for residue specific characterization of the folding process.

Dynamic Nuclear Polarization Enhanced Nuclear Spin Optical Rotation.

Nuclear spin optical rotation (NSOR) has been investigated as a magneto-optical effect, which holds the potential for applications, including hybrid optical-nuclear magnetic resonance (NMR) spectroscopy and gradientless imaging. The intrinsic nature of NSOR renders its detection relatively insensitive, which has prevented it moving from a proof of concept to a method supporting chemical characterizations. In this work, the dissolution dynamic nuclear polarization technique is introduced to provide nuclear spin polarization, increasing the signal-to-noise ratio by several thousand times. NSOR signals of 1 H and 19 F nuclei are observed in a single scan for diluted compounds, which has made this effect suitable for the determination of electronic transitions from a specific nucleus in a large molecule.

Dynamic Nuclear Polarization Using 3D Aromatic Boron Cluster Radicals.

A set of two dodecaborate [B12(OR)12]1- radical cluster anions containing a dense layer of fluorinated end-groups provides nuclear spin hyperpolarization via the dissolution dynamic nuclear polarization (D-DNP) technique. We show that these clusters can enhance 19F nuclear magnetic resonance (NMR) signals. Importantly, given the inherent radical delocalization in dodecaborate-based clusters, these species are compatible with reactive compounds such as Lewis acids, providing ∼1000-2000 times of signal enhancement for B(C6F5)3 in liquid state NMR spectroscopy experiments at 9.4 Tesla. This observation suggests that 3D aromatic radicals can provide advantages over the conventional radical species that are currently used for DNP such as 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) by showing superior chemical compatibility. The ability to hyperpolarize reactive compounds using [B12(OR)12]1- cluster radicals opens up new applications of reaction monitoring by D-DNP NMR spectroscopy, including the observation of catalytically active species in complex reaction mixtures.

Tunable iridium catalyst designs with bidentate N-heterocyclic carbene ligands for SABRE hyperpolarization of sterically hindered substrates.

A series of bidentate N-heterocyclic carbene (NHC) iridium catalysts, [Ir(κC,N-NHC)H2L2]BPh4, are proposed for SABRE hyperpolarization. The steric and electronic properties of the NHCs are used to tune substrate affinity and thereby SABRE efficiency. The sterically hindered substrates 2,4-diaminopyrimidine and trimethoprim yielded maximum proton NMR signal enhancements of ∼300-fold and ∼150-fold, respectively.

Indirect detection of intermediate in decarboxylation reaction of phenylglyoxylic acid by hyperpolarized 13C NMR.

The decarboxylation reaction of phenylglyoxylic acid with hydrogen peroxide is studied by real-time hyperpolarized carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy at room temperature. A non-observable reaction intermediate is identified using blind selective saturation pulses in the expected chemical shift range, thereby revealing information on the reaction mechanism.

Determination of protein-ligand binding modes using fast multi-dimensional NMR with hyperpolarization.

Elucidation of small molecule-protein interactions provides essential information for understanding biological processes such as cellular signaling, as well as for rational drug development. Here, multi-dimensional NMR with sensitivity enhancement by dissolution dynamic nuclear polarization (D-DNP) is shown to allow the determination of the binding epitope of folic acid when complexed with the target dihydrofolate reductase. Protein signals are selectively enhanced by polarization transfer from the hyperpolarized ligand. A pseudo three-dimensional data acquisition with ligand-side Hadamard encoding results in protein-side [13C, 1H] chemical shift correlations that contain intermolecular nuclear Overhauser effect (NOE) information. A scoring function based on this data is used to select pre-docked ligand poses. The top five poses are within 0.76 Å root-mean-square deviation from a reference structure for the encoded five protons, showing improvements compared with the poses selected by an energy-based scoring function without experimental inputs. The sensitivity enhancement provided by the D-DNP combined with multi-dimensional NMR increases the speed and potentially the selectivity of structure elucidation of ligand binding epitopes.

Amplification of Nuclear Overhauser Effect Signals by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins.

Immobilization of a target protein enhances the cross-relaxation rates for transfer of nuclear spin polarization but reduces the accessible target concentration. Hyperpolarization of ligand spins by dissolution dynamic nuclear polarization (D-DNP) is shown to increase sensitivity for observing the intraligand nuclear Overhauser effect (NOE). This effect, also known as the transferred NOE (trNOE), can be used for detection of binding and for obtaining binding-related structural information. The measurement of hyperpolarized trNOE signals is demonstrated for the ligand 4'-hydroxyazobenzene-2-carboxylic acid interacting with avidin protein immobilized on polystyrene beads. In a sample containing 63.5 µM ligands and 0.83 µM accessible protein binding sites, the signal enhancement provided by D-DNP leads to single-scan detection of the NOE buildup, despite that this signal peaks at only 2% of the total ligand signal. These buildup curves allow the confirmation of binding through a change in the sign of the NOE and the quantitative determination of cross-relaxation rates. The combination of the D-DNP technique and protein immobilization may facilitate the identification of intraligand NOEs in ligand screening for drug discovery. The same method may be applied to in vivo characterization of ligand interactions with cell surface proteins.

Nuclear Spin Hyperpolarization of NH2 - and CH3 -Substituted Pyridine and Pyrimidine Moieties by SABRE.

Hyperpolarization of N-heterocycles with signal amplification by reversible exchange (SABRE) induces NMR sensitivity gains for biological molecules. Substitutions with functional groups, in particular in the ortho-position of the heterocycle, however, result in low polarization using a typical Ir catalyst with a bis-mesityl N-heterocyclic carbene ligand for SABRE, presumably due to steric hindrance. With the addition of allylamine or acetonitrile as coligands to the precatalyst chloro(1,5-cyclooctadiene)[4,5-dimethyl-1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene] iridium, the 1 H signal enhancement increased in several substrates with ortho NH2 substitutions. For example, for a proton in 2,4-diaminopyrimidine, the enhancement factors increased from -7±1 to -210±20 with allylamine or to -160±10 with acetonitrile. CH3 substituted molecules yielded maximum signal enhancements of -25±7 with acetonitrile addition, which is considerably less than the corresponding NH2 substituted molecules, despite exhibiting similar steric size. With the more electron-donating NH2 substitution resulting in greater enhancement, it is concluded that steric hindrance is not the only dominant factor in determining the polarizability of the CH3 substituted compounds. The addition of allylamine increased the signal enhancement for the 290 Da trimethoprim, a molecule with a 2,4-diaminopyrimidine moiety serving as an antibacterial agent, to -70.