Heteronuclear Cross‐Relaxation Effects in the NMR Spectroscopy of Hyperpolarized Targets

Dissolution dynamic nuclear polarization (DNP) enables high‐sensitivity solution‐phase NMR experiments on long‐lived nuclear spin species such as ¹⁵N and ¹³C. This report explores certain features arising in solution‐state ¹H NMR upon polarizing low‐γ nuclear species. Following solid‐state hyperpolarization of both ¹³C and ¹H, solution‐phase ¹H NMR experiments on dissolved samples revealed transient effects, whereby peaks arising from protons bonded to the naturally occurring ¹³C nuclei appeared larger than the typically dominant ¹²C‐bonded ¹H resonances. This enhancement of the satellite peaks was examined in detail with respect to a variety of mechanisms that could potentially explain this observation. Both two‐ and three‐spin phenomena active in the solid state could lead to this kind of effect; still, experimental observations revealed that the enhancement originates from ¹³C→¹H polarization‐transfer processes active in the liquid state. Kinetic equations based on modified heteronuclear cross‐relaxation models were examined, and found to well describe the distinct patterns of growth and decay shown by the ¹³C‐bound ¹H NMR satellite resonances. The dynamics of these novel cross‐relaxation phenomena were determined, and their potential usefulness as tools for investigating hyperpolarized ensembles and for obtaining enhanced‐sensitivity ¹H NMR traces was explored.     

Progress in Hyperpolarized Ultrafast 2D NMR Spectroscopy

An important development in the field of NMR spectroscopy has been the advent of hyperpolarization approaches, capable of yielding nuclear spin states whose value exceeds by orders‐of‐magnitude what even the highest‐field spectrometers can afford under Boltzmann equilibrium. Included among these methods is an ex situ dynamic nuclear polarization (DNP) approach, which yields liquid‐phase samples possessing spin polarizations of up to 50 %. Although capable of providing an NMR sensitivity equivalent to the averaging of about 1 000 000 scans, this methodology is constrained to extract its “superspectrum” within a single—or at most a few—transients. This makes it a poor starting point for conventional 2D NMR acquisition experiments, which require a large number of scans that are identical to one another except for the increment of a certain t₁ delay. It has been recently suggested that by merging this ex situ DNP approach with spatially encoded “ultrafast” methods, a suitable starting point could arise for the acquisition of 2D spectra on hyperpolarized liquids. Herein, we describe the experimental principles, potential features, and current limitations of such integration between the two methodologies. For a variety of small molecules, these new hyperpolarized ultrafast experiments can, for equivalent overall durations, provide heteronuclear correlation spectra at significantly lower concentrations than those currently achievable by conventional 2D NMR acquisitions. A variety of challenges still remain to be solved before bringing the full potential of this new integrated 2D NMR approach to fruition; these outstanding issues are discussed.     

Sensitivity Enhancement in Transverse Relaxation Optimized NMR Spectroscopy

Dramatically shortened transverse relaxation times in transverse relaxation optimized spectroscopy (TROSY) result from interference between dipole–dipole interactions and the anisotropy of the chemical shift. Thus NMR spectroscopy becomes a suitable method for studying large biomolecules, with optimal performance when 1-GHz spectrometers become available. By using new phase cycles and data-processing methods, the sensitivity of the TROSY experiment was increased by a factor of √2, which is of considerable importance for applications in high-field NMR studies on large proteins.     

Effective PHIP Labeling of Bioactive Peptides Boosts the Intensity of the NMR Signal

A series of novel bioactive derivatives of the sunflower trypsin inhibitor‐1 (SFTI‐1) suitable for hyperpolarization by parahydrogen‐induced polarization (PHIP) was developed. The PHIP activity was achieved by labeling with L ‐propargylglycine, O‐propargyl‐L ‐tyrosine, or 4‐pentynoic acid. ¹H NMR signal enhancements (SE) of up to a factor of 70 were achieved in aqueous solution. We found that an isolated spatial location of the triple bond within the respective label and its accessibility for the hydrogenation catalyst are essential factors for the degree of signal enhancement.     

Implementation and Characterization of Flow Injection in Dissolution Dynamic Nuclear Polarization NMR Spectroscopy

The use of dissolution dynamic nuclear polarization (D ‐DNP) offers substantially increased signals in liquid‐state NMR spectroscopy. A challenge in realizing this potential lies in the transfer of the hyperpolarized sample to the NMR detector without loss of hyperpolarization. Here, the use of a flow injection method using high‐pressure liquid leads to improved performance compared to the more common gas‐driven injection, by suppressing residual fluid motions during the NMR experiment while still achieving a short injection time. Apparent diffusion coefficients are determined from pulsed field gradient echo measurements, and are shown to fall below 1.5 times the value of a static sample within 0.8 s. Due to the single‐scan nature of D ‐DNP, pulsed field gradients are often the only choice for coherence selection or encoding, but their application requires stationary fluid. Sample delivery driven by a high‐pressure liquid will improve the applicability of these types of D‐DNP advanced experiments.     

Recovering Invisible Signals by Two‐Field NMR Spectroscopy

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two‐field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low‐field dimension. Two‐field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.     

Harnessing Water to Enhance Quadrupolar NMR Spectroscopy and Imaging

¹⁷O and ¹⁴N are attractive targets for in vivo NMR spectroscopy and imaging, but low gyromagnetic ratios γ and fast spin relaxation complicate observations. This work explores indirect ways of detecting some of these sites with the help of proton-detected double resonance techniques. As standard coherence transfer methods are of limited use for such indirect detection, alternative routes for probing the quadrupolar spectra on ¹H were tested. These centered on modulating the broadening effects imparted onto protons adjacent to the low-γ species through J couplings through either continuous wave or spin-echo double-resonance decoupling/recoupling sequences. As in all cases, the changes imparted by these double-resonance strategies were small due to the fast relaxation undergone by the quadrupoles, the sensitivity of these approaches was amplified by transferring their effects onto the abundant water ¹H signal. These amplifications were mediated by the spontaneous exchanges that the labile ¹Hs bound to ¹⁷O or ¹⁴N undergo with the water protons. In experiments designed on the basis of double-resonance spin echoes, these enhancements were imparted by looping the transverse encodings together with multiple longitudinal storage periods, leading to decoupling-recoupling with exchange (D-REX) sequences. In experiments designed on the basis of continuous on/off quadrupolar decoupling, these solvent exchanges were incorporated into chemical-exchange saturation transfer schemes, leading to decoupling-recoupling with saturation transfer (D-REST) sequences. Both of these variants harnessed sizable proportions of the easily detectable water signals, in order to characterize the NMR spectra and/or to image with atomic-site specificity the ¹⁷O and ¹⁴N species.     

Longitudinal Relaxation Enhancement in 1H NMR Spectroscopy of Tissue Metabolites via Spectrally Selective Excitation

Nuclear magnetic resonance spectroscopy is governed by longitudinal (T₁) relaxation. For protein and nucleic acid experiments in solutions, it is well established that apparent T₁ values can be enhanced by selective excitation of targeted resonances. The present study explores such longitudinal relaxation enhancement (LRE) effects for molecules residing in biological tissues. The longitudinal relaxation recovery of tissue resonances positioned both down‐ and upfield of the water peak were measured by spectrally selective excitation/refocusing pulses, and compared with conventional water‐suppressed, broadband‐excited counterparts at 9.4 T. Marked LRE effects with up to threefold reductions in apparent T₁ values were observed as expected for resonances in the 6–9 ppm region; remarkably, statistically significant LRE effects were also found for several non‐exchanging metabolite resonances in the 1–4 ppm region, encompassing 30–50 % decreases in apparent T₁ values. These LRE effects suggest a novel means of increasing the sensitivity of tissue‐oriented experiments, and open new vistas to investigate the nature of interactions among metabolites, water and macromolecules at a molecular level.     

In Situ and Ex Situ Low‐Field NMR Spectroscopy and MRI Endowed by SABRE Hyperpolarization

By using 5.75 and 47.5 mT nuclear magnetic resonance (NMR) spectroscopy, up to 10⁵‐fold sensitivity enhancement through signal amplification by reversible exchange (SABRE) was enabled, and subsecond temporal resolution was used to monitor an exchange reaction that resulted in the buildup and decay of hyperpolarized species after parahydrogen bubbling. We demonstrated the high‐resolution low‐field proton magnetic resonance imaging (MRI) of pyridine in a 47.5 mT magnetic field endowed by SABRE. Molecular imaging (i.e. imaging of dilute hyperpolarized substances rather than the bulk medium) was conducted in two regimes: in situ real‐time MRI of the reaction mixture (in which pyridine was hyperpolarized), and ex situ MRI (in which hyperpolarization decays) of the liquid hyperpolarized product. Low‐field (milli‐Tesla range, e.g. 5.75 and 47.5 mT used in this study) parahydrogen‐enhanced NMR and MRI, which are free from the limitations of high‐field magnetic resonance (including susceptibility‐induced gradients of the static magnetic field at phase interfaces), potentially enables new imaging applications as well as differentiation of hyperpolarized chemical species on demand by exploiting spin manipulations with static and alternating magnetic fields.     

Strategies for 1H-Detected Dynamic Nuclear Polarization Magic-Angle Spinning NMR Spectroscopy

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic-angle spinning NMR spectroscopy. Herein, the feasibility of proton-detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected ¹⁴N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.     

Hyperpolarized NMR Spectroscopy: d‐DNP,PHIP, and SABRE Techniques

The intensity of NMR signals can be enhanced by several orders of magnitude by using various techniques for the hyperpolarization of different molecules. Such approaches can overcome the main sensitivity challenges facing modern NMR/magnetic resonance imaging (MRI) techniques, whilst hyperpolarized fluids can also be used in a variety of applications in material science and biomedicine. This Focus Review considers the fundamentals of the preparation of hyperpolarized liquids and gases by using dissolution dynamic nuclear polarization (d‐DNP) and parahydrogen‐based techniques, such as signal amplification by reversible exchange (SABRE) and parahydrogen‐induced polarization (PHIP), in both heterogeneous and homogeneous processes. The various new aspects in the formation and utilization of hyperpolarized fluids, along with the possibility of observing NMR signal enhancement, are described.     

Shift Reagents in NMR Spectroscopy

The number of possible applications of NMR spectroscopy has rapidly increased during the past few years. New fields of applications have been opened by the development of supraconducting solenoids and various spin-decoupling techniques and by the method of “pulsed Fourier transform NMR-spectroscopy”. These methods originate mainly from progress in instrumentation. Recently, another “technique” has been introduced into NMR spectroscopy, which—principally on the basis of chemical and spectroscopic experience—is much less expensive but nevertheless useful. The basic principles, background, and most important applications of this method, known as the “NMR-shift-reagent technique”, form the subject of this paper.