Spin Hyperpolarization in Modern Magnetic Resonance.

Magnetic resonance techniques are successfully utilized in a broad range of scientific disciplines and in various practical applications, with medical magnetic resonance imaging being the most widely known example. Currently, both fundamental and applied magnetic resonance are enjoying a major boost owing to the rapidly developing field of spin hyperpolarization. Hyperpolarization techniques are able to enhance signal intensities in magnetic resonance by several orders of magnitude, and thus to largely overcome its major disadvantage of relatively low sensitivity. This provides new impetus for existing applications of magnetic resonance and opens the gates to exciting new possibilities. In this review, we provide a unified picture of the many methods and techniques that fall under the umbrella term "hyperpolarization" but are currently seldom perceived as integral parts of the same field. Specifically, before delving into the individual techniques, we provide a detailed analysis of the underlying principles of spin hyperpolarization. We attempt to uncover and classify the origins of hyperpolarization, to establish its sources and the specific mechanisms that enable the flow of polarization from a source to the target spins. We then give a more detailed analysis of individual hyperpolarization techniques: the mechanisms by which they work, fundamental and technical requirements, characteristic applications, unresolved issues, and possible future directions. We are seeing a continuous growth of activity in the field of spin hyperpolarization, and we expect the field to flourish as new and improved hyperpolarization techniques are implemented. Some key areas for development are in prolonging polarization lifetimes, making hyperpolarization techniques more generally applicable to chemical/biological systems, reducing the technical and equipment requirements, and creating more efficient excitation and detection schemes. We hope this review will facilitate the sharing of knowledge between subfields within the broad topic of hyperpolarization, to help overcome existing challenges in magnetic resonance and enable novel applications.

Rapid hyperpolarization and purification of the metabolite fumarate in aqueous solution.

Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30-45%.

Cyclometallated Imides as Templates for the H-Bond Directed Iridium-Catalyzed Asymmetric Hydrogenation of N-Methyl, N-Alkyl and N-Aryl Imines.

A combined computational and experimental approach allowed us to develop overall the most selective catalyst for the direct hydrogenation of N-methyl, N-alkyl and N-aryl imines described to date. Iridium catalysts with a cyclometallated cyclic imide group provide selectivity of up to 99 % enantiomeric excess. Computational studies show that the selectivity results from the combined effect of H-bonding of the imide C=O with the substrate iminium ion and a stabilizing π-π interaction with the cyclometallated ligand. The cyclometallated ligand thus exhibits a unique mode of action, serving as a template for the H-bond directed approach of the substrate which results in enhanced selectivity. The catalyst (2) has been synthesized and isolated as a crystalline air-stable solid. X-ray analysis of 2 confirmed the structure of the catalyst and the correct position of the imide C=O groups to engage in an H-bond with the substrate. 19F NMR real-time monitoring showed the hydrogenation of N-methyl imines catalyzed by 2 is very fast, with a TOF of approx. 3500 h-1.

Hyperpolarizing Small Molecules using Parahydrogen and Solid-State Spin Diffusion.

Parahydrogen-induced polarization (PHIP) is an inexpensive way to produce hyperpolarized molecules with polarization levels of >10% in the solution-state, but is strongly limited in generality since it requires chemical reactions/interactions with H2. Here we report a new method to widen the scope of PHIP hyperpolarization: a source molecule is produced via PHIP with high 13C polarization, and precipitated out of solution together with a target species. Spin diffusion within the solid carries the polarization onto 13C spins of the target, which can then be dissolved for solution-state applications. We name this method PHIP-SSD (PHIP with solid-state spin diffusion) and demonstrate it using PHIP-polarized [1-13C]-fumarate as the source molecule, to polarize different 13C-labelled target molecules. 13C polarizations of between 0.01 and 3% were measured on [1-13C]-benzoic acid, depending on the molar ratio of fumarate:benzoate in the solid state. We also show that PHIP-SSD does not require specific co-crystallization conditions by grinding dry powders of target molecules together with solid fumarate crystals, and obtain 13C signal enhancements of between 100 and 200 on [13C,15N2]-urea, [1-13C]-pyruvate, and [1-13C]-benzoic acid. This approach appears to be a promising new strategy for facile hyperpolarization based on PHIP.

Parahydrogen-Polarized Fumarate for Preclinical in Vivo Metabolic Magnetic Resonance Imaging.

We present a versatile method for the preparation of hyperpolarized [1-13C]fumarate as a contrast agent for preclinical in vivo MRI, using parahydrogen-induced polarization (PHIP). To benchmark this process, we compared a prototype PHIP polarizer to a state-of-the-art dissolution dynamic nuclear polarization (d-DNP) system. We found comparable polarization, volume, and concentration levels of the prepared solutions, while the preparation effort is significantly lower for the PHIP process, which can provide a preclinical dose every 10 min, opposed to around 90 min for d-DNP systems. With our approach, a 100 mM [1-13C]-fumarate solution of volumes up to 3 mL with 13-20% 13C-hyperpolarization after purification can be produced. The purified solution has a physiological pH, while the catalyst, the reaction side products, and the precursor material concentrations are reduced to nontoxic levels, as confirmed in a panel of cytotoxicity studies. The in vivo usage of the hyperpolarized fumarate as a perfusion agent in healthy mice and the metabolic conversion of fumarate to malate in tumor-bearing mice developing regions with necrotic cell death is demonstrated. Furthermore, we present a one-step synthesis to produce the 13C-labeled precursor for the hydrogenation reaction with high yield, starting from 13CO2 as a cost-effective source for 13C-labeled compounds.

Zero- to Ultralow-Field Nuclear Magnetic Resonance Enhanced with Dissolution Dynamic Nuclear Polarization.

Zero- to ultralow-field nuclear magnetic resonance is a modality of magnetic resonance experiment which does not require strong superconducting magnets. Contrary to conventional high-field nuclear magnetic resonance, it has the advantage of allowing high-resolution detection of nuclear magnetism through metal as well as within heterogeneous media. To achieve high sensitivity, it is common to couple zero-field nuclear magnetic resonance with hyperpolarization techniques. To date, the most common technique is parahydrogen-induced polarization, which is only compatible with a small number of compounds. In this article, we establish dissolution dynamic nuclear polarization as a versatile method to enhance signals in zero-field nuclear magnetic resonance experiments on sample mixtures of [13C]sodium formate, [1-13C]glycine, and [2-13C]sodium acetate, and our technique is immediately extendable to a broad range of molecules with >1 s relaxation times. We find signal enhancements of up to 11,000 compared with thermal prepolarization in a 2 T permanent magnet. To increase the signal in future experiments, we investigate the relaxation effects of the TEMPOL radicals used for the hyperpolarization process at zero- and ultralow-fields.

Enzymatic Reactions Observed with Zero- and Low-Field Nuclear Magnetic Resonance.

We demonstrate that enzyme-catalyzed reactions can be observed in zero- and low-field NMR experiments by combining recent advances in parahydrogen-based hyperpolarization methods with state-of-the-art magnetometry. Specifically, we investigated two model biological processes: the conversion of fumarate into malate, which is used in vivo as a marker of cell necrosis, and the conversion of pyruvate into lactate, which is the most widely studied metabolic process in hyperpolarization-enhanced imaging. In addition to this, we constructed a microfluidic zero-field NMR setup to perform experiments on microliter-scale samples of [1-13C]fumarate in a lab-on-a-chip device. Zero- to ultralow-field (ZULF) NMR has two key advantages over high-field NMR: the signals can pass through conductive materials (e.g., metals), and line broadening from sample heterogeneity is negligible. To date, the use of ZULF NMR for process monitoring has been limited to studying hydrogenation reactions. In this work, we demonstrate this emerging analytical technique for more general reaction monitoring and compare zero- vs low-field detection.

Direct Production of a Hyperpolarized Metabolite on a Microfluidic Chip.

Microfluidic systems hold great potential for the study of live microscopic cultures of cells, tissue samples, and small organisms. Integration of hyperpolarization would enable quantitative studies of metabolism in such volume limited systems by high-resolution NMR spectroscopy. We demonstrate, for the first time, the integrated generation and detection of a hyperpolarized metabolite on a microfluidic chip. The metabolite [1-13C]fumarate is produced in a nuclear hyperpolarized form by (i) introducing para-enriched hydrogen into the solution by diffusion through a polymer membrane, (ii) reaction with a substrate in the presence of a ruthenium-based catalyst, and (iii) conversion of the singlet-polarized reaction product into a magnetized form by the application of a radiofrequency pulse sequence, all on the same microfluidic chip. The microfluidic device delivers a continuous flow of hyperpolarized material at the 2.5 µL/min scale, with a polarization level of 4%. We demonstrate two methods for mitigating singlet-triplet mixing effects which otherwise reduce the achieved polarization level.

Constant-adiabaticity ultralow magnetic field manipulations of parahydrogen-induced polarization: application to an AA'X spin system.

The field of magnetic resonance imaging with hyperpolarized contrast agents is rapidly expanding, and parahydrogen-induced polarization (PHIP) is emerging as an inexpensive and easy-to-implement method for generating the required hyperpolarized biomolecules. Hydrogenative PHIP delivers hyperpolarized proton spin order to a substrate via chemical addition of H2 in the spin-singlet state, but it is typically necessary to transfer the proton polarization to a heteronucleus (usually 13C) which has a longer spin lifetime. Adiabatic ultralow magnetic field manipulations can be used to induce the polarization transfer, but this is necessarily a slow process, which is undesirable since the spins continually relax back to thermal equilibrium. Here we demonstrate two constant-adiabaticity field sweep methods, one in which the field passes through zero, and one in which the field is swept from zero, for optimal polarization transfer on a model AA'X spin system, [1-13C]fumarate. We introduce a method for calculating the constant-adiabaticity magnetic field sweeps, and demonstrate that they enable approximately one order of magnitude faster spin-order conversion compared to linear sweeps. The present method can thus be utilized to manipulate nonthermal order in heteronuclear spin systems.

Chemical Reaction Monitoring using Zero-Field Nuclear Magnetic Resonance Enables Study of Heterogeneous Samples in Metal Containers.

We demonstrate that heterogeneous/biphasic chemical reactions can be monitored with high spectroscopic resolution using zero-field nuclear magnetic resonance spectroscopy. This is possible because magnetic susceptibility broadening is negligible at ultralow magnetic fields. We show the two-step hydrogenation of dimethyl acetylenedicarboxylate with para-enriched hydrogen gas in conventional glass NMR tubes, as well as in a titanium tube. The low frequency zero-field NMR signals ensure that there is no significant signal attenuation arising from shielding by the electrically conductive sample container. This method paves the way for in situ monitoring of reactions in complex heterogeneous multiphase systems and in reactors made of conductive materials while maintaining resolution and chemical specificity.

Real-Time Nuclear Magnetic Resonance Detection of Fumarase Activity Using Parahydrogen-Hyperpolarized [1-13C]Fumarate.

Hyperpolarized fumarate can be used as a probe of real-time metabolism in vivo, using carbon-13 magnetic resonance imaging. Dissolution dynamic nuclear polarization is commonly used to produce hyperpolarized fumarate, but a cheaper and faster alternative is to produce hyperpolarized fumarate via PHIP (parahydrogen-induced polarization). In this work, we trans-hydrogenate [1-13C]acetylene dicarboxylate with para-enriched hydrogen using a commercially available Ru catalyst in water to produce hyperpolarized [1-13C]fumarate. We show that fumarate is produced in 89% yield, with succinate as a side product in 11% yield. The proton polarization is converted into 13C magnetization using a constant adiabaticity field cycle, and a polarization level of 24% is achieved using 86% para-enriched hydrogen gas. We inject the hyperpolarized [1-13C]fumarate into cell suspensions and track the metabolism. This work opens the path to greatly accelerated preclinical studies using fumarate as a biomarker.

High-Resolution Nuclear Magnetic Resonance Spectroscopy with Picomole Sensitivity by Hyperpolarization on a Chip.

We show that high-resolution NMR can reach picomole sensitivity for micromolar concentrations of analyte by combining parahydrogen-induced hyperpolarization (PHIP) with a high-sensitivity transmission line microdetector. The para-enriched hydrogen gas is introduced into solution by diffusion through a membrane integrated into a microfluidic chip. NMR microdetectors, operating with sample volumes of a few µL or less, benefit from a favorable scaling of mass sensitivity. However, the small volumes make it very difficult to detect species present at less than millimolar concentrations in microfluidic NMR systems. In view of overcoming this limitation, we implement PHIP on a microfluidic device with a 2.5 µL detection volume. Integrating the hydrogenation reaction into the chip minimizes polarization losses to spin-lattice relaxation, allowing the detection of picomoles of substance. This corresponds to a concentration limit of detection of better than 1µMs , unprecedented at this sample volume. The stability and sensitivity of the system allow quantitative characterization of the signal dependence on flow rates and other reaction parameters and permit homo- (1H-1H) and heteronuclear (1H-13C) 2D NMR experiments at natural 13C abundance.

Polarization transfer via field sweeping in parahydrogen-enhanced nuclear magnetic resonance.

We show that in a spin system of two magnetically inequivalent protons coupled to a heteronucleus such as 13C, an adiabatic magnetic field sweep, passing through zero field, transfers the proton singlet order into magnetization of the coupled heteronucleus. This effect is potentially useful in parahydrogen-enhanced nuclear magnetic resonance and is demonstrated on singlet-hyperpolarized [1-13C]maleic acid, which is prepared via the reaction between [1-13C]acetylene dicarboxylic acid and para-enriched hydrogen gas. The magnetic field sweeps are of microtesla amplitudes and have durations on the order of seconds. We show a polarization enhancement by a factor of 104 in the 13C spectra of [1-13C]maleic acid in a 1.4 T magnetic field.

Preservation of Nuclear Spin Order by Precipitation.

We demonstrate that non-equilibrium nuclear spin order survives precipitation from solution and redissolution. The effect is demonstrated on 13 C- and 2 H-labeled sodium fumarate, with precipitation and dissolution achieved by altering the pH. The lifetime of the spin magnetization in the precipitate suspension is found to be much longer than in solution. Our preliminary results show an extension of the effective relaxation time T1 for the metabolite fumarate by a factor of ≈6. We show that when the free radical agent TEMPO is present in the solution, it is not incorporated into the precipitate, suggesting that this procedure may provide a means to store and transport agents polarized by dynamic nuclear polarization. Although the relaxation time, T1 , of the precipitate suspension is longer than that of the same molecules in solution, it is significantly shorter than that observed in the immobilized solid state.

Combined homogeneous and heterogeneous hydrogenation to yield catalyst-free solutions of parahydrogen-hyperpolarized [1-13C]succinate.

We show that catalyst-free aqueous solutions of hyperpolarized [1-13C]succinate can be produced using parahydrogen-induced polarization (PHIP) and a combination of homogeneous and heterogeneous catalytic hydrogenation reactions. We generate hyperpolarized [1-13C]fumarate via PHIP using para-enriched hydrogen gas with a homogeneous ruthenium catalyst, and subsequently remove the toxic catalyst and reaction side products via a purification procedure. Following this, we perform a second hydrogenation reaction using normal hydrogen gas to convert the fumarate into succinate using a solid Pd/Al2O3 catalyst. This inexpensive polarization protocol has a turnover time of a few minutes, and represents a major advance for in vivo applications of [1-13C]succinate as a hyperpolarized contrast agent.

Over 20% Carbon-13 Polarization of Perdeuterated Pyruvate Using Reversible Exchange with Parahydrogen and Spin-Lock Induced Crossing at 50 µT.

Carbon-13 hyperpolarized pyruvate is about to become the next-generation contrast agent for molecular magnetic resonance imaging of cancer and other diseases. Here, efficient and rapid pyruvate hyperpolarization is achieved via signal amplification by reversible exchange (SABRE) with parahydrogen through synergistic use of substrate deuteration, alternating, and static microtesla magnetic fields. Up to 22 and 6% long-lasting 13C polarization (T1 = 3.7 ± 0.25 and 1.7 ± 0.1 min) is demonstrated for the C1 and C2 nuclear sites, respectively. The remarkable polarization levels become possible as a result of favorable relaxation dynamics at the microtesla fields. The ultralong polarization lifetimes will be conducive to yielding high polarization after purification, quality assurance, and injection of the hyperpolarized molecular imaging probes. These results pave the way to future in vivo translation of carbon-13 hyperpolarized molecular imaging probes prepared by this approach.

Magnetometer-Detected Nuclear Magnetic Resonance of Photochemically Hyperpolarized Molecules.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) enables nuclear spin ordering by irradiating samples with light. Polarized spins are conventionally detected via high-field chemical-shift-resolved NMR (above 0.1 T). In this Letter, we demonstrate in situ low-field photo-CIDNP measurements using a magnetically shielded fast-field-cycling NMR setup detecting Larmor precession via atomic magnetometers. For solutions comprising mM concentrations of the photochemically polarized molecules, hyperpolarized 1H magnetization is detected by pulse-acquired NMR spectroscopy. The observed NMR line widths are about 5 times narrower than normally anticipated in high-field NMR and are systematically affected by light irradiation during the acquisition period, reflecting a reduction of the transverse relaxation time constant, T2*, on the order of 10%. Magnetometer-detected photo-CIDNP spectroscopy enables straightforward observation of spin-chemistry processes in the ambient field range from a few nT to tens of mT. Potential applications of this measuring modality are discussed.

Synergies between Hyperpolarized NMR and Microfluidics: A Review.

Hyperpolarized nuclear magnetic resonance and lab-on-a-chip microfluidics are two dynamic, but until recently quite distinct, fields of research. Recent developments in both areas increased their synergistic overlap. By microfluidic integration, many complex experimental steps can be brought together onto a single platform. Microfluidic devices are therefore increasingly finding applications in medical diagnostics, forensic analysis, and biomedical research. In particular, they provide novel and powerful ways to culture cells, cell aggregates, and even functional models of entire organs. Nuclear magnetic resonance is a non-invasive, high-resolution spectroscopic technique which allows real-time process monitoring with chemical specificity. It is ideally suited for observing metabolic and other biological and chemical processes in microfluidic systems. However, its intrinsically low sensitivity has limited its application. Recent advances in nuclear hyperpolarization techniques may change this: under special circumstances, it is possible to enhance NMR signals by up to 5 orders of magnitude, which dramatically extends the utility of NMR in the context of microfluidic systems. Hyperpolarization requires complex chemical and/or physical manipulations, which in turn may benefit from microfluidic implementation. In fact, many hyperpolarization methodologies rely on processes that are more efficient at the micro-scale, such as molecular diffusion, penetration of electromagnetic radiation into a sample, or restricted molecular mobility on a surface. In this review we examine the confluence between the fields of hyperpolarization-enhanced NMR and microfluidics, and assess how these areas of research have mutually benefited one another, and will continue to do so.

Relayed hyperpolarization for zero-field nuclear magnetic resonance.

Zero- to ultralow-field nuclear magnetic resonance (ZULF NMR) is a rapidly developing form of spectroscopy that provides rich spectroscopic information in the absence of large magnetic fields. However, signal acquisition still requires a mechanism for generating a bulk magnetic moment for detection, and the currently used methods only apply to a limited pool of chemicals or come at prohibitively high cost. We demonstrate that the parahydrogen-based SABRE (signal amplification by reversible exchange)-Relay method can be used as a more general means of generating hyperpolarized analytes for ZULF NMR by observing zero-field J-spectra of [13C]-methanol, [1-13C]-ethanol, and [2-13C]-ethanol in both 13C-isotopically enriched and natural abundance samples. We explore the magnetic field dependence of the SABRE-Relay efficiency and show the existence of a second maximum at 19.0 ± 0.3 mT. Despite presence of water, SABRE-Relay is used to hyperpolarize ethanol extracted from a store-bought sample of vodka (%PH ~ 0.1%).