Rapid and Simple 13C-Hyperpolarization by 1H Dissolution Dynamic Nuclear Polarization Followed by an Inline Magnetic Field Inversion.

Dissolution dynamic nuclear polarization (dDNP) is a method of choice for preparing hyperpolarized 13C metabolites such as 1-13C-pyruvate used for in vivo applications, including the real-time monitoring of cancer cell metabolism in human patients. The approach consists of transferring the high polarization of electron spins to nuclear spins via microwave irradiation at low temperatures (1.0-1.5 K) and moderate magnetic fields (3.3-7 T). The solid sample is then dissolved and transferred to an NMR spectrometer or MRI scanner for detection in the liquid state. Common dDNP protocols use direct hyperpolarization of 13C spins reaching polarizations of >50% in ∼1-2 h. Alternatively, 1H spins are polarized before transferring their polarization to 13C spins using cross-polarization, reaching polarization levels similar to those of direct DNP in only ∼20 min. However, it relies on more complex instrumentation, requiring highly skilled personnel. Here, we explore an alternative route using 1H dDNP followed by inline adiabatic magnetic field inversion in the liquid state during the transfer. 1H polarizations of >70% in the solid state are obtained in ∼5-10 min. As the hyperpolarized sample travels from the dDNP polarizer to the NMR spectrometer, it goes through a field inversion chamber, which causes the 1H → 13C polarization transfer. This transfer is made possible by the J-coupling between the heteronuclei, which mixes the Zeeman states at zero-field and causes an antilevel crossing. We report liquid-state 13C polarization up to ∼17% for 3-13C-pyruvate and 13C-formate. The instrumentation needed to perform this experiment in addition to a conventional dDNP polarizer is simple and readily assembled.

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.

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.

Frémy's Salt as a Low-Persistence Hyperpolarization Agent: Efficient Dynamic Nuclear Polarization Plus Rapid Radical Scavenging.

Nuclear magnetic resonance (NMR) spectroscopy is a key technique for molecular structure determination in solution. However, due to its low sensitivity, many efforts have been made to improve signal strengths and reduce the required substrate amounts. In this regard, dissolution dynamic nuclear polarization (DDNP) is a versatile approach as signal enhancements of over 10 000-fold are achievable. Samples are signal-enhanced ex situ by transferring electronic polarization from radicals to nuclear spins before dissolving and shuttling the boosted sample to an NMR spectrometer for detection. However, the applicability of DDNP suffers from one major drawback, namely, paramagnetic relaxation enhancements (PREs) that critically reduce relaxation times due to the codissolved radicals. PREs are the primary source of polarization losses canceling the signal improvements obtained by DNP. We solve this problem by using potassium nitrosodisulfonate (Frémy's salt) as polarization agent (PA), which provides high nuclear spin polarization and allows for rapid scavenging under mild reducing conditions. We demonstrate the potential of Frémy's salt, (i) showing that both 1H and 13C polarization of ∼30% can be achieved and (ii) describing a hybrid sample shuttling system (HySSS) that can be used with any DDNP/NMR combination to remove the PA before NMR detection. This gadget mixes the hyperpolarized solution with a radical scavenger and injects it into an NMR tube, providing, within a few seconds, quantitatively radical-free, highly polarized solutions. The cost efficiency and broad availability of Frémy's salt might facilitate the use of DDNP in many fields of research.

Frozen water NMR lineshape analysis enables absolute polarization quantification.

Typical magnetic resonance experiments are routinely limited by weak signal responses. In some cases, the low intrinsic sensitivity can be alleviated by the implementation of hyperpolarization technologies. Dissolution-dynamic nuclear polarization offers a means of hyperpolarizing small molecules. Hyperpolarized water is employed in several dynamic nuclear polarization studies, and hence accurate and rapid quantification of the 1H polarization level is of utmost importance. The solid-state nuclear magnetic resonance spectrum of water acquired under dissolution-dynamic nuclear polarization conditions has revealed lineshapes which become asymmetric at high levels of 1H polarization, which is an interesting fundamental problem in itself, but also complicates data interpretation and can prevent correct estimations of polarization levels achieved. In previous studies, attempts to simulate the 1H spectral lineshape of water as a function of the 1H polarization led to significant disagreement with the experimental results. Here we propose and demonstrate that such simulations, and therefore polarization quantification, can be implemented accurately, in particular by taking into account the detector dead time during 1H signal acquisition that can lead to severe spectral distortions. Based on these findings, we employed an echo-based radiofrequency pulse sequence to achieve distortion-free 1H spectra of hyperpolarized water, and adequate simulations of these echo-based spectra were implemented to extract the absolute 1H polarization level from the hyperpolarized water signal only, thus alleviating the need for lengthy and insensitive measurements of thermal equilibrium signals.

Fine optimization of a dissolution dynamic nuclear polarization experimental setting for 13C NMR of metabolic samples.

NMR-based analysis of metabolite mixtures provides crucial information on biological systems but mostly relies on 1D 1H experiments for maximizing sensitivity. However, strong peak overlap of 1H spectra often is a limitation for the analysis of inherently complex biological mixtures. Dissolution dynamic nuclear polarization (d-DNP) improves NMR sensitivity by several orders of magnitude, which enables 13C NMR-based analysis of metabolites at natural abundance. We have recently demonstrated the successful introduction of d-DNP into a full untargeted metabolomics workflow applied to the study of plant metabolism. Here we describe the systematic optimization of d-DNP experimental settings for experiments at natural 13C abundance and show how the resolution, sensitivity, and ultimately the number of detectable signals improve as a result. We have systematically optimized the parameters involved (in a semi-automated prototype d-DNP system, from sample preparation to signal detection, aiming at providing an optimization guide for potential users of such a system, who may not be experts in instrumental development). The optimization procedure makes it possible to detect previously inaccessible protonated 13C signals of metabolites at natural abundance with at least 4 times improved line shape and a high repeatability compared to a previously reported d-DNP-enhanced untargeted metabolomic study. This extends the application scope of hyperpolarized 13C NMR at natural abundance and paves the way to a more general use of DNP-hyperpolarized NMR in metabolomics studies.

Practical dissolution dynamic nuclear polarization.

This review article intends to provide insightful advice for dissolution-dynamic nuclear polarization in the form of a practical handbook. The goal is to aid research groups to effectively perform such experiments in their own laboratories. Previous review articles on this subject have covered a large number of useful topics including instrumentation, experimentation, theory, etc. The topics to be addressed here will include tips for sample preparation and for checking sample health; a checklist to correctly diagnose system faults and perform general maintenance; the necessary mechanical requirements regarding sample dissolution; and aids for accurate, fast and reliable polarization quantification. Herein, the challenges and limitations of each stage of a typical dissolution-dynamic nuclear polarization experiment are presented, with the focus being on how to quickly and simply overcome some of the limitations often encountered in the laboratory.

Protonation tuned dipolar order mediated 1H→13C cross-polarization for dissolution-dynamic nuclear polarization experiments.

A strategy of dipolar order mediated nuclear spin polarization transfer has recently been combined with dissolution-dynamic nuclear polarization (dDNP) and improved by employing optimized shaped radiofrequency pulses and suitable molecular modifications. In the context of dDNP experiments, this offers a promising means of transferring polarization from high-gamma 1H spins to insensitive 13C spins with lower peak power and lower energy compared with state-of-the-art cross-polarization schemes. The role of local molecular groups and the glassing matrix protonation level are both postulated to play a key role in the polarization transfer pathway via an intermediary reservoir of dipolar spin order. To gain appreciation of the mechanisms involved in the dipolar order mediated polarization transfer under dDNP conditions, we investigate herein the influence of the pivotal characteristics of the sample makeup: (i) revising the protonation level for the constituents of the DNP glass; and (ii) utilizing deuterated molecular derivatives. Experimental demonstrations are presented for the case of [1-13C]sodium acetate. We find that the proton sample molarity has a large impact on both the optimal parameters and the performance of the dipolar order mediated cross-polarization sequence, with the 13C signal build-up time drastically shortened in the case of high solvent protonation levels. In the case of a deuterated molecular derivative, we observe that the nearby 2H substituted methyl group is deleterious to the 1H→13C transfer phenomenon (particularly at low levels of sample protonation). Overall, increased solvent protonation makes the dipolar order governed polarization transfer significantly faster and more efficient. This study sheds light on the influential sample formulation traits which govern the dipolar order-controlled transfer of polarization and indicates that the polarization transfer efficiencies of deuterated molecules can be boosted and reach high performances simply by adequate solvent protonation.

Porous functionalized polymers enable generating and transporting hyperpolarized mixtures of metabolites.

Hyperpolarization by dissolution dynamic nuclear polarization (dDNP) has enabled promising applications in spectroscopy and imaging, but remains poorly widespread due to experimental complexity. Broad democratization of dDNP could be realized by remote preparation and distribution of hyperpolarized samples from dedicated facilities. Here we show the synthesis of hyperpolarizing polymers (HYPOPs) that can generate radical- and contaminant-free hyperpolarized samples within minutes with lifetimes exceeding hours in the solid state. HYPOPs feature tunable macroporous porosity, with porous volumes up to 80% and concentration of nitroxide radicals grafted in the bulk matrix up to 285 µmol g-1. Analytes can be efficiently impregnated as aqueous/alcoholic solutions and hyperpolarized up to P(13C) = 25% within 8 min, through the combination of 1H spin diffusion and 1H → 13C cross polarization. Solutions of 13C-analytes of biological interest hyperpolarized in HYPOPs display a very long solid-state 13C relaxation times of 5.7 h at 3.8 K, thus prefiguring transportation over long distances.

Direct observation of hyperpolarization breaking through the spin diffusion barrier.

Dynamic nuclear polarization (DNP) is a widely used tool for overcoming the low intrinsic sensitivity of nuclear magnetic resonance spectroscopy and imaging. Its practical applicability is typically bounded, however, by the so-called "spin diffusion barrier," which relates to the poor efficiency of polarization transfer from highly polarized nuclei close to paramagnetic centers to bulk nuclei. A quantitative assessment of this barrier has been hindered so far by the lack of general methods for studying nuclear polarization flow in the vicinity of paramagnetic centers. Here, we fill this gap and introduce a general set of experiments based on microwave gating that are readily implemented. We demonstrate the versatility of our approach in experiments conducted between 1.2 and 4.2 K in static mode and at 100 K under magic angle spinning (MAS)-conditions typical for dissolution DNP and MAS-DNP-and directly observe the marked dependence of polarization flow on temperature.

Pulse sequence and sample formulation optimization for dipolar order mediated 1H→13C cross-polarization.

We have recently demonstrated the use of contactless radiofrequency pulse sequences under dissolution-dynamic nuclear polarization conditions as an attractive way of transferring polarization from sensitive 1H spins to insensitive 13C spins with low peak radiofrequency pulse powers and energies via a reservoir of dipolar order. However, many factors remain to be investigated and optimized to enable the full potential of this polarization transfer process. We demonstrate herein the optimization of several key factors by: (i) implementing more efficient shaped radiofrequency pulses; (ii) adapting 13C spin labelling; and (iii) avoiding methyl group relaxation sinks. Experimental demonstrations are presented for the case of [1-13C]sodium acetate and other relevant molecular candidates. By employing the range of approaches set out above, polarization transfer using the dipolar order mediated cross-polarization radiofrequency pulse sequence is improved by factors approaching ∼1.65 compared with previous results. Dipolar order mediated 1H→13C polarization transfer efficiencies reaching ∼76% were achieved using significantly reduced peak radiofrequency pulse powers relative to the performance of highly sophisticated state-of-the-art cross-polarization methods, indicating 13C nuclear spin polarization levels on the order of ∼32.1% after 10 minutes of 1H DNP. The approach does not require extensive pulse sequence optimization procedures and can easily accommodate high concentrations of 13C-labelled molecules.

Solid-state 1H spin polarimetry by 13CH3 nuclear magnetic resonance.

Dissolution dynamic nuclear polarization is used to prepare nuclear spin polarizations approaching unity. At present, 1H polarization quantification in the solid state remains fastidious due to the requirement of measuring thermal equilibrium signals. Line shape polarimetry of solid-state nuclear magnetic resonance spectra is used to determine several useful properties regarding the spin system under investigation. In the case of highly polarized nuclear spins, such as those prepared under the conditions of dissolution dynamic nuclear polarization experiments, the absolute polarization of a particular isotopic species within the sample may be directly inferred from the characteristics of the corresponding resonance line shape. In situations where direct measurements of polarization are complicated by deleterious phenomena, indirect estimates of polarization using coupled heteronuclear spins prove informative. We present a simple analysis of the 13C spectral line shape of [2-13C]sodium acetate based on the normalized deviation of the centre of gravity of the 13C peaks, which can be used to indirectly evaluate the proton polarization of the methyl group moiety and very likely the entire sample in the case of rapid and homogeneous 1H-1H spin diffusion. For the case of positive microwave irradiation, 1H polarization was found to increase with an increasing normalized centre of gravity deviation. These results suggest that, as a dopant, [2-13C]sodium acetate could be used to indirectly gauge 1H polarizations in standard sample formulations, which is potentially advantageous for (i) samples polarized in commercial dissolution dynamic nuclear polarization devices that lack 1H radiofrequency hardware, (ii) measurements that are deleteriously influenced by radiation damping or complicated by the presence of large background signals and (iii) situations where the acquisition of a thermal equilibrium spectrum is not feasible.

Hyperpolarized NMR Metabolomics at Natural 13C Abundance.

Metabolomics plays a pivotal role in systems biology, and NMR is a central tool with high precision and exceptional resolution of chemical information. Most NMR metabolomic studies are based on 1H 1D spectroscopy, severely limited by peak overlap. 13C NMR benefits from a larger signal dispersion but is barely used in metabolomics due to ca. 6000-fold lower sensitivity. We introduce a new approach, based on hyperpolarized 13C NMR at natural abundance, that circumvents this limitation. A new untargeted NMR-based metabolomic workflow based on dissolution dynamic nuclear polarization (d-DNP) for the first time enabled hyperpolarized natural abundance 13C metabolomics. Statistical analysis of resulting hyperpolarized 13C data distinguishes two groups of plant (tomato) extracts and highlights biomarkers, in full agreement with previous results on the same biological model. We also optimize parameters of the semiautomated d-DNP system suitable for high-throughput studies.

Application and methodology of dissolution dynamic nuclear polarization in physical, chemical and biological contexts.

Dissolution dynamic nuclear polarization (d-DNP) is a versatile method to enhance nuclear magnetic resonance (NMR) spectroscopy. It boosts signal intensities by four to five orders of magnitude thereby providing the potential to improve and enable a plethora of applications ranging from the real-time monitoring of chemical or biological processes to metabolomics and in-cell investigations. This perspectives article highlights possible avenues for developments and applications of d-DNP in biochemical and physicochemical studies. It outlines how chemists, biologists and physicists with various fields of interest can transform and employ d-DNP as a powerful characterization method for their research.

Transport of hyperpolarized samples in dissolution-DNP experiments.

Dissolution dynamic nuclear polarization (D-DNP) experiments rely on the transfer of a sample between two high-field magnets. During this transfer, samples might experience passage through regions where the stray fields of the magnets are very weak, can approach zero, and even change their sign. This can lead to unexpected spectral features in spin systems that undergo transitions from weak- to strong-coupling regimes and vice versa, much like in field cycling nuclear magnetic resonance experiments. We herein demonstrate that the spectral features observed in D-DNP experiments can be rationalized, provided the time-dependence of the spin Hamiltonian upon field cycling is sufficiently adiabatic. Under such conditions, a passage through a weak static field can lead to the emergence of a long-lived state (LLS) based on an imbalance between the populations of singlet and triplet states in pairs of nuclei that are strongly coupled during the passage through low field. The LLS entails the appearance of anti-phase multiplet components upon transfer to a high-field magnet for observation of NMR signals.

A cryogen-consumption-free system for dynamic nuclear polarization at 9.4 T.

A novel system for dissolution dynamic nuclear polarization based on a cost-effective "cryogen-free" magnet that can generate fields up to 9.4 T with a sample space that can reach temperatures below 1.4 K in a continuous and stable manner. Polarization levels up to P(1H) = 60 ±â€¯5% can be reached with TEMPOL in about 20 min, and P(13C) = 50 ±â€¯5% can be achieved using adiabatic cross polarization.

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.

Hyperpolarized long-lived nuclear spin states in monodeuterated methyl groups.

Monodeuterated methyl groups may support a long-lived nuclear spin state, with a relaxation time exceeding the conventional spin-lattice relaxation time T1. Dissolution-DNP (dynamic nuclear polarization) may be used to hyperpolarize such a long-lived spin state. This is demonstrated for the CH2D groups of a piperidine derivative. The polarized sample is manipulated in the ambient magnetic field of the laboratory, without destruction of the hyperpolarized singlet order. Strongly enhanced CH2D signals are observed more than one minute after dissolution, even in the presence of paramagnetic radicals, by which time the NMR signal from the hyperpolarized proton magnetization has completely disappeared.

Tailored Microstructured Hyperpolarizing Matrices for Optimal Magnetic Resonance Imaging.

Tailoring the physical features and the porous network architecture of silica-based hyperpolarizing solids containing TEMPO radicals, known as HYPSO (hybrid polarizing solids), enabled unprecedented performance of dissolution dynamic nuclear polarization (d-DNP). High polarization values up to P(1 H)=99 % were reached for samples impregnated with a mixture of H2 O/D2 O and loaded in a 6.7 T polarizer at temperatures around 1.2 K. These HYPSO materials combine the best performance of homogeneous DNP formulations with the advantages of solid polarizing matrices, which provide hyperpolarized solutions free of any-potentially toxic-additives (radicals and glass-forming agents). The hyperpolarized solutions can be expelled from the porous solids, filtered, and rapidly transferred either to a nuclear magnetic resonance (NMR) spectrometer or to a magnetic resonance imaging (MRI) system.

Dynamic Nuclear Polarization Opens New Perspectives for NMR Spectroscopy in Analytical Chemistry.

Dynamic nuclear polarization (DNP) can boost sensitivity in nuclear magnetic resonance (NMR) experiments by several orders of magnitude. This Feature illustrates how the coupling of DNP with both liquid- and solid-state NMR spectroscopy has the potential to considerably extend the range of applications of NMR in analytical chemistry.