Parahydrogen-induced Polarization Research Articles

Opportunities and challenges with hyperpolarized bioresponsive probes for functional imaging using magnetic resonance.

The development of hyperpolarized bioresponsive probes for magnetic resonance imaging (MRI) applications is an emerging and rapidly growing topic in chemistry. A wide range of hyperpolarized molecular biosensors for functional MRI have been developed in recent years. These probes comprise many different types of small-molecule reporters that can be hyperpolarized using dissolution dynamic nuclear polarization and parahydrogen-induced polarization or xenon-chelated macromolecular conjugates hyperpolarized using spin-exchange optical pumping. In this Perspective, we discuss how the amplified magnetic resonance signals of these agents are responsive to biologically relevant stimuli such as target proteins, reactive oxygen species, pH or metal ions. We examine how functional MRI using these systems allows a great number of biological processes to be monitored rapidly. Consequently, hyperpolarized bioresponsive probes may play a critical role in functional molecular imaging for observing physiology and pathology in real time.

Parahydrogen-induced polarization allows 2000-fold signal enhancement in biologically active derivatives of the peptide-based drug octreotide

Octreotide, a somatostatin analogue, has shown its efficacy for the diagnostics and treatment of various types of cancer, i.e., in octreotide scan, as radio-marker after labelling with a radiopharmaceutical. To avoid toxicity of radio-labeling, octreotide-based assays can be implemented into magnetic resonance techniques, such as MRI and NMR. Here we used a Parahydrogen-Induced Polarization (PHIP) approach as a cheap, fast and straightforward method. Introduction of l-propargyl tyrosine as a PHIP marker at different positions of octreotide by manual Solid-Phase Peptide Synthesis (SPPS) led to up to 2000-fold proton signal enhancement (SE). Cell binding studies confirmed that all octreotide variants retained strong binding affinity to the surface of human-derived cancer cells expressing somatostatin receptor 2. The hydrogenation reactions were successfully performed in methanol and under physiologically compatible mixtures of water with methanol or ethanol. The presented results open up new application areas of biochemical and pharmacological studies with octreotide.

13C and 15N Benchtop NMR Detection of Metabolites via Relayed Hyperpolarization**

AbstractParahydrogen‐based nuclear spin hyperpolarization allows various magnetic‐resonance applications, and it is particularly attractive because of its technical simplicity, low cost, and ability to quickly (in seconds) produce large volumes of hyperpolarized material. Although many parahydrogen‐based techniques have emerged, some of them remain unexplored due to the lack of careful optimization studies. In this work, we investigate and optimize a novel parahydrogen‐induced polarization (PHIP) technique that relies on proton exchange referred to below as PHIP‐relay. An INEPT (insensitive nuclei enhanced by polarization transfer) sequence is employed to transfer polarization from hyperpolarized protons to heteronuclei ( N and C) and nuclear signals are detected using benchtop NMR spectrometers (1 T and 1.4 T, respectively). We demonstrate the applicability of the PHIP‐relay technique for hyperpolarization of a wide range of biochemicals by examining such key metabolites as urea, ammonium, glucose, amino acid glycine, and a drug precursor benzamide. By optimizing chemical and NMR parameters of the PHIP‐relay, we achieve a 17,100‐fold enhancement of N signal of [ C, N ]‐urea compared to the thermal signal measured at 1 T. We also show that repeated measurements with shorter exposure to parahydrogen provide a higher effective signal‐to‐noise ratio compared to longer parahydrogen bubbling.

Quantum-enhanced MRI Sensitivity: Dissolution-dynamic Nuclear and Parahydrogen-induced Polarization

Contemporarily, magnetic resonance imaging (MRI) has been widely utilized in medical diagnostic. Among various features, the signal intensities serve as the key role in resolution of the detection results. In general, the MRI signal intensities can be substantially increased by several orders of magnitude via dissolution-dynamic nuclear polarization (d-DNP) and parahydrogen-induced polarization (PHIP). This study exhibits the general principles and components of the preparation of these two methods, as well as illustrates the current applications and limitations of d-DNP and PHIP both theoretically and analytically. The experimental conditions, including temperature, magnetic field strength, and whether microwave irradiation is required, are described and contrasted. According to the analysis, the advantages and drawbacks of these two approaches have been compared, along with expectations and outlooks of the future development of quantum-enhanced MRI techniques, in terms of the MRI signal sensitivity. Overall, these results shed light on guiding further exploration of enhancing the resolution of MRI.

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.

Real-Time Pyruvate Chemical Conversion Monitoring Enabled by PHIP.

In recent years, parahydrogen-induced polarization side arm hydrogenation (PHIP-SAH) has been applied to hyperpolarize [1-13C]pyruvate and map its metabolic conversion to [1-13C]lactate in cancer cells. Developing on our recent MINERVA pulse sequence protocol, in which we have achieved 27% [1-13C]pyruvate carbon polarization, we demonstrate the hyperpolarization of [1,2-13C]pyruvate (∼7% polarization on each 13C spin) via PHIP-SAH. By altering a single parameter in the pulse sequence, MINERVA enables the signal enhancement of C1 and/or C2 in [1,2-13C]pyruvate with the opposite phase, which allows for the simultaneous monitoring of different chemical reactions with enhanced spectral contrast or for the same reaction via different carbon sites. We first demonstrate the ability to monitor the same enzymatic pyruvate to lactate conversion at 7T in an aqueous solution, in vitro, and in-cell (HeLa cells) via different carbon sites. In a second set of experiments, we use the C1 and C2 carbon positions as spectral probes for simultaneous chemical reactions: the production of acetate, carbon dioxide, bicarbonate, and carbonate by reacting [1,2-13C]pyruvate with H2O2 at a high temperature (55 °C). Importantly, we detect and characterize the intermediate 2-hydroperoxy-2-hydroxypropanoate in real time and at high temperature.

Understanding Heterogeneous Catalytic Hydrogenation by Parahydrogen-Induced Polarization NMR Spectroscopy

Parahydrogen-induced polarization (PHIP) is an NMR hyperpolarization technique with the ability to increase the NMR sensitivity by serval orders. The generation of hyperpolarization in hydrogenation reactions is associated with a pairwise addition behavior, which is sensitive to the properties of catalysts and the reaction process, making it a powerful tool for the characterization of catalyst properties and elucidation of reaction mechanisms. In this Perspective, we provide an in-depth overview of the heterogeneous PHIP (HET-PHIP) technique and its application in heterogeneous catalytic hydrogenation. The fundamentals of HET-PHIP are introduced. We describe how HET-PHIP can be used to obtain information about the morphology and electronic properties of supported monometallic catalysts, bimetallic catalysts, and zeolites as well as the facet effect and strong metal–support interaction (SMSI) effect in hydrogenation. Advances in the mechanistic understanding of catalytic hydrogenations by using HET-PHIP are discussed with representative work. We propose the current limitations and prospects of the HET-PHIP technique in catalytic hydrogenation reactions.

Radio-Frequency Sweeps at Microtesla Fields for Parahydrogen-Induced Polarization of Biomolecules

Magnetic resonance imaging of 13C-labeled metabolites enhanced by parahydrogen-induced polarization (PHIP) enables real-time monitoring of processes within the body. We introduce a robust, easily implementable technique for transferring parahydrogen-derived singlet order into 13C magnetization using adiabatic radio frequency sweeps at microtesla fields. We experimentally demonstrate the applicability of this technique to several molecules, including some molecules relevant for metabolic imaging, where we show significant improvements in the achievable polarization, in some cases reaching above 60% nuclear spin polarization. Furthermore, we introduce a site-selective deuteration scheme, where deuterium is included in the coupling network of a pyruvate ester to enhance the efficiency of the polarization transfer. These improvements are enabled by the fact that the transfer protocol avoids relaxation induced by strongly coupled quadrupolar nuclei.

Nanomaterials for hyperpolarized nuclear magnetic resonance and magnetic resonance imaging.

Nanomaterials play an important role in the development and application of hyperpolarized materials for magnetic resonance imaging (MRI). In this context they can not only act as hyperpolarized materials which are directly imaged but also play a role as carriers for hyperpolarized gases and catalysts for para-hydrogen induced polarization (PHIP) to generate hyperpolarized substrates for metabolic imaging. Those three application possibilities are discussed, focusing on carbon-based materials for the directly imaged particles. An overview over recent developments in all three fields is given, including the early developments in each field as well as important steps towards applications in MRI, such as making the initially developed methods more biocompatible and first imaging experiments with spatial resolution in either phantoms or in vivo studies. Focusing on the important features nanomaterials need to display to be applicable in the MRI context, a wide range of different approaches to that extent is covered, giving the reader a general idea of different possibilities as well as recent developments in those different fields of hyperpolarized magnetic resonance. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Mechanistic Insights into Molecular Crystalline Organometallic Heterogeneous Catalysis through Parahydrogen-Based Nuclear Magnetic Resonance Studies.

The heterogeneous solid-gas reactions of crystals of [Rh(L2)(propene)][BArF4] (1, L2 = tBu2PCH2CH2PtBu2) with H2 and propene, 1-butene, propyne, or 1-butyne are explored by gas-phase nuclear magnetic resonance (NMR) spectroscopy under batch conditions at 25 °C. The temporal evolution of the resulting parahydrogen-induced polarization (PHIP) effects measures catalytic flux and thus interrogates the efficiency of catalytic pairwise para-H2 transfer, speciation changes in the crystalline catalyst at the molecular level, and allows for high-quality single-scan 1H, 13C NMR gas-phase spectra for the products to be obtained, as well as 2D-measurements. Complex 1 reacts with H2 to form dimeric [Rh(L2)(H)(μ-H)]2[BArF4]2 (4), as probed using EXAFS; meanwhile, a single-crystal of 1 equilibrates NMR silent para-H2 with its NMR active ortho isomer, contemporaneously converting into 4, and 1 and 4 each convert para-H2 into ortho-H2 at different rates. Hydrogenation of propene using 1 and para-H2 results in very high initial polarization levels in propane (>85%). Strong PHIP was also detected in the hydrogenation products of 1-butene, propyne, and 1-butyne. With propyne, a competing cyclotrimerization deactivation process occurs to afford [Rh(tBu2PCH2CH2PtBu2)(1,3,4-Me3C6H3)][BArF4], while with 1-butyne, rapid isomerization of 1-butyne occurs to give a butadiene complex, which then reacts with H2 more slowly to form catalytically active 4. Surprisingly, the high PHIP hydrogenation efficiencies allow hyperpolarization effects to be seen when H2 is taken directly from a regular cylinder at 25 °C. Finally, changing the chelating phosphine to Cy2PCH2CH2PCy2 results in initial high polarization efficiencies for propene hydrogenation, but rapid quenching of the catalyst competes to form the zwitterion [Rh(Cy2PCH2CH2PCy2){η6-(CF3)2(C6H3)}BArF3].

Anomalously Large Antiphase Signals from Hyperpolarized Orthohydrogen Using a MOF‐Based SABRE Catalyst

AbstractHyperpolarized orthohydrogen (o‐H2) is a frequent product of parahydrogen‐based hyperpolarization approaches like signal amplification by reversible exchange (SABRE), where the hyperpolarized o‐H2 signal is usually absorptive. We describe a novel manifestation of this effect wherein large antiphase o‐H2 signals are observed, with 1H enhancements up to ≈500‐fold (effective polarization PH≈1.6 %). This anomalous effect is attained only when using an intact heterogeneous catalyst constructed using a metal–organic framework (MOF) and is qualitatively independent of substrate nature. This seemingly paradoxical observation is analogous to the “partial negative line” (PNL) effect recently explained in the context of Parahydrogen Induced Polarization (PHIP) by Ivanov and co‐workers. The two‐spin order of the o‐H2 resonance is manifested by a two‐fold higher Rabi frequency, and the lifetime of the antiphase HP o‐H2 resonance is extended by several‐fold.

Anomalously Large Antiphase Signals from Hyperpolarized Orthohydrogen Using a MOF-Based SABRE Catalyst.

Hyperpolarized orthohydrogen (o-H2 ) is a frequent product of parahydrogen-based hyperpolarization approaches like signal amplification by reversible exchange (SABRE), where the hyperpolarized o-H2 signal is usually absorptive. We describe a novel manifestation of this effect wherein large antiphase o-H2 signals are observed, with 1 H enhancements up to ≈500-fold (effective polarization PH ≈1.6 %). This anomalous effect is attained only when using an intact heterogeneous catalyst constructed using a metal-organic framework (MOF) and is qualitatively independent of substrate nature. This seemingly paradoxical observation is analogous to the "partial negative line" (PNL) effect recently explained in the context of Parahydrogen Induced Polarization (PHIP) by Ivanov and co-workers. The two-spin order of the o-H2 resonance is manifested by a two-fold higher Rabi frequency, and the lifetime of the antiphase HP o-H2 resonance is extended by several-fold.

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.

Real-time cell metabolism assessed repeatedly on the same cells via para-hydrogen induced polarization.

Signal-enhanced or hyperpolarized nuclear magnetic resonance (NMR) spectroscopy stands out as a unique tool to monitor real-time enzymatic reactions in living cells. The singlet state of para-hydrogen is thereby one source of spin order that can be converted into largely enhanced signals of e.g. metabolites. Here, we have investigated a parahydrogen-induced polarization (PHIP) approach as a biological assay for in vitro cellular metabolic characterization. Here, we demonstrate the possibility to perform consecutive measurements yielding metabolic information on the same sample. We observed a strongly reduced pyruvate-to-lactate conversion rate (flux) of a Hodgkin's lymphoma cancer cell line L1236 treated with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT) affecting the amount of NAD+ and thus NADH in cells. In the consecutive measurement the flux was recovered by NADH to the same amount as in the single-measurement-per-sample and provides a promising new analytical tool for continuous real-time studies combinable with bioreactors and lab-on-a-chip devices in the future.

Parahydrogen-Induced Hyperpolarization of Unsaturated Phosphoric Acid Derivatives

Parahydrogen-induced nuclear polarization offers a significant increase in the sensitivity of NMR spectroscopy to create new probes for medical diagnostics by magnetic resonance imaging. As precursors of the biocompatible hyperpolarized probes, unsaturated derivatives of phosphoric acid, propargyl and allyl phosphates, are proposed. The polarization transfer to 1H and 31P nuclei of the products of their hydrogenation by parahydrogen under the ALTADENA and PASADENA conditions, and by the PH-ECHO-INEPT+ pulse sequence of NMR spectroscopy, resulted in a very high signal amplification, which is among the largest for parahydrogen-induced nuclear polarization transfer to the 31P nucleus.

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.

Parahydrogen-induced polarization and spin order transfer in ethyl pyruvate at high magnetic fields

Nuclear magnetic resonance has experienced great advances in developing and translating hyperpolarization methods into procedures for fundamental and clinical studies. Here, we propose the use of a wide-bore NMR for large-scale (volume- and concentration-wise) production of hyperpolarized media using parahydrogen-induced polarization. We discuss the benefits of radio frequency-induced parahydrogen spin order transfer, we show that 100% polarization is theoretically expected for homogeneous B0 and B1 magnetic fields for a three-spin system. Moreover, we estimated that the efficiency of spin order transfer is not significantly reduced when the B1 inhomogeneity is below ± 5%; recommendations for the sample size and RF coils are also given. With the latest breakthrough in the high-yield synthesis of 1-13C-vinyl pyruvate and its deuterated isotopologues, the high-field PHIP-SAH will gain increased attention. Some remaining challenges will be addressed shortly.

Polarizing insensitive nuclei at ultralow magnetic fields using parahydrogen: A facile route to optimize adiabatic magnetic field sweeps.

Parahydrogen induced polarization (PHIP) provides a powerful tool to enhance inherently weak nuclear magnetic resonance signals, particularly in biologically relevant compounds. The initial source of PHIP is the non-equilibrium spin order of parahydrogen, i.e., dihydrogen, where the two protons make up a singlet spin state. Conversion of this spin order into net magnetization of magnetic heteronuclei, e.g., 13C, provides one of the most efficient ways to exploit PHIP. We propose a facile route to increase the performance of PHIP transfer in experiments with adiabatic sweeps of the ultralow magnetic field. To date, this technique yields the highest efficiency of PHIP transfer, yet, it has been mostly utilized with linear field sweeps, which does not consider the underlying spin dynamics, resulting in sub-optimal polarization. This issue was previously addressed by using the "constant" adiabaticity method, which, however, requires extensive calculations for large spin systems. In this work, the field sweep is optimized by utilizing the field dependence of the average 13C polarization. Both the experimental detection and the numerical simulation of this dependence are straightforward, even for complex multi-spin systems. This work provides a comprehensive survey of PHIP transfer dynamics at ultralow fields for two molecular systems that are relevant for PHIP, namely, maleic acid and allyl pyruvate. The proposed optimization allowed us to increase the resulting 13C polarization in 13C-allyl pyruvate from 6.8% with a linear profile to 8.7% with an "optimal" profile. Such facile optimization routines are valuable for adiabatic experiments in complex spin systems undergoing rapid relaxation or chemical exchange.

Synthesis of 13 C and 2 H Labeled Vinyl Pyruvate and Hyperpolarization of Pyruvate.

The hyperpolarization of nuclear spins has enabled unique applications in chemistry, biophysics, and particularly metabolic imaging. Parahydrogen-induced polarization (PHIP) offers a fast and cost-efficient way of hyperpolarization. Nevertheless, PHIP lags behind dynamic nuclear polarization (DNP), which is already being evaluated in clinical studies. This shortcoming is mainly due to problems in the synthesis of the corresponding PHIP precursor molecules. The most widely used DNP tracer in clinical studies, particularly for the detection of prostate cancer, is 1-13 C-pyruvate. The ideal derivative for PHIP is the deuterated vinyl ester because the spin physics allows for 100 % polarization. Unfortunately, there is no efficient synthesis for vinyl esters of β-ketocarboxylic acids in general and pyruvate in particular. Here, we present an efficient new method for the preparation of vinyl esters, including 13 C labeled, fully deuterated vinyl pyruvate using a palladium-catalyzed procedure. Using 50 % enriched parahydrogen and mild reaction conditions, a 13 C polarization of 12 % was readily achieved; 36 % are expected with 100 % pH2 . Higher polarization values can be potentially achieved with optimized reaction conditions.

Deuteron-Decoupled Singlet NMR in Low Magnetic Fields: Application to the Hyperpolarization of Succinic Acid.

The reaction of unsaturated substrates with hydrogen gas enriched in the para spin isomer leads to products with a high degree of nuclear singlet spin order. This leads to greatly enhanced NMR signals, with important potential applications such as magnetic resonance imaging (MRI) of metabolic processes. Although parahydrogen-induced polarization has the advantage of being cheap, compact, and mobile, especially when performed in ultralow magnetic fields, efficiency is lost when more than a few protons are involved. This strongly restricts the range of compatible substances. We show that these difficulties may be overcome by a combination of deuteration with the application of a sinusoidally modulated longitudinal field as a well as a transverse rotating magnetic field. We demonstrate a six-fold enhancement in the 13 C hyperpolarization of [1-13 C, 2,3-d2 ]-succinic acid, as compared with standard hyperpolarization methods, applied in the same ultralow field regime.