Hyperpolarizing DNA Nucleobases via NMR Signal Amplification by Reversible Exchange.

The present work investigates the potential for enhancing the NMR signals of DNA nucleobases by parahydrogen-based hyperpolarization. Signal amplification by reversible exchange (SABRE) and SABRE in Shield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) of selected DNA nucleobases is demonstrated with the enhancement (ε) of 1H, 15N, and/or 13C spins in 3-methyladenine, cytosine, and 6-O-guanine. Solutions of the standard SABRE homogenous catalyst Ir(1,5-cyclooctadeine)(1,3-bis(2,4,6-trimethylphenyl)imidazolium)Cl ("IrIMes") and a given nucleobase in deuterated ethanol/water solutions yielded low 1H ε values (≤10), likely reflecting weak catalyst binding. However, we achieved natural-abundance enhancement of 15N signals for 3-methyladenine of ~3300 and ~1900 for the imidazole ring nitrogen atoms. 1H and 15N 3-methyladenine studies revealed that methylation of adenine affords preferential binding of the imidazole ring over the pyrimidine ring. Interestingly, signal enhancements (ε~240) of both 15N atoms for doubly labelled cytosine reveal the preferential binding of specific tautomer(s), thus giving insight into the matching of polarization-transfer and tautomerization time scales. 13C enhancements of up to nearly 50-fold were also obtained for this cytosine isotopomer. These efforts may enable the future investigation of processes underlying cellular function and/or dysfunction, including how DNA nucleobase tautomerization influences mismatching in base-pairing.

Mechanisms of Methylenecyclobutane Hydrogenation over Supported Metal Catalysts Studied by Parahydrogen-Induced Polarization Technique.

In this work the mechanism of methylenecyclobutane hydrogenation over titania-supported Rh, Pt and Pd catalysts was investigated using parahydrogen-induced polarization (PHIP) technique. It was found that methylenecyclobutane hydrogenation leads to formation of a mixture of reaction products including cyclic (1-methylcyclobutene, methylcyclobutane), linear (1-pentene, cis-2-pentene, trans-2-pentene, pentane) and branched (isoprene, 2-methyl-1-butene, 2-methyl-2-butene, isopentane) compounds. Generally, at lower temperatures (150-350 °C) the major reaction product was methylcyclobutane while higher temperature of 450 °C favors the formation of branched products isoprene, 2-methyl-1-butene and 2-methyl-2-butene. PHIP effects were detected for all reaction products except methylenecyclobutane isomers 1-methylcyclobutene and isoprene implying that the corresponding compounds can incorporate two atoms from the same parahydrogen molecule in a pairwise manner in the course of the reaction in particular positions. The mechanisms were proposed for the formation of these products based on PHIP results.

Low-Cost High-Pressure Clinical-Scale 50% Parahydrogen Generator Using Liquid Nitrogen at 77 K.

We report on a robust and low-cost parahydrogen generator design employing liquid nitrogen as a coolant. The core of the generator consists of catalyst-filled spiral copper tubing, which can be pressurized to 35 atm. Parahydrogen fraction >48% was obtained at 77 K with three nearly identical generators using paramagnetic hydrated iron oxide catalysts. Parahydrogen quantification was performed on the fly via benchtop NMR spectroscopy to monitor the signal from residual orthohydrogen-parahydrogen is NMR silent. This real-time quantification approach was also used to evaluate catalyst activation at up to 1.0 standard liter per minute flow rate. The reported inexpensive device can be employed for a wide range of studies employing parahydrogen as a source of nuclear spin hyperpolarization. To this end, we demonstrate the utility of this parahydrogen generator for hyperpolarization of concentrated sodium [1-13C]pyruvate, a metabolic contrast agent under investigation in numerous clinical trials. The reported pilot optimization of SABRE-SHEATH (signal amplification by reversible exchange-shield enables alignment transfer to heteronuclei) hyperpolarization yielded 13C signal enhancement of over 14,000-fold at a clinically relevant magnetic field of 1 T corresponding to approximately 1.2% 13C polarization-if near 100% parahydrogen would have been employed, the reported value would be tripled to 13C polarization of 3.5%.

Heterogeneous Parahydrogen-Induced Polarization of Diethyl Ether for Magnetic Resonance Imaging Applications.

Magnetic resonance imaging (MRI) with the use of hyperpolarized gases as contrast agents provides valuable information on lungs structure and function. While the technology of 129 Xe hyperpolarization for clinical MRI research is well developed, it requires the expensive equipment for production and detection of hyperpolarized 129 Xe. Herein we present the 1 H hyperpolarization of diethyl ether vapor that can be imaged on any clinical MRI scanner. 1 H nuclear spin polarization of up to 1.3 % was achieved using heterogeneous hydrogenation of ethyl vinyl ether with parahydrogen over Rh/TiO2 catalyst. Liquefaction of diethyl ether vapor proceeds with partial preservation of hyperpolarization and prolongs its lifetime by ≈10 times. The proof-of-principle 2D 1 H MRI of hyperpolarized diethyl ether was demonstrated with 0.1×1.1 mm2 spatial and 120 ms temporal resolution. The long history of use of diethyl ether for anesthesia is expected to facilitate the clinical translation of the presented approach.

Low-Flammable Parahydrogen-Polarized MRI Contrast Agents.

Many MRI contrast agents formed with the parahydrogen-induced polarization (PHIP) technique exhibit biocompatible profiles. In the context of respiratory imaging with inhalable molecular contrast agents, the development of nonflammable contrast agents would nonetheless be highly beneficial for the biomedical translation of this sensitive, high-throughput and affordable hyperpolarization technique. To this end, we assess the hydrogenation kinetics, the polarization levels and the lifetimes of PHIP hyperpolarized products (acids, ethers and esters) at various degrees of fluorine substitution. The results highlight important trends as a function of molecular structure that are instrumental for the design of new, safe contrast agents for in vivo imaging applications of the PHIP technique, with an emphasis on the highly volatile group of ethers used as inhalable anesthetics.

Synthesis and 15 N NMR Signal Amplification by Reversible Exchange of [15 N]Dalfampridine at Microtesla Magnetic Fields.

Signal Amplification by Reversible Exchange (SABRE) technique enables nuclear spin hyperpolarization of wide range of compounds using parahydrogen. Here we present the synthetic approach to prepare 15 N-labeled [15 N]dalfampridine (4-amino[15 N]pyridine) utilized as a drug to reduce the symptoms of multiple sclerosis. The synthesized compound was hyperpolarized using SABRE at microtesla magnetic fields (SABRE-SHEATH technique) with up to 2.0 % 15 N polarization. The 7-hour-long activation of SABRE pre-catalyst [Ir(IMes)(COD)Cl] in the presence of [15 N]dalfampridine can be remedied by the use of pyridine co-ligand for catalyst activation while retaining the 15 N polarization levels of [15 N]dalfampridine. The effects of experimental conditions such as polarization transfer magnetic field, temperature, concentration, parahydrogen flow rate and pressure on 15 N polarization levels of free and equatorial catalyst-bound [15 N]dalfampridine were investigated. Moreover, we studied 15 N polarization build-up and decay at magnetic field of less than 0.04 µT as well as 15 N polarization decay at the Earth's magnetic field and at 1.4 T.

Heterogeneous 1 H and 13 C Parahydrogen-Induced Polarization of Acetate and Pyruvate Esters.

Magnetic resonance imaging of [1-13 C]hyperpolarized carboxylates (most notably, [1-13 C]pyruvate) allows one to visualize abnormal metabolism in tumors and other pathologies. Herein, we investigate the efficiency of 1 H and 13 C hyperpolarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties using heterogeneous hydrogenation of corresponding vinyl, allyl and propargyl precursors in isotopically unlabeled and 1-13 C-enriched forms with parahydrogen over Rh/TiO2 catalysts in methanol-d4 and in D2 O. The maximum obtained 1 H polarization was 0.6±0.2 % (for propyl acetate in CD3 OD), while the highest 13 C polarization was 0.10±0.03 % (for ethyl acetate in CD3 OD). Hyperpolarization of acetate esters surpassed that of pyruvates, while esters with a triple carbon-carbon bond in unsaturated alcoholic moiety were less efficient as parahydrogen-induced polarization precursors than esters with a double bond. Among the compounds studied, the maximum 1 H and 13 C NMR signal intensities were observed for propyl acetate. Ethyl acetate yielded slightly less intense NMR signals which were dramatically greater than those of other esters under study.

Mechanistic in situ investigation of heterogeneous hydrogenation over Rh/TiO2 catalysts: selectivity, pairwise route and catalyst nature.

The selectivity of product formation is strongly correlated with the nature of the catalyst active centers. Therefore, the selective synthesis of active sites with certain structure is a big challenge in modern catalysis. Here synthetic procedures are adopted for the formation of 1% Rh/TiO2 catalysts with different properties. It is shown that the nature of the precursor used for catalyst preparation is important, and that the use of a solution of rhodium acetate instead of rhodium nitrate leads to the selective formation of butenes during 1,3-butadiene hydrogenation. The use of parahydrogen in the reaction results in the enhancement of NMR signals via parahydrogen-induced polarization (PHIP) for all synthesized catalysts, and this signal enhancement increases with increasing catalyst calcination temperature. This effect is explained by the decoration of rhodium nanoparticles with titania which restricts hydrogen mobility on the surface, leading to the highest reported to date selectivity toward the pairwise hydrogen addition route of 7% for supported metal catalysts.

15 N NMR Hyperpolarization of Radiosensitizing Antibiotic Nimorazole by Reversible Parahydrogen Exchange in Microtesla Magnetic Fields.

Nimorazole belongs to the imidazole-based family of antibiotics to fight against anaerobic bacteria. Moreover, nimorazole is now in Phase 3 clinical trial in Europe for potential use as a hypoxia radiosensitizer for treatment of head and neck cancers. We envision the use of [15 N3 ]nimorazole as a theragnostic hypoxia contrast agent that can be potentially deployed in the next-generation MRI-LINAC systems. Herein, we report the first steps to create long-lasting (for tens of minutes) hyperpolarized state on three 15 N sites of [15 N3 ]nimorazole with T1 of up to ca. 6 minutes. The nuclear spin polarization was boosted by ca. 67000-fold at 1.4 T (corresponding to P15N of 3.2 %) by 15 N-15 N spin-relayed SABRE-SHEATH hyperpolarization technique, relying on simultaneous exchange of [15 N3 ]nimorazole and parahydrogen on polarization transfer Ir-IMes catalyst. The presented results pave the way to efficient spin-relayed SABRE-SHEATH hyperpolarization of a wide range of imidazole-based antibiotics and chemotherapeutics.

Pulse-Programmable Magnetic Field Sweeping of Parahydrogen-Induced Polarization by Side Arm Hydrogenation.

Among the hyperpolarization techniques geared toward in vivo magnetic resonance imaging, parahydrogen-induced polarization (PHIP) shows promise due to its low cost and fast speed of contrast agent preparation. The synthesis of 13C-labeled, unsaturated precursors to perform PHIP by side arm hydrogenation has recently opened new possibilities for metabolic imaging owing to the biological compatibility of the reaction products, although the polarization transfer between the parahydrogen-derived protons and the 13C heteronucleus must yet be better understood, characterized, and eventually optimized. In this realm, a new experimental strategy incorporating pulse-programmable magnetic field sweeping and in situ detection has been developed. The approach is evaluated by measuring the 13C polarization of ethyl acetate-1-13C, i.e., the product of pairwise addition of parahydrogen to vinyl acetate-1-13C, resulting from zero-crossing magnetic field ramps of various durations, amplitudes, and step sizes. The results demonstrate (i) the profound effect these parameters have on the 1H to 13C polarization transfer efficiency and (ii) the high reproducibility of the technique.

Parahydrogen-Induced Polarization of Diethyl Ether Anesthetic.

The growing interest in magnetic resonance imaging (MRI) for assessing regional lung function relies on the use of nuclear spin hyperpolarized gas as a contrast agent. The long gas-phase lifetimes of hyperpolarized 129 Xe make this inhalable contrast agent acceptable for clinical research today despite limitations such as high cost, low throughput of production and challenges of 129 Xe imaging on clinical MRI scanners, which are normally equipped with proton detection only. We report on low-cost and high-throughput preparation of proton-hyperpolarized diethyl ether, which can be potentially employed for pulmonary imaging with a nontoxic, simple, and sensitive overall strategy using proton detection commonly available on all clinical MRI scanners. Diethyl ether is hyperpolarized by pairwise parahydrogen addition to vinyl ethyl ether and characterized by 1 H NMR spectroscopy. Proton polarization levels exceeding 8 % are achieved at near complete chemical conversion within seconds, causing the activation of radio amplification by stimulated emission radiation (RASER) throughout detection. Although gas-phase T1 relaxation of hyperpolarized diethyl ether (at partial pressure of 0.5 bar) is very efficient, with T1 of ca. 1.2 second, we demonstrate that, at low magnetic fields, the use of long-lived singlet states created via pairwise parahydrogen addition extends the relaxation decay by approximately threefold, paving the way to bioimaging applications and beyond.

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.

Parahydrogen-Induced Hyperpolarization of Gases.

Imaging of gases is a major challenge for any modality including MRI. NMR and MRI signals are directly proportional to the nuclear spin density and the degree of alignment of nuclear spins with applied static magnetic field, which is called nuclear spin polarization. The level of nuclear spin polarization is typically very low, i.e., one hundred thousandth of the potential maximum at 1.5 T and a physiologically relevant temperature. As a result, MRI typically focusses on imaging highly concentrated tissue water. Hyperpolarization methods transiently increase nuclear spin polarizations up to unity, yielding corresponding gains in MRI signal level of several orders of magnitude that enable the 3D imaging of dilute biomolecules including gases. Parahydrogen-induced polarization is a fast, highly scalable, and low-cost hyperpolarization technique. The focus of this Minireview is to highlight selected advances in the field of parahydrogen-induced polarization for the production of hyperpolarized compounds, which can be potentially employed as inhalable contrast agents.

Clinical-Scale Batch-Mode Production of Hyperpolarized Propane Gas for MRI.

NMR spectroscopy and imaging (MRI) are two of the most important methods to study structure, function, and dynamics from atom to organism scale. NMR approaches often suffer from an insufficient sensitivity, which, however, can be transiently boosted using hyperpolarization techniques. One of these techniques is parahydrogen-induced polarization, which has been used to produce catalyst-free hyperpolarized propane gas with proton polarization that is 3 orders of magnitude greater than equilibrium thermal polarization at a 1.5 T field of a clinical MRI scanner. Here we show that more than 0.3 L of hyperpolarized propane gas can be produced in 2 s. This production rate is more than an order of magnitude greater than that demonstrated previously, and the reported production rate is comparable to that employed for in-human MRI using HP noble gas (e.g., 129Xe) produced via a spin exchange optical pumping (SEOP) hyperpolarization technique. We show that high polarization values can be retained despite the significant increase in the production rate of hyperpolarized propane. The enhanced signals of produced hyperpolarized propane gas were revealed by stopped-flow MRI visualization at 4.7 T. Achieving this high production rate enables the future use of this compound (already approved for unlimited use in foods by the corresponding regulating agencies, e.g., FDA in the USA, and more broadly as an E944 food additive) as a new inhalable contrast agent for diagnostic detection via MRI.

Single-Site Heterogeneous Catalysts: From Synthesis to NMR Signal Enhancement.

Catalysts with well-defined, single, active centers are of great importance and their utilization allows the gap between homo- and heterogeneous catalysis to be bridged and, importantly, the main selectivity problem of heterogeneous catalysis and the main separation challenge of homogeneous catalysis to be overcome. Moreover, the use of single-site catalysts allows the NMR signal to be significantly enhanced through the pairwise addition of two hydrogen atoms from a parahydrogen molecule to an unsaturated substrate. This review covers the fundamentals of the synthesis of single-site catalysts and shows the new aspects of their applications in both modern catalysis and the field of parahydrogen-based hyperpolarization. The different novel aspects of the formation and utilization of single-site catalysts, along with the possibility of NMR signal enhancement observations are described.

15 N Hyperpolarization of Dalfampridine at Natural Abundance for Magnetic Resonance Imaging.

Signal Amplification by Reversible Exchange (SABRE) is a promising method for NMR signal enhancement and production of hyperpolarized molecules. As nuclear spin relaxation times of heteronuclei are usually much longer than those of protons, SABRE-based hyperpolarization of heteronuclei in molecules is highly important in the context of biomedical applications. In this work, we demonstrate that the SLIC-SABRE technique can be successfully used to hyperpolarize 15 N nuclei in dalfampridine. The high polarization level of ca. 8 % achieved in this work made it possible to acquire 15 N MR images at natural abundance of the 15 N nuclei for the first time.

15 N MRI of SLIC-SABRE Hyperpolarized 15 N-Labelled Pyridine and Nicotinamide.

Magnetic Resonance Imaging (MRI) is a powerful non-invasive diagnostic method extensively used in biomedical studies. A significant limitation of MRI is its relatively low signal-to-noise ratio, which can be increased by hyperpolarizing nuclear spins. One promising method is Signal Amplification By Reversible Exchange (SABRE), which employs parahydrogen as a source of hyperpolarization. Recent studies demonstrated the feasibility to improve MRI sensitivity with this hyperpolarization technique. Hyperpolarized 15 N nuclei in biomolecules can potentially retain their spin alignment for tens of minutes, providing an extended time window for the utilization of the hyperpolarized compounds. In this work, we demonstrate for the first time that radio-frequency-based SABRE hyperpolarization techniques can be used to obtain 15 N MRI of biomolecule 1-15 N-nicotinamide. Two image acquisition strategies were utilized and compared: Single Point Imaging (SPI) and Fast Low Angle SHot (FLASH). These methods demonstrated opportunities of high-field SABRE for biomedical applications.

Hyperpolarizing Concentrated Metronidazole 15 NO2 Group over Six Chemical Bonds with More than 15 % Polarization and a 20†Minute Lifetime.

The NMR hyperpolarization of uniformly 15 N-labeled [15 N3 ]metronidazole is demonstrated by using SABRE-SHEATH. In this antibiotic, the 15 NO2 group is hyperpolarized through spin relays created by 15 N spins in [15 N3 ]metronidazole, and the polarization is transferred from parahydrogen-derived hydrides over six chemical bonds. In less than a minute of parahydrogen bubbling at approximately 0.4 µT, a high level of nuclear spin polarization (P15N ) of around 16 % is achieved on all three 15 N sites. This product of 15 N polarization and concentration of 15 N spins is around six-fold better than any previous value determined for 15 N SABRE-derived hyperpolarization. At 1.4 T, the hyperpolarized state persists for tens of minutes (relaxation time, T1 ≈10 min). A novel synthesis of uniformly 15 N-enriched metronidazole is reported with a yield of 15 %. This approach can potentially be used for synthesis of a wide variety of in vivo metabolic probes with potential uses ranging from hypoxia sensing to theranostic imaging.

Heterogeneous hydrogenation of phenylalkynes with parahydrogen: hyperpolarization, reaction selectivity, and kinetics.

Parahydrogen-induced polarization (PHIP) is a powerful technique for studying hydrogenation reactions in gas and liquid phases. Pairwise addition of parahydrogen to the hydrogenation substrate imparts nuclear spin order to reaction products, manifested as enhanced 1H NMR signals from the nascent proton sites. Nanoscale metal catalysts immobilized on supports comprise a promising class of catalysts for producing PHIP effects; however, on such catalysts the percentage of substrates undergoing the pairwise addition route-a necessary condition for observing PHIP-is usually low. In this paper, we present a systematic study of several metal catalysts (Rh, Pt, Pd, and Ir) supported on TiO2 in liquid-phase hydrogenation of different prototypical phenylalkynes (phenylacetylene, 1-phenyl-1-propyne, and 3-phenyl-1-propyne) with parahydrogen. Catalyst activity and selectivity were found to be affected by both the nature of the active metal and the percentage of metal loading. It was demonstrated that the optimal catalyst for production of hyperpolarized products is Rh/TiO2 with 4 wt% metal loading, whereas Pd/TiO2 provided the greatest selectivity for semihydrogenation of phenylalkynes. In a study of liquid-phase hydrogenation reaction kinetics, it was shown that reaction order with respect to hydrogen is nearly the same for pairwise and non-pairwise H2 addition-consistent with a similar nature of the catalytically active sites for these reaction pathways.

A versatile synthetic route to the preparation of 15 N heterocycles.

A robust medium-scale (approximately 3 g) synthetic method for 15 N labeling of pyridine (15 N-Py) is reported based on the Zincke reaction. 15 N enrichment in excess of 81% was achieved with approximately 33% yield. 15 N-Py serves as a standard substrate in a wide range of studies employing a hyperpolarization technique for efficient polarization transfer from parahydrogen to heteronuclei; this technique, called SABRE (signal amplification by reversible exchange), employs a simultaneous chemical exchange of parahydrogen and a to-be-hyperpolarized substrate (e.g., pyridine) on metal centers. In studies aimed at the development of hyperpolarized contrast agents for in vivo molecular imaging, pyridine is often employed either as a model substrate (for hyperpolarization technique development, quality assurance, and phantom imaging studies) or as a co-substrate to facilitate more efficient hyperpolarization of a wide range of emerging contrast agents (e.g., nicotinamide). Here, the produced 15 N-Py was used for the feasibility study of spontaneous 15 N hyperpolarization at high magnetic (HF) fields (7 T and 9.4 T) of an NMR spectrometer and an MRI scanner. SABRE hyperpolarization enabled acquisition of 2D MRI imaging of catalyst-bound 15 N-pyridine with 75 × 75 mm2 field of view (FOV), 32 × 32 matrix size, demonstrating the feasibility of 15 N HF-SABRE molecular imaging with 2.4 × 2.4 mm2 spatial resolution.