Signal Amplification By Reversible Exchange Hyperpolarization Research Articles

Adiabatic approach for heteronuclear SABRE hyperpolarization at high magnetic field

Signal Amplification By Reversible Exchange (SABRE) is a technique aimed at enhancing weak NMR signals of heteronuclei by utilizing the non-equilibrium spin order of parahydrogen. SABRE polarization transfer takes place by means of metalorganic complexes that interact with parahydrogen and the substrate to be polarized in a reversible manner. To achieve substrate hyperpolarization in the high magnetic field of an NMR magnet, radiofrequency (RF) excitation is required. There are two general options for the RF field amplitude: constant or modulated. To date, there has been limited optimization of the adiabatic SABRE conditions. In SABRE, the presence of chemical exchange significantly complicates the spin dynamics involved in polarization transfer and the optimization of adiabatic RF sweeps. We conducted a comprehensive analysis of high-field SABRE pulse sequences with RF sweeps on the heteronuclear channel, specifically 15N. We proposed a simple method for optimizing the amplitude modulation profile of the RF field, which is efficient for systems undergoing chemical exchange. Our approach involved utilizing the dependence of 15N polarization on the amplitude of the constant RF field on the 15N channel. By employing the "optimal" adiabatic RF profile, we achieved a 2.5-fold increase in 15N SABRE-derived polarization at high magnetic field compared to a linear sweep. We theoretically assessed the benefit of RF sweeps over constant RF fields for SABRE at high magnetic field. We demonstrated experimentally that at temperatures −5∘C - +10∘C RF sweeps are more efficient than constant RF field. Maximal increase in 15N polarization achieved was 1.7-fold for bound and 1.4-fold for free substrate. We attribute this increase in polarization to the adiabaticity of the polarization transfer process. This behavior was explained via numerical solution of SABRE master equation for different dissociation rate constants.

Modern Manufacturing Enables Magnetic Field Cycling Experiments and Parahydrogen-Induced Hyperpolarization with a Benchtop NMR.

Benchtop NMR (btNMR) spectrometers are revolutionizing the way we use NMR and lowering the cost drastically. Magnetic field cycling (MFC) experiments with precise timing and control over the magnetic field, however, were hitherto not available on btNMRs, although some systems exist for high-field, high-resolution NMR spectrometers. Still, the need and potential for btNMR MFC is great─e.g., to perform and analyze parahydrogen-induced hyperpolarization, another method that has affected analytical chemistry and NMR beyond expectations. Here, we describe a setup that enables MFC on btNMRs for chemical analysis and hyperpolarization. Taking full advantage of the power of modern manufacturing, including computer-aided design, three-dimensional printing, and microcontrollers, the setup is easy to reproduce, highly reliable, and easy to adjust and operate. Within 380 ms, the NMR tube was shuttled reliably from the electromagnet to the NMR isocenter (using a stepper motor and gear rod). We demonstrated the power of this setup by hyperpolarizing nicotinamide using signal amplification by reversible exchange (SABRE), a versatile method to hyperpolarize a broad variety of molecules including metabolites and drugs. Here, the standard deviation of SABRE hyperpolarization was between 0.2 and 3.3%. The setup also allowed us to investigate the field dependency of the polarization and the effect of different sample preparation protocols. We found that redissolution of the activated and dried Ir catalyst always reduced the polarization. We anticipate that this design will greatly accelerate the ascension of MFC experiments for chemical analysis with btNMR─adding yet another application to this rapidly developing field.

Enhancing 19F Benchtop NMR Spectroscopy by Combining para-Hydrogen Hyperpolarization and Multiplet Refocusing.

Benchtop NMR spectrometers provide a promising alternative to high-field NMR for applications that are limited by instrument size and/or cost. 19F benchtop NMR is attractive due to the larger chemical shift range of 19F relative to 1H and the lack of background signal in most applications. However, practical applications of benchtop 19F NMR are limited by its low sensitivity due to the relatively weak field strengths of benchtop NMR spectrometers. Here we present a sensitivity-enhancement strategy that combines SABRE (Signal Amplification By Reversible Exchange) hyperpolarization with the multiplet refocusing method SHARPER (Sensitive, Homogeneous, And Resolved PEaks in Real time). When applied to a range of fluoropyridines, SABRE-SHARPER achieves overall signal enhancements of up to 5700-fold through the combined effects of hyperpolarization and line-narrowing. This approach can be generalized to the analysis of mixtures through the use of a selective variant of the SHARPER sequence, selSHARPER. The ability of SABRE-selSHARPER to simultaneously boost sensitivity and discriminate between two components of a mixture is demonstrated, where selectivity is achieved through a combination of selective excitation and the choice of polarization transfer field during the SABRE step.

Screening of Protein-Ligand Binding Using a SABRE Hyperpolarized Reporter.

Hyperpolarization through signal amplification by reversible exchange (SABRE) provides a facile means to enhance nuclear magnetic resonance (NMR) signals of small molecules containing an N-heterocycle or other binding site for a polarization transfer catalyst. A purpose-designed reporter ligand, which is capable of binding both to a target protein and to the catalyst, makes the sensitivity enhancement by this technique compatible with the measurement of a range of biomolecular interactions. The 1H polarization of the reporter ligand 4-amidinopyridine, which is targeting trypsin, is used to screen ligands that are not themselves hyperpolarizable by SABRE. The respective protein-ligand dissociation constants (KD) are determined by an observed change in the R2 relaxation rate of the reporter. A calculation of expected signal changes indicates that the accessible ligand KD values extend over several orders of magnitude, while the concentrations of target proteins and ligands can be reduced considering the sensitivity gains from hyperpolarization. In general, the design of a single, weakly binding ligand for a target protein enables the use of SABRE hyperpolarization for ligand screening or other biophysical studies involving macromolecular interactions.

Improving SABRE hyperpolarization with highly nonintuitive pulse sequences: Moving beyond avoided crossings to describe dynamics.

Signal amplification by reversible exchange (SABRE) creates “hyperpolarization” (large spin magnetization) using a transition metal catalyst and parahydrogen, addressing the sensitivity limitations of magnetic resonance. SABRE and its heteronuclear variant X-SABRE are simple, fast, and general, but to date have not produced polarization levels as large as more established methods. We show here that the commonly used theoretical framework for these applications, which focuses on avoided crossings (also called level anticrossings or LACs), steer current SABRE and X-SABRE experiments away from optimal solutions. Accurate simulations show astonishingly rich and unexpected dynamics in SABRE/X-SABRE, which we explain with a combination of perturbation theory and average Hamiltonian approaches. This theoretical picture predicts simple pulse sequences with field values far from LACs (both instantaneously and on average) using different terms in the effective Hamiltonian to strategically control evolution and improve polarization transfer. Substantial signal enhancements under such highly nonintuitive conditions are verified experimentally.

Phase coherent excitation of SABRE permits simultaneous hyperpolarization of multiple targets at high magnetic field.

Hyperpolarization methods in magnetic resonance overcome sensitivity limitations, especially for low-γ nuclei such as 13C and 15N. Signal Amplification By Reversible Exchange (SABRE) and extended SABRE (X-SABRE) are efficient and low-cost methods for generating large polarizations on a variety of nuclei, but they most commonly use low magnetic fields (μT-mT). High field approaches, where hyperpolarization is generated directly in the spectrometer, are potentially much more convenient but have been limited to selectively hyperpolarize single targets. Here we introduce a new pulse sequence-based approach that affords broadband excitation of SABRE hyperpolarization at high magnetic fields without having to tailor pulse sequence parameters to specific targets. This permits simultaneous hyperpolarization of multiple targets for the first time at high field and offers a direct approach to integration of high-field SABRE hyperpolarization into routine NMR applications, such as NMR-based metabonomics and biomolecular NMR.

15 N SABRE Hyperpolarization of Metronidazole at Natural Isotope Abundance.

Signal Amplification By Reversible Exchange (SABRE) is gaining increased attention as a tool to enhance weak Nuclear Magnetic Resonance (NMR) signals. In SABRE, spin order is transferred from parahydrogen (H2 in its nuclear singlet spin state) to a substrate molecule in a transient Ir-based complex. In recent years, SABRE polarization of biologically active substrates has been demonstrated, notably of metronidazole - an antibiotic and antiprotozoal drug. In this work, we study 15 N SABRE polarization of metronidazole at natural isotope abundance. We are able to demonstrate significant 15 N polarization reaching 15 %, which corresponds to a signal enhancement of 46,000 at 9.4 T for the nitrogen atom with lone electron pair. Additionally, the other two N-atoms can be polarized, although less efficiently. We present a detailed study of the field dependence of polarization and explain the maxima in the field dependence using the concept of coherent polarization transfer at level anti-crossings in the SABRE complex. A study of spin relaxation phenomena presented here enables optimization of the magnetic field for efficient storage of non-thermal polarization.

Hyperpolarization Effects in Parahydrogen Activation with Pnictogen Biradicaloids: Metal-free PHIP and SABRE.

Biradicaloids attract attention as a novel class of reagents that can activate small molecules such as H2, ethylene and CO2. Herein, we study activation of parahydrogen (nuclear spin‐0 isomer of H2) by a number of 4‐ and 5‐membered pnictogen biradicaloids based on hetero‐cyclobutanediyl [X(μ‐NTer)2Z] and hetero‐cyclopentanediyl [X(μ‐NTer)2ZC(NDmp)] moieties (X,Z=P,As; Ter=2,6‐Mes2−C6H3, Dmp=2,6‐Me2−C6H3). The concerted mechanism of this reaction allowed observing strong nuclear spin hyperpolarization effects in 1H and 31P NMR experiments. Signal enhancements from two to four orders of magnitude were detected at 9.4 T depending on the structure. It is demonstrated that 4‐membered biradicaloids activate H2 reversibly, leading to SABRE (signal amplification by reversible exchange) hyperpolarization of biradicaloids themselves and their H2 adducts. In contrast, the 5‐membered counterparts demonstrate rather irreversible parahydrogen activation resulting in hyperpolarized H2 adducts only. Kinetic measurements provided parameters to support experimental observations.

Using SABRE Hyperpolarized 13C NMR Spectroscopy to Interrogate Organic Transformations of Pyruvate.

Signal amplification by reversible exchange (SABRE) is a hyperpolarization technique that uses a metal complex to catalytically transfer magnetization from parahydrogen to molecules of interest. SABRE is used here to monitor the decarboxylation of sodium pyruvate-1,2-[13C2] at a 15 mM concentration to form ethanoic acid and CO2 upon reaction with hydrogen peroxide (150 mM). The rate constant of this reaction is determined by hyperpolarized 13C SABRE-NMR spectroscopy as 0.056 ± 0.003 dm3 mol-1 s-1 at 298 K and is comparable to that determined from thermal 1H NMR (k = 0.050 ± 0.003 dm3 mol-1 s-1) and UV measurements (k = 0.053 ± 0.001 dm3 mol-1 s-1). The hyperpolarized reaction intermediate 2-hydroperoxy-2-hydroxypropanoate is detected in a single scan hyperpolarized 13C NMR spectrum. This work highlights how SABRE hyperpolarization can be used as a tool for the precise monitoring of chemical transformations by hyperpolarized NMR spectroscopy.

The application of novel Ir-NHC polarization transfer complexes by SABRE

In recent years, the hyperpolarization method Signal Amplification By Reversible Exchange (SABRE) has developed into a powerful technique to enhance Nuclear Magnetic Resonance (NMR) signals of organic substrates in solution (mostly via binding to the nitrogen lone pair of N-heterocyclic compounds) by several orders of magnitude. In order to establish the application and development of SABRE as a hyperpolarization method for medical imaging, the separation of the Ir-N-Heterocyclic Carbene (Ir-NHC) complex, which facilitates the hyperpolarization of the substrates in solution, is indispensable. Here, we report for the first time the use of novel Ir-NHC complexes with a polymer unit substitution in the backbone of N-Heterocyclic Carbenes (NHC) for SABRE hyperpolarization, which permits the removal of the complexes from solution after the hyperpolarization of a target substrate has been generated.

In Situ SABRE Hyperpolarization with Earth’s Field NMR Detection

Hyperpolarization methods, which increase the sensitivity of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI), have the potential to expand the range of applications of these powerful analytical techniques and to enable the use of smaller and cheaper devices. The signal amplification by reversible exchange (SABRE) method is of particular interest because it is relatively low-cost, straight-forward to implement, produces high-levels of renewable signal enhancement, and can be interfaced with low-cost and portable NMR detectors. In this work, we demonstrate an in situ approach to SABRE hyperpolarization that can be achieved using a simple, commercially-available Earth’s field NMR detector to provide 1H polarization levels of up to 3.3%. This corresponds to a signal enhancement over the Earth’s magnetic field by a factor of ε > 2 × 108. The key benefit of our approach is that it can be used to directly probe the polarization transfer process at the heart of the SABRE technique. In particular, we demonstrate the use of in situ hyperpolarization to observe the activation of the SABRE catalyst, the build-up of signal in the polarization transfer field (PTF), the dependence of the hyperpolarization level on the strength of the PTF, and the rate of decay of the hyperpolarization in the ultra-low-field regime.

Low-Concentration Measurements of Nuclear Spin-Induced Optical Rotation Using SABRE Hyperpolarization.

Nuclear spin-induced optical rotation (NSOR) is a promising phenomenon for molecular structure elucidation due to its sensitivity to electronic structure near atomic nuclei. It is the only experimentally verified nuclear magneto-optic effect (NMOE), so far observed usually in neat liquids or in concentrated binary mixtures, with the proportion of the minor component at least 10%. We report a method to extend the lower-concentration range of NSOR measurements by 2 orders of magnitude by employing continuous-flow SABRE (signal amplification by reversible exchange) hyperpolarization. This approach significantly increases the sensitivity of NSOR and enables its detection in dilute samples, as demonstrated with measurements of NSOR of 90 mmol/L solutions of pyridine and pyrazine. The results are compared with first-principles calculations, and good agreement is found. The possibility to measure low-concentration solutions significantly extends the pool of samples available for further studies of NMOEs.

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.

Quantitative quantum mechanical approach to SABRE hyperpolarization at high magnetic fields.

A theoretical approach is proposed for quantitative modeling of SABRE (Signal Amplification by Reversible Exchange) experiments performed using an NMR spectrometer at a high magnetic field. SABRE is a method that exploits the spin order of parahydrogen (the H2 molecule in its nuclear singlet state) for hyper-polarizing the spins of various substrates to enhance their NMR signals. An important feature of SABRE is that the substrate is not modified chemically; instead, spin order transfer takes place in a transient complex with parahydrogen. In high-field SABRE experiments, such a transfer is achieved by using suitable NMR excitation schemes. The approach presented here can explicitly treat the spin dynamics in the SABRE complex as well as the kinetics of substrate exchange (between the free and bound form) and complex interplay of spin evolution and chemical processes. One more important effect included in the model is the alteration of the spin state of parahydrogen giving rise to the formation of anti-phase spin order from the initial singlet order. Such a treatment enables a detailed analysis of known high-field SABRE schemes, quantitative comparison with experiments, and elucidation of the key factors that limit the resulting NMR signal enhancement.

High-throughput continuous-flow system for SABRE hyperpolarization.

Signal Amplification By Reversible Exchange (SABRE) is a versatile method for hyperpolarizing small organic molecules that helps to overcome the inherent low signal-to-noise ratio of nuclear magnetic resonance (NMR) measurements. It offers orders of magnitude enhanced signal strength, but the obtained nuclear polarization usually rapidly relaxes, requiring a quick transport of the sample to the spectrometer. Here we report a new design of a polarizing system, which can be used to prepare a continuous flow of SABRE-hyperpolarized sample with a considerable throughput of several millilitres per second and a rapid delivery into an NMR instrument. The polarizer performance under different conditions such as flow rate of the hydrogen or liquid sample is tested by measuring a series of NMR spectra and magnetic resonance images (MRI) of hyperpolarized pyridine in methanol. Results show a capability to continuously produce sample with dramatically enhanced signal over two orders of magnitude. The constant supply of hyperpolarized sample can be exploited, e.g., in experiments requiring multiple repetitions, such as 2D- and 3D-NMR or MRI measurements, and also naturally allows measurements of flow maps, including systems with high flow rates, for which the level of achievable thermal polarization might not be usable any more. In addition, the experiments can be viably carried out in a non-deuterated solvent, due to the effective suppression of the thermal polarization by the fast sample flow. The presented system opens the possibilities for SABRE experiments requiring a long-term, stable and high level of nuclear polarization.

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

A robust medium-scale (approximately 3g) 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 (7T and 9.4T) of an NMR spectrometer and an MRI scanner. SABRE hyperpolarization enabled acquisition of 2D MRI imaging of catalyst-bound 15 N-pyridine with 75×75mm2 field of view (FOV), 32×32 matrix size, demonstrating the feasibility of 15 N HF-SABRE molecular imaging with 2.4×2.4mm2 spatial resolution.

Quasi-Resonance Signal Amplification by Reversible Exchange.

Here we present the feasibility of NMR signal amplification by reversible exchange (SABRE) using radio frequency irradiation at low magnetic field (0.05 T) in the regime where the chemical shifts of free and catalyst-bound species are similar. In SABRE, the 15N-containing substrate and parahydrogen perform simultaneous chemical exchange on an iridium hexacoordinate complex. A shaped spin-lock induced crossing (SLIC) radio frequency pulse sequence followed by a delay is applied at quasi-resonance (QUASR) conditions of 15N spins of a 15N-enriched substrate. As a result of this pulse sequence application, 15N z-magnetization is created from the spin order of parahydrogen-derived hyperpolarized hydrides. The repetition of the pulse sequence block consisting of a shaped radio frequency pulse and the delay leads to the buildup of 15N magnetization. The modulation of this effect by the irradiation frequency, pulse duration and amplitude, delay duration, and number of pumping cycles was demonstrated. Pyridine-15N, acetonitrile-15N, and metronidazole-15N2-13C2 substrates were studied representing three classes of compounds (five- and six-membered heterocycles and nitrile), showing the wide applicability of the technique. Metronidazole-15N2-13C2 is an FDA-approved antibiotic that can be injected in large quantities, promising noninvasive and accurate hypoxia sensing. The 15N hyperpolarization levels attained with QUASR-SABRE on metronidazole-15N2-13C2 were more than 2-fold greater than those with SABRE-SHEATH (SABRE in shield enables alignment transfer to heteronuclei), demonstrating that QUASR-SABRE can deliver significantly more efficient means of SABRE hyperpolarization.

Facile Removal of Homogeneous SABRE Catalysts for Purifying Hyperpolarized Metronidazole, a Potential Hypoxia Sensor.

We report a simple and effective method to remove IrIMes homogeneous polarization transfer catalysts from solutions where NMR Signal Amplification By Reversible Exchange (SABRE) has been performed, while leaving intact the substrate's hyperpolarized state. Following microTesla SABRE hyperpolarization of 15N spins in metronidazole, addition of SiO2 microparticles functionalized with 3-mercaptopropyl or 2-mercaptoethyl ethyl sulfide moieties provides removal of the catalyst from solution well within the hyperpolarization decay time at 0.3 T (T 1>3 mins)-and enabling transfer to 9.4 T for detection of enhanced 15N signals in the absence of catalyst within the NMR-detection region. Successful catalyst removal from solution is supported by the inability to "re-hyperpolarize" 15N spins in subsequent attempts, as well as by 1H NMR and ICP-MS. Record-high 15N nuclear polarization of up to ~34% was achieved, corresponding to >100,000-fold enhancement at 9.4 T, and approximately 5/6th of the 15N hyperpolarization is retained after ~20-second-long purification procedure. Taken together, these results help pave the way for future studies involving in vivo molecular imaging using agents hyperpolarized via rapid and inexpensive parahydrogen-based methods.

Direct enhancement of nitrogen-15 targets at high-field by fast ADAPT-SABRE

Signal Amplification by Reversible Exchange (SABRE) is an attractive nuclear spin hyperpolarization technique capable of huge sensitivity enhancement in nuclear magnetic resonance (NMR) detection. The resonance condition of SABRE hyperpolarization depends on coherent spin mixing, which can be achieved naturally at a low magnetic field. The optimum transfer field to spin-1/2 heteronuclei is technically demanding, as it requires field strengths weaker than the earth’s magnetic field for efficient spin mixing. In this paper, we illustrate an approach to achieve strong 15N SABRE hyperpolarization at high magnetic field by a radio frequency (RF) driven coherent transfer mechanism based on alternate pulsing and delay to achieve polarization transfer. The presented scheme is found to be highly robust and much faster than existing related methods, producing ∼3 orders of magnitude 15N signal enhancement within 2 s of RF pulsing.

NMR Signal Amplification by Reversible Exchange of Sulfur-Heterocyclic Compounds Found In Petroleum.

NMR hyperpolarization via Signal Amplification by Reversible Exchange (SABRE) was employed to investigate the feasibility of enhancing the NMR detection sensitivity of sulfur-heterocycles (specifically 2-methylthiophene and dibenzothiophenes), a family of compounds typically found in petroleum and refined petroleum products. SABRE hyperpolarization of sulfur-heterocycles (conducted in seconds) offers potential advantages of providing structural information about sulfur-containing contaminants in petroleum, thereby informing petroleum purification and refining to minimize sulfur content in refined products such as gasoline. Moreover, NMR spectroscopy sensitivity gains endowed by hyperpolarization potentially allows for performing structural assays using inexpensive, low-magnetic-field (ca. 1 T) high-resolution NMR spectrometers ideally suited for industrial applications in the field.