Signal Amplification By Reversible Exchange Research Articles

SABRE-hyperpolarization dynamics of [1-13C]pyruvate monitored by in situ zero- to ultra-low field NMR

Hyperpolarized [1-13C]pyruvate is the leading metabolite used in the emerging field of hyperpolarization-enhanced MRI. Signal amplification by reversible exchange (SABRE) is a straight forward hyperpolarization method that has recently been shown to hyperpolarize [1-13C]pyruvate at low (microtesla and below) magnetic fields. Here, we show that commercial optical magnetometers with Rb-vapor media can be used to readily monitor the build-up and decay of the hyperpolarized MR signal. In addition, we measure ZULF-NMR spectra in various conditions, ranging from a J-coupling-dominated regime transitioning into a Zeeman-dominated regime when going from a sub-nT field to a µT field. The experimentally acquired spectra are matched well by numerical simulations.

13C MRI of hyperpolarized pyruvate at 120 µT

Nuclear spin hyperpolarization increases the sensitivity of magnetic resonance dramatically, enabling many new applications, including real-time metabolic imaging. Parahydrogen-based signal amplification by reversible exchange (SABRE) was employed to hyperpolarize [1-13C]pyruvate and demonstrate 13C imaging in situ at 120 µT, about twice Earth’s magnetic field, with two different signal amplification by reversible exchange variants: SABRE in shield enables alignment transfer to heteronuclei (SABRE-SHEATH), where hyperpolarization is transferred from parahydrogen to [1-13C]pyruvate at a magnetic field below 1 µT, and low-irradiation generates high tesla (LIGHT-SABRE), where hyperpolarization was prepared at 120 µT, avoiding magnetic field cycling. The 3-dimensional images of a phantom were obtained using a superconducting quantum interference device (SQUID) based magnetic field detector with submillimeter resolution. These 13C images demonstrate the feasibility of low-field 13C metabolic magnetic resonance imaging (MRI) of 50 mM [1-13C]pyruvate hyperpolarized by parahydrogen in reversible exchange imaged at about twice Earth’s magnetic field. Using thermal 13C polarization available at 120 µT, the same experiment would have taken about 300 billion years.

A Straightforward Method for the Generation of Hyperpolarized Orthohydrogen with a Partially Negative Line.

The hydrogen molecule, which exists in two spin isomers (ortho- and parahydrogen), is a highly studied system due to its fundamental properties and practical applications. Parahydrogen is used for Nuclear Magnetic Resonance signal enhancement, which is hyperpolarization of other molecules, including biorelevant ones. Hyperpolarization can be achieved by using Signal Amplification by Reversible Exchange (SABRE). SABRE can also convert parahydrogen into orthohydrogen, and surprisingly, in some cases, it has been discovered that orthohydrogen's resonance has the Partially Negative Line (PNL) pattern. Here, an approach for obtaining orthohydrogen with a PNL signal is presented for two catalysts: Ir-IMes, and Ir-IMesBn. The type of solvent in which SABRE is conducted is crucial for the observation of PNL. Specifically, a PNL signal can be easily generated in benzene using both catalysts, but it is more intense for Ir-IMesBn. In acetone, PNL is observed only for Ir-IMesBn. In methanol, no PNL is detected. The PNL effect is only detectable during the initial steps of pre-catalyst activation, and disappears as the activation process progresses. We have proposed a working hypothesis that explains our results. The presented data may facilitate the further investigation of PNL and its applications in material science and catalysis.

A Straightforward Method for the Generation of Hyperpolarized Orthohydrogen with a Partially Negative Line

AbstractThe hydrogen molecule, which exists in two spin isomers (ortho‐ and parahydrogen), is a highly studied system due to its fundamental properties and practical applications. Parahydrogen is used for Nuclear Magnetic Resonance signal enhancement, which is hyperpolarization of other molecules, including biorelevant ones. Hyperpolarization can be achieved by using Signal Amplification by Reversible Exchange (SABRE). SABRE can also convert parahydrogen into orthohydrogen, and surprisingly, in some cases, it has been discovered that orthohydrogen's resonance has the Partially Negative Line (PNL) pattern. Here, an approach for obtaining orthohydrogen with a PNL signal is presented for two catalysts: Ir−IMes, and Ir−IMesBn. The type of solvent in which SABRE is conducted is crucial for the observation of PNL. Specifically, a PNL signal can be easily generated in benzene using both catalysts, but it is more intense for Ir−IMesBn. In acetone, PNL is observed only for Ir−IMesBn. In methanol, no PNL is detected. The PNL effect is only detectable during the initial steps of pre‐catalyst activation, and disappears as the activation process progresses. We have proposed a working hypothesis that explains our results. The presented data may facilitate the further investigation of PNL and its applications in material science and catalysis.

Magnetic field dependence of the para-ortho conversion rate of molecular hydrogen in SABRE experiments

The use of parahydrogen – the isomer of molecular hydrogen with zero nuclear spin – is important for promising and actively developing methods for spin hyperpolarization of nuclei called parahydrogen induced polarization (PHIP). However, the dissolved parahydrogen in PHIP experiments quickly loses its spin order, resulting in the formation of orthohydrogen and reduction of the overall nuclear polarization of the substrate. This process is due to the difference of chemical shifts of hydride protons, as well as spin–spin couplings between nuclei, in the intermediate catalytic complexes, and it has not been rigorously explained so far. We proposed a new experimental technique based on magnetic field cycling for measuring the rate of molecular hydrogen para–ortho conversion in solution and applied it for non-hydrogenative PHIP Signal Amplification By Reversible Exchange (SABRE) experiments. The para–ortho conversion rate was measured over a wide range of magnetic field from 0.5 mT to 9.4 T. It was found that the conversion rate strongly depends on the magnetic field in which the reaction occurs, as well as on the concentrations of reactants. The rate decreases with increasing the concentration of pyridine ligand and increases with increasing the concentration of iridium catalyst. The model, which takes into account the reversible exchange of molecular hydrogen with the catalyst, nuclear spin–spin interaction of hydride protons with nuclei of ligands within catalytic complex and nuclear Zeeman interactions, qualitatively describes the experimental data. Two types of complexes with different spin system symmetry contribute to the molecular hydrogen conversion. In asymmetric complexes possessing hydride protons with different chemical shifts due to the presence of chlorine anion ligand the para–ortho conversion rate increases with magnetic field, while for symmetric complexes this mechanism is not operable. In the magnetic field where level anti-crossing occurs the resonant feature for the rate of para–ortho conversion is found. The results of this work can be utilized for finding the optimal conditions for obtaining the maximum hyperpolarization in the experiments employing parahydrogen.

J Coupling Constants of

Observing pyruvate metabolism in vivo has become a focal point of molecular magnetic resonance imaging. Signal amplification by reversible exchange (SABRE) has recently emerged as a versatile hyperpolarization technique. Tuning of the spin order transfer (SOT) in SABRE is challenging as the small 1H-13C J couplings, in the 13C-pyruvate case, result in SOT being not readily discernible. We demonstrate an experimental method using frequency-selective excitation of parahydrogen-derived polarization SOT sequence (SEPP-SPINEPT); its application led to up to 5700-fold 13C signal gain. In this way, we estimated the lifetime of two Ir-pyruvate SABRE complexes alongside the individual probing of eight small 1H-13C J couplings that connect the hydride protons in these complexes to 1- and 2-13C pyruvate spins, affording values between 0 and 2.69 Hz. Using electronic structure calculations, we define the low-energy structure of the corresponding complexes. Hence, this study demonstrates a novel approach to analyzing the spin topology of short-lived organometallic complexes.

LC-Photo-CIDNP hyperpolarization of biomolecules bearing a quasi-isolated spin pair: Magnetic-Field dependence via a rapid-shuttling device

Liquid-state low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an emerging technology tailored to enhance the sensitivity of NMR spectroscopy via LED- or laser-mediated optical irradiation. LC-photo-CIDNP is particularly useful to detect solvent-exposed aromatic residues (Trp, Tyr), either in isolation or within polypeptides and proteins. This study investigates the magnetic-field dependence of the LC-photo-CIDNP of Trp-α-13C-β,β,2,4,5,6,7-d7, a Trp isotopolog bearing a quasi-isolated 1Hα-13Cαspin pair (QISP). We employed a new rapid-shuttling side-illumination field-cycling device that enables ultra-fast (90–120 ms) vertical movements of NMR samples within the bore of a superconducting magnet. Thus, LC-photo-CIDNP hyperpolarization occurs at low field, while hyperpolarized signals are detected at high field (700 MHz). Resonance lineshapes were excellent, and the effect of several fields (1.18–7.08 T range) on hyperpolarization efficiency could be readily explored. Remarkably, unprecedented LC-photo-CIDNP enhancements ε ≅ 1,200 were obtained at 50 MHz (1.18 T), suggesting exciting avenues to hypersensitive LED-enhanced NMR in liquids at low field.

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.

Photo‐SABRE: Nuclear Spin Hyperpolarization of cis‐trans Photoswitchable Molecules by Parahydrogen**

AbstractThis work reports the 23000‐ and 9200‐fold enhancement of the 15N nuclear spin polarization of cis and trans isomers of azobenzene, correspondingly, as compared to the thermal NMR signals at 9.4 T by exploiting signal amplification by reversible exchange (SABRE) and parahydrogen molecules at 400 nT simultaneously with light irradiation. Only cis‐azobenzene gains substantial hyperpolarization of its 15N spins directly from parahydrogen in SABRE through coherent polarization transfer. Consequently cis‐trans photoisomerization at ultralow magnetic field preserves the SABRE‐derived nuclear hyperpolarization of cis‐azobenzene resulting in hyperpolarization of trans‐azobenzene as well, despite its direct coordination with the SABRE Ir‐complex being sterically hindered. Moreover, the proposed approach, which we called photo‐SABRE, allows to hyperpolarize the long‐lived spin order of trans‐isomer 15N spins with a lifetime of about 25 minutes, which greatly exceeds their relaxation times at high (10 seconds) and low (200 seconds) magnetic field. Since this spin order in 15N2‐trans‐azobenzene is collectively formed by nuclei of different kinds, it can be detected by both 15N or 1H NMR spectroscopy.

Water-Compatible and Recyclable Heterogeneous SABRE Catalyst for NMR Signal Amplification.

A water-compatible and recyclable catalyst for nuclear magnetic resonance (NMR) hyperpolarization via signal amplification by reversible exchange (SABRE) was developed. The [Ir(COD)(IMes)Cl] catalyst was attached to a polymeric resin of bis(2-pyridyl)amine (heterogeneous SABRE catalyst, HET-SABRE catalyst), and it amplified the 1H NMR signal of pyridine up to (-) 4455-fold (43.2%) at 1.4 T in methanol and (-) 50-fold (0.5%) in water. These are the highest amplification factors ever reported among HET-SABRE catalysts and for the first time in aqueous media. Moreover, the HET-SABRE catalyst demonstrated recyclability by retaining its activity in water after more than three uses. This newly designed polymeric resin-based heterogeneous catalyst shows great promise for NMR signal amplification for biomedical NMR and MRI applications in the future.

Essential tools of linear algebra for calculating nuclear spin dynamics of chemically exchanging systems

In this work, we describe essential tools of linear algebra necessary for calculating the effect of chemical exchange on spin dynamics and polarization transfer in various nuclear magnetic resonance (NMR) experiments. We show how to construct matrix representations of Hamiltonian, relaxation, and chemical exchange superoperators in both Hilbert and Liouville space, as well as demonstrate corresponding codes in Python. Examples of applying the code are given for problems involving chemical exchange between NH3 and NH4+ at zero and high magnetic field and polarization transfer from parahydrogen relevant in SABRE (signal amplification by reversible exchange) at low magnetic field (0-20mT). The presented methodology finds utility for describing the effect of chemical exchange on NMR spectra and can be extended further by taking into account non-linearities in the master equation.

In Vivo Metabolic Imaging of [1-13 C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra-fast and low-cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1-13 C]pyruvate-d3 for injection in 6 minutes. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13 C]pyruvate-d3 polarized to ≈11 % (residual 250 mM methanol and 20 μM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Facile hyperpolarization chemistry for molecular imaging and metabolic tracking of [1–13C]pyruvate in vivo

Hyperpolarization chemistry based on reversible exchange of parahydrogen, also known as Signal Amplification By Reversible Exchange (SABRE), is a particularly simple approach to attain high levels of nuclear spin hyperpolarization, which can enhance NMR and MRI signals by many orders of magnitude. SABRE has received significant attention in the scientific community since its inception because of its relative experimental simplicity and its broad applicability to a wide range of molecules, however, in vivo detection of molecular probes hyperpolarized by SABRE has remained elusive. Here we describe a first demonstration of SABRE-hyperpolarized contrast detected in vivo, specifically using hyperpolarized [1–13C]pyruvate. Biocompatible formulations of hyperpolarized [1–13C]pyruvate in, both, methanol-water, and ethanol-water mixtures followed by dilution with saline and catalyst filtration were prepared and injected into healthy Sprague Dawley and Wistar rats. Effective hyperpolarization-catalyst removal was performed with silica filters without major losses in hyperpolarization. Metabolic conversion of pyruvate to lactate, alanine, and bicarbonate was detected in vivo. Pyruvate-hydrate was also observed as a minor byproduct. Measurements were performed on the liver and kidney at 4.7 T via time-resolved spectroscopy and chemical-shift-resolved MRI. In addition, whole-body metabolic measurements were obtained using a cryogen-free 1.5 T MRI system, illustrating the utility of combining lower-cost MRI systems with simple, low-cost hyperpolarization chemistry to develop safe and scalable molecular imaging.

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

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

Parahydrogen in Reversible Exchange Induces Long-Lived 15N Hyperpolarization of Anticancer Drugs Anastrozole and Letrozole.

Hyperpolarization modalities overcome the sensitivity limitations of NMR and unlock new applications. Signal amplification by reversible exchange (SABRE) is a particularly cheap, quick, and robust hyperpolarization modality. Here, we employ SABRE for simultaneous chemical exchange of parahydrogen and nitrile-containing anticancer drugs (letrozole or anastrozole) to enhance 15N polarization. Distinct substrates require unique optimal parameter sets, including temperature, magnetic field, or a shaped magnetic field profile. The fine tuning of these parameters for individual substrates is demonstrated here to maximize 15N polarization. After optimization, including the usage of pulsed μT fields, the 15N nuclei on common anticancer drugs, letrozole and anastrozole, can be polarized within 1-2 min. The hyperpolarization can exceed 10%, corresponding to 15N signal enhancement of over 280,000-fold at a clinically relevant magnetic field of 1 T. This sensitivity gain enables polarization studies at naturally abundant 15N enrichment level (0.4%). Moreover, the nitrile 15N sites enable long-lasting polarization storage with [15N]T1 over 9 min, enabling signal detection from a single hyperpolarization cycle for over 30 min.

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.

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.

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.

Real-Time Reaction Monitoring of Azide-Alkyne Cycloadditions Using Benchtop NMR-Based Signal Amplification by Reversible Exchange (SABRE).

Rufinamide, possessing a triazole ring, is a new antiepileptic drug (AED) relatively well-absorbed in the lower dose range (10 mg/kg per day) and is currently being used in antiepileptic medications. Triazole derivatives can interact with various enzymes and receptors in biological systems via diverse non-covalent interactions, thus inducing versatile biological effects. Strain-promoted azide-alkyne cycloaddition (SPAAC) is a significant method for obtaining triazoles, even under physiological conditions, in the absence of a copper catalyst. To confirm the progress of chemical reactions under biological conditions, research on reaction monitoring at low concentrations is essential. This promising strategy is gaining acceptance for applications in fields such as drug development and nanoscience. We investigated the optimum Ir catalyst and magnetic field for achieving maximum proton hyperpolarization transfer in triazole derivatives. These reactions were analyzed using signal amplification by reversible exchange (SABRE) to overcome the limitations of low sensitivity in nuclear magnetic resonance spectroscopy, when monitoring copper-free click reactions in real time. Finally, a more versatile copper-catalyzed click reaction was monitored in real time, using a 60 MHz benchtop NMR system, in order to analyze the reaction mechanism.